In order to reveal the effect of parathyroid hormone-like hormone (PTHLH) gene on the reproductive ability of buffalo,buffalo PTHLH gene was cloned,and its nucleotide and amino acid sequences were analyzed by bioinformatics.Primers were designed by CE Design according to the sequence of cattle PTHLH gene in GenBank (accession No.:NM_001290949).The coding region of buffalo PTHLH gene was cloned by PCR amplification and sequencing technology.The primary structure,secondary structure,tertiary structure and physicochemical properties of PTHLH protein,and homology were analyzed,and phylogenetic tree of PTHLH was constructed by softwares such as DNAMAN,ProtParam,SOPMA and PSORTⅡ Prediction,et al.The results showed that PTHLH gene in buffalo was cloned,including a 534 bp length coding region and 177 amino acids.Compared with cattle,pigs,horses,goats,sheep and camels,the nucleotide sequence of gene coding region homology of PTHLH gene in buffalo were 98.3%,90.4%,90.1%,98.1%,97.5% and 89.2%,respectively.Thus,there was a high homology among different species,and the phyletic evolution was the same as their genetic relationship.The research indicated that PTHLH gene was conservative in the course of evolution.The physicochemical properties analysis result showed that the molecular formula of PTHLH protein was C₈₉₅H₁₄₅₁N₂₇₁O₂₆₆S₂,the molecular weight was 2 885 u,the half-life was 30 h,the theoretical electrical points (pI) was 10.00,the extinction coefficient of water solution at 280 nm was 23 950,the peptide chain N-terminal was Met,the unstable coefficient was 60.04,thus PTHLH protein belong to the basic unstable protein.The fat coefficient of PTHLH protein was 72.15,and the total average hydrophilicity was -0.928,so it belonged to insoluble protein.The protein subpopulations were located in the nucleus,cytoplasm and mitochondria.The structural domain prediction results showed that buffalo PTHLH protein contained a PTH region and a low complexity region.The secondary structure analysis showed that buffalo PTHLH protein contained 83 alpha helix (46.89%),17 extended chain (9.60%),10 beta turn (5.66%) and 67 random coil (37.85%),and consistent with the tertiary structure prediction.In this study,the eukaryotic expression vector was constructed,and the accuracy of the carrier was verified by electrophoresis and sequencing.In summary,the successful cloning of PTHLH gene and the successful construction of its eukaryotic expression vector provide materials for the future study of the function and genetic characteristics of PTHLH gene in buffalo.

This experiment was aimed to express recombinant neuraminidase(NA) protein of H6N6 subtype avian influenza virus (AIV) isolated from Guizhou province,and prepared polyclonal antibody against NA protein of AIV.The specific primers were designed according to N6 gene of AIV in GenBank(accession No.:MG434500),and used for amplification of N6 gene by PCR.PCR product was cloned into the expression vector pET-32a(+).The soluble recombinant protein was expressed in E.coli BL21(DE3) with IPTG.The expression product was sonicated and purified by nickel column.The recombinant proteins were identified by SDS-PAGE and Western blotting.The polyclonal antibody was prepared from the rabbit immunized with purified recombinant protein.The titer of the antibody was detected by indirect ELISA.The results showed that coding region of N6 gene was 1 380 bp,which encoded 459 amino acids.There were 33 nucleotides deletion from position 175 to 207 bp at N6 gene stalk.The recombinant plasmid pET-32a-N6 was identified by double enzyme digestion,it appeared about 5 900 bp vector band and about 1 380 bp target band,pET-32a-N6 recombinant plasmid was successfully constructed;SDS-PAGE analysis result showed the molecular weight of the recombinant N6 protein was about 70 ku which consistented with expected result.It showed that the target gene was successfully expressed in the form of inclusion body.The purified recombinant protein was double-stained by SDS-PAGE and Western blotting,and showed a band of 70 ku in size,indicating that the purified protein was the recombinant protein pET-32a-N6.The purified protein of denaturation and renaturation could be specifically recognized by His-antibody and rabbit-anti-N6 hyperimmune serum,respectively.The titer of the antibody was about 1:3 200 by detection of indirect ELISA.All the results indicated that N6 gene of H6N6 subtype AIV was successfully cloned and expressed,the prepared anti-N6 showed good immune activity,and the protein purified could be specifically recognized by His-antibody and rabbit-anti-N6 hyperimmune serum.

In order to investigate the effect of glycosylation modification on the immunogenicity of Brucella P39 protein,the expression and purification of Brucella P39 protein were carried out in this experiment,and the expressed protein was modified with mannose hexasaccharide.The primers were designed according to the Brucella P39 gene sequence published in GenBank,and the P39 gene fragment was cloned from the Brucella 16M genome and ligated into pMD19-T vector to transform E.coli DH5α competent cells.The positive strain plasmid was extracted and identified by enzyme digestion.After correct identification,the recombinant plasmid pGEX-6P-1-P39 was constructed,and the induction expression and optimization of recombinant protein were analyzed.The target protein induced was analyzed by SDS-PAGE and Western blotting,and the correct protein was purified by GST affinity chromatography.The purified P39 protein was modified by mannose hexasaccharide using EDC/NHS method to study the effect of the target protein on the phagocytosis of macrophages after modification of mannose hexose.The results showed that the target gene with a fragment size of 1 206 bp was successfully cloned,and the prokaryotic expression vector pGEX-6P-1-P39 was constructed.The P39 protein was successfully expressed in E.coli,and the protein was mainly soluble.Western blotting results showed a specific band at approximately 65 ku.After purification,the target protein size was 43 ku,the target protein P39 was successfully glycosylated,and the molar ratio of the modified product to the protein was 2.3:1.In addition,glycosylation modification could significantly advance the phagocytosis time of mouse macrophages by protein activation.This study could provide a reference for research the mechanism of mannose hexose modification affecting the immunogenicity of P39 protein.

This study was aimed to establish a loop-mediated isothermal amplification (LAMP) to detect Klebsiella pneumoniae in food.The virulence gene region unique to Klebsiella pneumoniae was selected,and two pairs of primers (one pair of inner primers and one pair of external primers) were designed to perform an in vitro constant temperature amplification reaction.The betaine,nucleic acid material (dNTPs),internal and external primer ratio,Mg²⁺ concentration,reaction time and temperature in the reaction system were optimized and selected,and specificity and sensitivity tests were performed.At the same time,the established LAMP method was used to detect the artificial contamination of the sterilized chicken.Through optimization experiments,the optimal reaction system and reaction conditions were screened.Using the optimized LAMP reaction system,specific detection tests were performed on 18 common microorganisms including Klebsiella pneu- monia,the results showed that the method was specific.The minimum detection limit for the genome of Klebsiella pneumonia LAMP reaction system was 0.875 pg/μL,which was 100 times more sensitive than traditional PCR method.The artificial chicken positive samples were tested and the detection limit was 30 CFU/mL.This experiment successfully established a LAMP detection method for detecting pathogenic Klebsiella pneumonia in food.Two pairs of primers were used to thermostatically amplify the target DNA in 6 regions of Klebsiella pneumoniae with staircase product in vitro,which was more sensitive with shorter reaction time and lower testing cost.

The study was aimed to clone and bioinformatics analysis of lmo2192 gene of Listeria monocytogenes LM90SB2 isolated from diseased sheep in Xinjiang.Specific primers were designed according to the lmo2192 gene sequence in GenBank (accession No.:CAD00270).The PCR method was used to amplify lmo2192 gene.The PCR products were purified and cloned into pMD19-T vector,and then the positive colonies were screened and sequenced,and the nucleotide sequence of lom2192 gene was sequenced and analyzed.The secondary and tertiary structures of the encoded proteins were predicted,and homology and genetic variation analysis were performed.The results showed that LM90SB2 lmo2192 gene was 1 277 bp in length,containing a 969 bp open reading frame (ORF) that encoded 322 amino acids.The nucleotide sequence homology of lmo2192 gene of LM90SB2 strain was 100.0% with CⅡMS-PH-1;99.8% to 99.9% with 81-0861,10-0809,81-0592,81-0558,NTSN,F2365 and WSLC1033;And 97.0% and 96.9% with L2074 and NH1,respectively.The putative amino acid sequence homology was 91.6% to 100.0% with above strains.The phylogenetic tree showed that the lmo2192 gene of LM90SB2 strain was closely related to the serotype 4b strain and clustered into the same branch.The protein secondary structure prediction result showed that LM90SB2 lmo2192 protein was a hydrophilic protein without signal peptide and transmembrane structure.The protein domain predicted result showed that the lmo2192 protein was an ATPase component.The lmo2192 gene of LM90SB2 strain was successfully cloned,it provided theoretical basis for further study of the lmo2192 gene function of LM90SB2.

To investigate the mechanism of Mycobacterium bovis inhibiting apoptosis,proteins interacted with human TNFR1 associated death domain (TRADD) were screened by yeast two-hybrid system.In order to construct the genome library,the genome of Mycobacterium bovis was digested by Sau3AⅠ,and then inserted into the vector pGADT7,and transformed into E.coli DH5α.tradd gene was amplified by PCR and inserted into the vector pGBKT7.The recombinant vector named pGBKT7-tradd was transformed into yeast Y2HGold.The genome library of Mycobacterium bovis was screened to obtain positive clones interacting with human TRADD.The positive clones were confirmed by DNA sequencing and BLAST.The results showed that the titer of library was 2×10⁶ CFU with the average of inserted fragments about 1.5 kb,and the recombinant rate was >95%.TRADD expression without toxicity and autoactivation in yeast was detected by Western blotting.Seven proteins interacting with TRADD were obtained by homology analysis from 20 positive colonies.The candidate proteins in the study were provided the clue to research the infection mechanism of Mycobacterium bovis.

In order to analyze the expression of Myomaker gene in skeletal muscle of adult mice,the total RNA was extracted from the skeletal muscle of three randomly selected healthy adult mice,and the coding sequence (CDS) of Myomaker gene was amplified by PCR method and analyzed by bioinformatics software.In addition,immunohistochemical and Western blotting methods were used to determine the localization and expression of Myomaker protein in skeletal muscle.The results showed that the CDS region of Myomaker gene was 666 bp,encoding 221 amino acids.The sequence homology of the nucleotide of Myomaker gene in mice were 71.1%,80.7%,83.7%,83.2%,84.1%,86.4%,80.6% and 84.0% comparing with Danio rerio,Gallus gallus,Felis catus,Bos taurus,Canis lupus familiaris,Homo sapiens,Nestor notabilis,Sus scrofa,respectively,and the sequence homology of the amino acids were 75.9%,79.3%,85.5%,85.5%,88.3%,86.9%,81.4% and 93.2%,respectively.The formula of Myomaker protein was C₁₁₅₄H₁₇₆₉N₂₇₅O₂₉₅S₁₈,and the molecular weight,theory isoelectric point,instability index and grand average of hydrophobicity was 24 ku,9.14,39.22 and 0.501,respectively.Immunohistochemical and Western blotting analysis showed that the Myomaker protein expressed in skeletal muscle of healthy adult mice,but it just existed in partial myocyte membrane.This study laid a foundation for the further study of Myomaker gene.

The experiment was aimed to predict the metabolic protein production of different corn by-products based on the NRC-2001 model,and analyze the molecular functional group characteristics of different corn by-products and the correlation between them based on the spectral technique,and establish a regression equation.The nutrient value of dairy cow protein was evaluated by NRC-2001 model,and the spectral data was collected and analyzed by Fourier transform infrared (FTIR) technique.The results showed that the metabolic parameters of different corn by-products with significant differences (P<0.05) predicted by the NRC-2001 model.Among them,the total amount of truly absorbable metabolic protein in the small intestine had the highest value in corn gluten meal,followed by corn germ cake,corn germ,corn gluten feed and corn kernel.There was a similar trend in feed milk value (FMV).In the spectral structure analysis,there were significant differences in the molecular functional group parameters of the feed (P<0.05).There was a correlation between the predicted yield of protein metabolites and the molecular functional group parameters.The regression model established was better.Among them,the value of Amide Ⅰ_CELC in corn kernel was significantly higher than other feeds (P<0.05), the values of Amide Ⅰ_SCHO and Amide Ⅰ_CELC were significantly lower in corn germ (P<0.05), the values of Amide Ⅱ_SCHO and Amide Ⅱ_CELC were significantly lower in corn gluten feed than that others (P<0.05),and the value of Amide Ⅱ_TNSCHO of the corn germ cake was significantly higher than that of other feeds (P<0.05).There was no significant correlation between the contents of MCP,AMCP and Amide Ⅰ_TNSCHO in corn and its by-products (P>0.05).However,there was a significant negative correlation between the characteristic parameters and other spectral molecular functional groups (P<0.05). Amide Ⅰ_TCHO and Amide Ⅱ_SCHO could be used as predictors to estimate ARUP,MP and FMV,with a coefficient of determination of 0.997.In conclusion,there was a relationship between the predicted metabolic parameters of protein and biomolecular spectroscopic features in different corn by-products and the nutritional value of feed could be quickly analyzed and estimated by the Fourier transform infrared spectroscopy.

This experiment was conducted to investigate the silage quality and nutritive value of whole-plant corn silage of different varieties.Five different varieties of corn were harvested at 1/2 to 3/4 milk line and silaged in plastic bags (50 cm×80 cm) for 60 d (15 to 20℃),then sensory evaluation and fermentation status were analyzed.Chemical and digestion analysis were carried out after the samples were air-dried.The results showed that the overall sensory scores for whole-plant corn silage were good except for Jiyuan 128 which was fair.Lactic acid concentration of Wofeng 9 (6.05%DM) was significantly higher than Jiyuan 128(5.13%DM) and Zhengdan 958 (5.13%DM)(P<0.05),while acetic acid and propionic acid concentrations were extremely significantly lower than Luyu 36,Zhengdan 958 and Jingke 25 (P<0.01).The V-scores were all above 97.00 of whole-plant corn silage.The contents of CP of Jingke 25,EE of Wofeng 9,WSC of Zhengdan 958 and starch of Jiyuan 128 was the highest among the five varieties of corn silage,which were 9.24%DM,3.11%DM,2.84%DM and 30.33%DM,respectively.The RFV of different varieties corn silage from high to low were:Wofeng 9(163.00) > Jiyuan 128(160.93) > Zhengdan 958 (156.30) > Jingke 25(152.70) > Luyu 36(151.77).The 24 h NDFD of Zhengdan 958 and Wofeng 9 (17.84%DM;18.28%DM) were extremely significantly lower than that of Jingke 25 and Jiyuan 128 (24.87%DM;22.97%DM) (P<0.01).In conclusion,the RFV of Wofeng 9 was the highest and its sensory evaluation,lactic acid concentration,V-score,EE and WSC contents were higher,while NH₃-N/TN,NDF and ADF contents were lower,therefore,Wofeng 9 performed better than others under this experimental condition.The second was Jiyuan 128 whose RFV and 48 h NDFD were higher,while ADF and ADL contents were the lowest.

The aim of this experiment was to investigate the effects of dietary vitamin D and calcium levels on growth performance,nutrient digestibility and metabolisms of nitrogen,calcium and phosphorus of winter fur-growing mink at a fixed ratio of calcium to phosphorus.One hundred and seventeen healthy male minks at the age of (135±5) days were randomly divided into 9 groups with 13 replicates per group and 1 mink per replicate.Nine experimental diets were formulated by using 3×3 double factorial experiment design.Dietary calcium to phosphorus ratio was fixed at 1.7:1,and three vitamin D levels were 2 300,4 300 and 6 300 IU/kg,respectively,while three calcium levels were 2.0%,2.4% and 2.8%,respectively.The levels of vitamin D and calcium in the 9 diets were 2 300 IU/kg and 2.0% (group Ⅰ),4 300 IU/kg and 2.0% (group Ⅱ),6 300 IU/kg and 2.0% (group Ⅲ),2 300 IU/kg and 2.4% (group Ⅳ),4 300 IU/kg and 2.4%(group Ⅴ),6 300 IU/kg and 2.4% (group Ⅵ),2 300 IU/kg and 2.8% (group Ⅶ),4 300 IU/kg and 2.8% (group Ⅷ),6 300 IU/kg and 2.8% (group Ⅸ).The pretrial lasted for 13 days,and the formal trial lasted for 60 days.The results showed as follows:①There was a extremely significant difference of F/G in mink among groups (P<0.01),and that in group Ⅱ was the lowest.The calcium levels had a significant effect on the F/G (P<0.05),while the vitamin D levels had an extremely significant effect on the F/G (P<0.01).②The calcium levels had an extremely significant effect on fat digestibility (P<0.01),and that of 2.8% calcium level was extremely significantly lower than 2.0% and 2.4% calcium levels (P<0.01).②The dietary levels of vitamin D and calcium had no significant effect on nitrogen metabolism indexes of mink (P>0.05).④There was an extremely significant difference in the fecal calcium,fecal phosphorus,calcium digestibility and phosphorus digestibility of mink among the groups (P<0.01).The digestibility of calcium and phosphorus were the highest in group Ⅱ,followed by group Ⅲ.The calcium level had an extremely significant effect on fecal calcium,fecal phosphorus,digestibilities of calcium and phosphorus (P<0.01),and 2.0% calcium level extremely significantly decreased fecal calcium and fecal phosphorus contents (P<0.01),and increased calcium and phosphorus digestibilities (P<0.01).Vitamin D levels had a significant effect on calcium digestibility (P<0.05), and had an extremely significant effect on fecal phosphorus and phosphorus digestibility (P<0.01).Vitamin D levels of 2 300 to 4 300 IU/kg could increase digestibilities of calcium and phosphorus,reduce the fecal phosphorus content.Comprehensive analysis of various indicators,when the dietary calcium to phosphorus ratio was 1.7:1,the vitamin D level was 2 300 to 4 300 IU/kg,and the calcium level was 2.0%,minks during winter fur-growing period could get a lower F/G,calcium and phosphorus emissions,higher EE,calcium and phosphorus digestibility.

In order to explore the growth profiles and growth rates of Danzhou chicken at different stages,three nonlinear analysis models (Gompertz,Logistic and Bertalanffy) were employed to fit and analyze the body weight growth curves of Danzhou chickens with black and mahua colors feather at the age of 0 to 18 weeks.200 Danzhou chickens with black feather (100 males and 100 females) and 200 Danzhou chickens with mahua colors feather (100 males and 100 females) were selected and weighed at 0,2,4,6,8,10,12,14,16 and 18 weeks of age,and the body weight growth curves were fitted and analyzed by Gompertz,Logistic and Bertalanffy models,respectively.The results showed that the growth curve of Danzhou chickens demonstrated a "S" shape,consistent with normal growth and development.The measured body weights of female Danzhou chickens during 0 to 18 weeks and male Danzhou chickens during 0 to 16 weeks showed no significant difference (P>0.05),and that of male Danzhou chickens with mahua colors feather were significantly higher than that with black feather at 16 to 18 weeks (P<0.05).The growth curves predicted by three nonlinear models all fitted well with the measured body weight curves of Danzhou chickens with black and mahua colors feather (R²>0.992),and the prediction curve of the model was basically consistent with the actual observation curve.the Gompertz model fitted better than Logistic or Bertalanffy models,and it fitted better in female chickens than male chickens.In summary,the present study revealled the growth profiles and growth rates of Danzhou chicken at different stages,which may provide scientific support for optimizing breeding production and industrialization of Danzhou chicken.

The experiment was conducted to study the effects of Macleaya cordata extracts on growth performance,serum immune indexes and antioxidant indexes of weaned piglets.120 "Duroc×Landrace×Yorkshire" hybrid pigs with 35 days of age were selected and randomly divided into 2 treatment groups with 5 replicates per group and 12 pigs per replicate,according to similar body weight and the same ratio of gender.The experiment lasted for 30 days with 5 d for preliminary trial.The weaned piglets in control group fed with the basal diet,and that in experimental group fed with basal diet supplemented 0.1% Macleaya cordata extracts.During the experiment,the growth performance was measured (ADG,ADFI and F/G),and at the end of the experiment,two piglets were chosen from each replicate for blood sampling and then the immune and antioxidant indexes were determined.The results showed as follow:①The ADG of weaned piglets supplemented with Macleaya cordata extracts was increased by 3.89% than that of the control group (P>0.05),and the F/G was decreased by 4.32% (P>0.05),meanwhile the diarrhea rate had a significantly decreasing trend(P=0.076).②Compared with the control group,the serum IgG and TNF-α contents of weaned piglets supplemented with Macleaya cordata extracts were significantly increased (P<0.05),the contents of IgA and IgM were increased by 4.59% and 4.60%,respectively (P>0.05).③Adding Macleaya cordata extracts in the diet could significantly reduce the serum MDA content (P<0.05),and significantly increase the T-SOD activity (P<0.05).In conclusion,the addition of 0.1% Macleaya cordata extract in the diet could reduce the diarrhea rate,increase the body immunity and antioxidant capacity,and had no adverse effect on growth performance of weaned piglets.

Clostridium butyricum is an anaerobic heterotrophic gram-positive Bacillus,which produces butyric acid,lactic acid and acetic acid.Clostridium butyricum has the biological characteristics of heat resistance,acid resistance and tolerance to various antibiotics.Clostridium butyricum is a kind of green additive with extensive development potential which can be colonized in the intestines of livestock and poultry,inhibit the growth of intestinal harmful bacteria and promote the multiplication of beneficial bacteria,can effectively improve the digestion and absorption of nutrients and the immunity of livestock and poultry,increase the economic benefit of livestock and poultry breeding.Clostridium butyricum can be found in soil or healthy animals feces,which is green,non-polluting and no residue additive with extensive development potential to protect the animals health.This paper introduced the biochemical characteristics of Clostridium butyricum,summarized its effects on intestinal morphology,intestinal barrier,intestinal flora,and immunity of livestock and poultry.The research looked back on the application progress of Clostridium butyricum in livestock and poultry production in recent ten years,and discussed the problems in the practical application in order to provide references for further exploitation of Clostridium butyricum using in animal safety production.

In order to improve the growth performance,Hainan Black goats were fed with different ratios of Stylosanthes and kinggrass silage in this experiment,so as to select the optimum proportion of the two kinds of roughage.Sixteen Hainan Black goats with similar age and weight were randomly divided into 4 groups:Control group (CK group,roughage was kinggrass) and three test groups (S-KS2,S-KS3 and S-KS4 groups,Stylosanthes and kinggrass silage were combined at dry matter ratios of 20:80,30:70 and 40:60,respectively).After 60 days of feeding,the growth performance,serum total protein (TP),albumin (ALB),globulin (GLB),alanine aminotransferase (ALT),aspartate aminotransferase (AST),urea nitrogen (BUN),triglyceride (TG),glucose (GLU),total cholesterol (TC) and nutrient apparent digestibility of goats were measured.The results showed that the average daily gain (ADG) and the BUN content of goats in S-KS2 group were significantly lower than those in S-KS3 group (P<0.05),the feed/gain (F/G) was significantly higher than that in S-KS3 group (P<0.05),but there was no significant difference between S-KS2 group and CK groups (P>0.05).The ADG and the average daily feed intake (ADFI) of goats in S-KS3 group were significantly higher than those in CK group (P<0.05).The serum TP and GLB contents in group S-KS4 were significantly higher than those in the control group (P<0.05),but the apparent digestibility of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were significantly lower than that of S-KS2 and S-KS3 groups (P<0.05).In conclusion,under the condition of this experiment,the mixed feeding of Stylosanthes and kinggrass silage did not cause adverse effects on goats compared with the control group,the ADG and ADFI of goats were improved when the proportion of Stylosanthes and kinggrass silage was 30:70.It was recommended that Stylosanthes and kinggrass silage could be mixed and fed in a ratio of 30:70.

This experiment was conducted to investigate suitable Cu and Zn contents for finishing pigs.One hundred and eight Su Jiang pigs with about 55 kg body weight were randomly divided into 6 groups with 3 replicates per group and 6 pigs per replicate by 2×3 factor design.The low-,medium-,and high-dose Cu and Zn combined with phytase addition and no addition were assigned to 6 diets,one for each group.The trial lasted for 42 d.The growth performance,nutrients apparent digestibility,serum biochemical indexes,and Cu and Zn contents in serum and metacarpals of finishing pigs were tested.The results showed that the growth performance of finishing pigs were not significantly affected by reducing Cu and Zn contents or supplementing with phytase (P>0.05).The apparent digestibility of Cu and Zn were extremely significantly increased (P<0.01) and the serum Cu content was extremely significantly decreased from high dose to medium dose or low dose (P<0.01).The activity of alkline phosphatase (AKP) in serum was extremely significantly decreased by decreasing the content of Cu and Zn from high to medium dose (P<0.01).The apparent digestibility of crude protein was extremely significantly increased (P<0.01),and the Zn apparent digestibility and AKP activity were significantly increased (P<0.05) by the addition of phytase.Zn content in serum and metacarpals,the activities of ceruloplasmin and lactate dehydrogenase (LDH) in serum were not significantly affected by the content of Cu and Zn and phytase addition (P>0.05).In conclusion,reducing Cu and Zn contents to medium level in high phytate diet could increase the apparent digestibility of Cu and Zn,but had no adverse impact on growth performance.Adding phytase in the diet could increase Zn and crude protein apparent digestibility.Therefore,moderate contents of Cu and Zn was 25 and 40 mg/kg,and phytase were recommended to add in diet of Su Jiang finishing pig.

This study was aimed to explore the effects of fermented Fuzheng jiedu oral liquid on the production performance,immune index and meat quality of broilers.Using single factor to select 90 one-day-old healthy broilers,randomly divided into three groups (3 replicates per group,10 birds per replicate):Control group (basal diet),group Ⅰ (basal diet+centralizer Fuzheng jiedu oral liquid) and group Ⅱ (basal diet +fermented Fuzheng jiedu oral liquid).The production performance and immune organ development of broilers were measured at 21 and 42 days of age,the meat quality was determined at 42 days of age.The results showed that compared with control group,at 1 to 42 days of age,the average daily gain of groups Ⅰ and Ⅱ were significantly increased by 8.09% and 13.76% (P<0.05),the feed conversion rate was significantly reduced by 2.23% and 10.27% (P<0.05),respectively.At 21 and 42 days of age,compared with control group,the spleen index of groups Ⅰ and Ⅱ were increased by 9.09% (P>0.05) and 15.15%(P<0.05),34.38% and 53.13% (P<0.05),the bursa of Fabricius index were increased by 1.22% (P>0.05) and 20.41% (P<0.05),8.72% and 18.97% (P<0.05),respectively,and the liver index were increased.At 42 days of age,the pectorales moisture content of groups Ⅰ and Ⅱ were significantly increased by 0.57% and 2.10% (P<0.05) than that of control group,the crude protein content of groups Ⅰ and Ⅱ were improved by 1.61% (P>0.05) and 4.25% (P<0.05),the crude fat content of groups Ⅰ and Ⅱ were raised by 49.89% and 43.85% than that of control group (P>0.05),a^* value of groups Ⅰ and Ⅱ were increased by 7.88% and 58.62% than that of control group (P>0.05),the inosine acid of groups Ⅰ and Ⅱ were significantly raised by 24.50% and 31.13% (P<0.05) than that of control group.What's more,the drip loss of pectoralis muscle and b^* value of the meat were reduced by 21.91% and 32.24% (P<0.05),4.55% and 21.73% (P>0.05),respectively.The contents of muscle moisture,crude protein,crude fat and inosidine of leg muscles of group Ⅱ were significantly increased than group Ⅰ and control group (P<0.05),the loss of water were significantly dropped (P<0.05).The a^* value was higher than group Ⅰ and control group,the L^* and b^* values were lower than group Ⅰ and control group.The results suggested that fermented Fuzheng jiedu oral liquid was able to significantly increase production performance and immune indexes,and improve meat quality of broilers.

In order to determine the behavior of cows and improve the management level of fine feed,a Real-time identification system for cows' movement behaviors based on the semi-supervised fuzzy clustering algorithm was developed and the wireless sensor node was designed on the principle of low power consumption,high sensitivity and high operational stability.At the same time,the transmission performance of wireless sensor nodes was tested to determine the optimal transmission distance and the optimal node fixed height,in which the transmission distance were set at 10,20 and 30 m,while the fixed height were 10,20 and 30 cm,respectively.And the accuracy,precision and sensitivity of K-means algorithm,BP neural networks algorithm and semi-supervised fuzzy clustering algorithm were compared.The results showed that the wireless sensor node consist with three-axis accelerometer ADXL345,processor MSP430-F149,and wireless transceiver CC1101 which could collect data automatically of cows' movement behaviors,and meet the long-term reliable data transmission and other work requirements.The test results showed that the optimal transmission distance was 10 m and the optimal fixed node height was 30 cm.The accuracy,precision and sensitivity of semi-supervised fuzzy clustering algorithm were 95.4%,53.0% and 60.6%,respectively,that of K-means algorithm,BP neural network were 90.3%,39.9%,45.6% and 93.7%,45.5%,47.0%,respectively.In conclusion,the accuracy of semi-supervised fuzzy clustering algorithm was high,implementation was simple,learning complexity was low and running speed was fast,and it also had strong optimization ability.

This study was aimed to explore and analyze the polymorphisms of growth hormone (GH) gene in Wumeng crested chickens.Taking Wumeng crested chickens as the research materials,the blood genomic DNA was extracted to construct DNA pool,all exons and partial introns of GH gene were amplified by PCR,the polymorphisms of GH gene was detected by sequencing,the secondary structure,tertiary structure and basic properties (physicochemical properties,hydrophobicity,signal peptide,transmembrane region,coiled-coil region and conserved domain) of GH protein were analyzed using bioinformatics softwares.The results showed that there were 8 SNPs(T1453C,A1489G,A1512G,G2152A,G3206A,G3347A,C3452A and C3453G) of GH gene in Wumeng crested chickens,there were 3 SNPs(G2152A,C3452A and C3453G) only appeared in flathead Wumeng crested chickens.The polymorphic sites were in different regions,C3452A and C3453G were located in the non-coding region,T1453C,A1489G,A1512G,G2152A and G3206A were located in introns region,and G3347A was located in exon 5.After mutation,the mRNA structure of GH gene were changed,which made the minimum free energy increases,and the stability decreases.Bioinformatics analysis result indicated that GH protein was secreted proteins,which had signal peptide sequence,a typical helical coil structure and a hydrophobic region,there was also a conserved domains in 10-214 amino acids.However,it was not a trans-membrane protein,and the stability was low.The results suggested that GH gene in Wumeng crested chickens had high genetic diversity,which could provide a reference for the conservation breeding of Wumeng crested chickens.

The author performed a review on relative studies of rabbit genetics and breeding including traditional breeding,molecular breeding and reproductive techniques in 2017,the points of domestic and international studies were different when compared to previous years.In China,studies of traditional breeding mainly included breed selection and trait evaluation,and had made good progresses.In addition,more studies including growth,meat quality,fur,reproduction and disease resistance were found in molecular breeding,which selected and filtered relative genes and molecular markers related to targeted traits.There were few and scattered reproductive technology studies mainly related to artificial insemination and environmental effect including mating season and light-controlled administration which influenced the health and productivity of rabbit.Compared with domestic studies,similar studies including genetic evaluation of traits,interaction between heredity and environment,and evaluation methods were shown in foreign researches,receiving boosts in improving growth performance and meat quality.Studies of molecular breeding mainly related to performances on growth,meat quality,disease resistance and reproduction,which also filtered and researched molecular makers but insufficient in fur traits,and researches of reproduction techniques focused on the effect of environment and additives on reproduction and semen freezing.This review could provide references for the later research and production of rabbit breeding.

To investigate the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR) on in vitro developmental potency of handmade clone (HMC) reconstructed embryos in Debao Black pigs,this study was performed from donor cells and reconstructed embryos,compared with developmental effects of 5-Aza-CdR treatment with HMC reconstructed embryos of five different concentrations (0,5,10,20 and 40 nmol/L) and five different treatment times (0,24,48,72,and 96 h),and screened for the optimal treatment concentration and time;Compared with in vitro developmental potency of four different concentrations (0,0.25,0.5 and 1 μmol/L) 5-Aza-CdR,combined with optimal concentration and optimal time to treatment with donor cells and reconstructed embryos.The results showed that there was no significant difference in the cleavage rate of reconstructed embryos between 5,10,20 and 40 nmol/L 5-Aza-CdR for 72 h compared with blank control group (P>0.05),20 nmol/L 5-Aza-CdR group significantly increased the blastocyst rate of reconstructed embryos (P<0.05).Both 10 and 20 nmol/L 5-Aza-CdR significantly increased the number of blastocysts (P<0.05),20 nmol/L 5-Aza-CdR was the best.Compared with blank control group,treatment with 20 nmol/L 5-Aza-CdR for 72 h significantly increased the blastocyst rate and blastocyst number of the reconstructed embryos (P<0.05),the remaining treatment time had no significant effect on the cleavage rate,blastocyst rate and blastocyst number of reconstructed embryos (P>0.05).Under optimal treatment concentration (20 nmol/L) and optimal treatment time(72 h),combined with four treatment concentrations (0,0.25,0.5 and 1 μmol/L) of the donor cells and treated with the reconstructed embryos and the donor cells simultaneously,the developmental potency of HMC reconstructed embryos in each treatment group was promoted,but the effect was not significant (P>0.05),which 0.25 to 0.5 μmol/L 5-Aza-CdR was better.In summary,the appropriate concentration (0.25 to 0.5 μmol/L) treatment of donor cells with DNA methylase inhibitor 5-Aza-CdR and treatment of reconstructed embryos with 20 nmol/L 5-Aza-CdR for 72 h could effectively improve in vitro developmental potency of HMC reconstructed embryos in Debao Black pig,which laid a foundation for the future study of DNA methylation regulation mechanism of HMC embryo in Debao Black pig.

This study was aimed to explore the effect of the polymorphism of acetaldehyde dehydrogenase 1A1 (ALDH1A1) gene intron 4 on meat traits in Yanhuang cattle.99 Yanhuang cattle at 18 months old were selected,the single nucleotide polymorphism (SNP) of ALDH1A1 gene intron 4 in Yanhuang cattle were detected by direct sequencing of PCR amplification products,and the correlation between ALDH1A1 gene intron 4 SNP and meat traits (body height,body length,cross height,chest circumference,abdominal circumference,tube circumference,body weight,back fat thickness,loin eye area and intramuscular fat) were analyzed using SPSS 19.0 software.The results showed that a C/T mutation had been found in ALDH1A1 gene intron 4,there were three genotypes (TT,TC and CC) and two alleles (T and C),TC was the dominant genotype,and C was the dominant allele.The chi-square test result showed that Yanhuang cattle population was in Hardy-Weinberg equilibrium (P>0.05).Population genetic parameter analysis showed the heterozygosity (He) of the mutation site was relatively low,there was a low varitation in Yanhuang cattle population,it was a moderately polymorphic (0.25<PIC<0.5),and the genetic marker could provide genetic information.There was no significant difference between the different genotypes and meat traits in Yanhuang cattle (P>0.05).The results indicated that the polymorphism of ALDH1A1 gene intron 4 was found in Yanhuang cattle,it needed to expand the number of sample for further explore whether the site could be considered as genetic markers for meat traits in Yanhuang cattle or not.

This study was designed to compare the myocardial enzymes activity,liver metabolism,kidney metabolism and some other biochemical indicators related to body antioxidant capacity,energy metabolism and lipid metabolism between yaks at two different altitudes.49 blood samples of yaks were collected in Datong,Qinghai (higher altitude) and 29 blood samples of yaks were collected in Hongsongwa farm,Hebei (lower altitude).25 blood biochemical indicators were tested and the results of the two groups were compared with independent t-tests.The results showed that,compared with yaks at lower altitude,the activities of blood ALT,AST,CK-MB,α-HBDH and LDH,the levels of blood TBiL,DBiL,TP,GLB,CRE,UA,MDA,and GLU in yaks at higher altitude were extremely significantly higher (P<0.01),and the activity of ALP were significantly higher (P<0.05) as well.While the SOD activity,NO,ALB and TG levels,and A/G were extremely significantly decreased (P<0.01).The rest of the indicators showed no significant difference (P>0.05).In conclusion,there were certain differences in myocardial enzyme activity,metabolic function and antioxidant capacity of yaks at two different altitudes,and yaks at higher altitude had been on the plateau for a long time and gradually developed the adaptability to the plateau hypoxia environment.

This study was conducted to compare the effects of calving season on the parameters of lactation curve of Holstein dairy cow for parity 1 to 3 in Beijing 28 farm based on the dairy herd improvement (DHI)data from 1998 to 2016.Wood model was used to fit the lactation curve of parity 1 to 3 in different calving season groups and individuals,and obtain lactation curve parameters of dairy cows in different calving seasons under different parity.Lactation parameters include lactation potential (a),the rate of milk production increased to peak (b),milk production decline rate (c),lactation persistence (Per),lactation peak (PY) and estimated 305 d milk yield (305MY).The NLIN modules in SAS 9.2 were used for fitting Wood model lactation curve at group and individual level.The parameters of Wood curve were analyzed using a mixed linear model.The results showed that the calving season had a significant effect on parameters of lactation curve,derived parameters of PY and 305MY in the Wood lactation curve (P<0.05),but had no significant effect on derived parameters of Per (P>0.05).At the same time,for different calving seasons,the lactation curve of calves produced in summer was lower than that of other calving seasons,and the higher the parity,the more obvious the trend was.The impact on the first parity cow was relatively small.From the analysis of the parity,the first lactation persistence was significantly higher than that of multiple parity cows (P<0.05).In the first parity cow,the 305MY of summer calving was 274.33~490.17 kg lower than that of other calving season cows,while for the delivered cows,it was lower than the other calving seasons by 440.76~930.68 kg.Based on the above analysis results,dairy farms in Beijing should pay more attention to multiple lactation cows and fresh cows to prevent heat stress,while adjust breeding plan to avoid serious loss due to too many cows calving in the summer.

Cashmere fineness is the key quantitative trait to evaluate cashmere quality and cashmere price.In recent years,with the intensive study of cashmere fineness functional marker genes and through the genome-wide association analysis,the candidate genes of fineness traits of cashmere were gradually screened out.But the key regulated genes,their expression levels and molecular regulation mechanisms of cashmere fineness have not been resolved.Gene expression is regulated by a variety of regulatory factors.The regulation of non-coding RNA is more active,it mainly includes microRNA,lncRNA and circRNA.High throughput sequencing and biological information analysis discovered that non-coding RNA is differential expressed and regulated of related target genes of skin,secondary hair follicle and the development of secondary hair follicle in periodic growth in different cashmere fineness of individual in cashmere goat breeds and varieties.At present,the related researchs of using SNP marker,transcriptional screening,high throughput analytical non-coding RNA and genome-wide association analysis to selected differential genes of cashmere fineness have found that the same cashmere fineness target genes focus on two pathways were more,but it was very rare to cover two or more passages.In this paper,the authors mainly discussed the research progress on molecular regulation mechanism of cashmere fineness candidate genes,and microRNA,lncRNA and circRNA on cashmere fineness related target genes at transcriptional level.

The aim of this study was to investigate the effect of LPS on the expression of TLR4 and related immune factors in embryo implantation stage of sheep.The constructed pcDNA3.1-TLR4 overexpression vector was used to transfect sheep endometrial stromal cells,and Western blotting was used to identify the transfection effect.Then the endometrial stromal cells were stimulated with 1 μg/L LPS,and the inflammatory model of endometrial stromal cells was established.The TLR4 and related immune factors IL-1β and IL-6 of cell treated with LPS were detected after cultured for 12,24,48 and 72 h by Real-time quantitative PCR and Western blotting methods with untreated cell as the control.The results showed that compared with the control group,LPS promoted the release of IL-1β and IL-6,but the expression of IL-6 in the cells gradually decreased with time,and the expression of IL-1β increased gradually,which made Th1/Th2 biased to the Th1 direction.The expressions of TLR4 mRNA at 12,24 and 72 h were all significantly higher than that in control group (P<0.05),and the expression at 48 h was extremely significantly higher than that in control group (P<0.01),and the expression of TLR4 protein after LPS treatment was always higher than that of the control group.In conclusion,the pcDNA3.1-TLR4 overexpression vector was successfully transferred into the endometrial stromal cells of sheep.LPS effectively activated the TLR4 signaling pathway in endometrial cells and promoted the expression of downstream factors.The TLR4 protein in the decidua tissue also played an important role in maintaining the balance of early pregnancy microenvironment.

In order to understand the prevalence of Bluetongue virus type 16 (BTV-16) strains from Jiangcheng county in Yunnan province in recent years,and the genetic evolution relationship between L2 gene and foreign strains,in this study,300 bovine heparin sodium anticoagulated bloods were taken from Jiangcheng county for extraction of red blood cells,and 10 days old chicken embryos were inoculated intravenously.The collected chicken embryo livers were crushed and centrifuged,and the supernatant was inoculated into C6/36 and BHK21 cells for passage.For the cytopathic samples,the group-specific VP7 fragment primers were used for RT-PCR detection.The BTV-16 L2 gene-specific primers were used to perform RT-PCR amplification and sequencing of the BTV nucleic acid positive samples.The nucleotide and amino acid homology alignments and genetic evolution analysis of the obtained L2 gene coding region sequences were performed using DNAStar and Mega 6.0 softwares.At the same time,the isolated virus was neutralized by BTV-16 standard positive serum.The results showed that 30 cytopathic samples were found in Jiangcheng county,17 of which were initially identified as BTV by RT-PCR;6 of them were identified as BTV-16 strains by L2 gene sequence analysis and neutralization test;The nucleotide and amino acid homology alignment analysis showed that the nucleotide and amino acid homology of the 6 strains were 93.4% to 98.0% and 94.2% to 99.1%,respectively;Genetic evolution analysis found that 5 of them were closely related to the BTV-16 strains isolated in Japan from 2001 to 2008 and in India from 1982 to 2011,one strain was closely related to the BTV-16 strain isolated from Japan in 1985 to 1990.This study found that the BTV-16 strain in Jiangcheng county of Yunnan province showed a cross-continuous epidemic of new and old strains,but the genetic variation in natural evolution was small and had certain stability.This study at the molecular level clarified the local popular BTV from Jiangcheng county in Yunnan province.The genetics and differences between the BTV-16 L2 gene provided a scientific basis for further research of BTV molecular epidemiology and detection.

In order to identify the agent of a serious disease occurred in Ictalurus punctatus,a dominant bacteria (GZTL2017) was isolated from diseased Ictalurus punctatus.It was identified by clinical anatomy observation,bacterial isolation and culture,gram stain microscopy,animal regression test,biochemical test,16S rDNA sequence analysis,partial virulence gene detection and drug susceptibility analysis.Clinical anatomical observations showed that the diseased Ictalurus punctatus showed surface ulcers,fin root bleeding,gills and symptoms of intestinal congestion.The isolate showed milky white colonies with moist surface,smooth and translucent edges,and uniform morphology in the medium.Gram stain microscopy showed that the isolate was single or double-existent,with straight-negative bacilli at both ends.Animal regression tests showed that the isolate had strong pathogenicity.Biochemical tests showed that the isolate was mobilized,with positive reactions such as oxidase,V-P and phenylalanine deaminase,and negative reactions such as arginine dihydrolase and H₂S.The 16S rDNA sequence of the isolate was more than 99% homology with that of Aeromonas veronii,and the isolate formed a cluster with other Aeromonas veronii reference strains by phylogenetic tree.Three virulence genes were detected from the isolate,including aerolysin (Aer),adhesion (Aha) and outer membrane protein A (OmpA) genes.The drug sensitivity test showed that the strain was sensitive to 14 drugs such as florfenicol,doxycycline and ofloxacin;It was moderately sensitive to 7 drugs such as norfloxacin,neomycin and vancomycin;It was resistant to 8 drugs such as medimycin,oxacillin and cefradine;And the minimal inhibitory concentrations of florfenicol and doxycycline were 1 and 2 μg/mL,respectively.This experiment successfully isolated a single strain of Aeromonas veronii,which provided a scientific basis for the prevention and treatment of Ictalurus punctatus infected with Aeromonas veronii.

In order to investigate the main pathogenic bacteria of mastitis and biological characteristics from a large-scale donkey farm in Xinjiang,routine microbiological methods were used to isolate pathogens,biochemical identification and 16S rRNA PCR amplification methods were used to identify pathogens,K-B susceptibility paper method was used to determine their drug resistance,D_{600 nm} value was measured,bacterial growth curves were drawn,the homology was analyzed,and the phylogenetic tree was constructed.The results showed that the isolates were short gram-negative bacilli,could grow pink and metallic luster colonies on MacConkey culture medium and Eosin-Meilan medium,respectively.The biochemical identification of isolates of phosphohydrazine,lysine,ornithine,raffinose,sorbitol,side ginsenoside and xyloglucan tested positively,while the phenylalanine and hydrogen sulfide tests were negative,and after the PCR amplification,the 205 bp No.1 strain and the 203 bp No.2 strain were obtained,their characteristics accorded with the characteristics of Escherichia coli(E.coli).Antibiotic sensitive test showed that the isolates were highly sensitive to streptomycin and chloramphenicol and resistant to penicillin,amoxicillin and gentamicin.The growth curve showed that the isolates were highly prolificated at 2 to 16 h,stabilized at 16 to 32 h,and entered the recession after 32 h.The homology of No.1 and No.2 strains was as high as 99%.The homology with E.coli 11J and RCB800 was the highest (100%);The No.2 strain had the highest homology with E.coli 675SK2 and DTU-1 (100%).From the phylogenetic tree,No.1 strain was in the same small branch with E.coli 11J and RCB800,and No.2 strain was in the same small branch with E.coli 675SK2 and DTU-1.Therefore,the pathogen of the donkey mastitis was E.coli,and its biological characteristics laid a solid theoretical basis for the further systematic study of the pathogenic mechanism of donkey mastitis E.coli.

The test was designed to identify the antifungal substances of Bacillus amyloliquefaciens SSY2 and their physicochemical properties.Identification and extraction of antibacterial substances were carried out by active component analysis,ammonium sulfate precipitation,ultrafiltration and salt removal;The physical and chemical properties were initially determined by detecting the time,enzyme activity,temperature,pH,stability and storage temperature of the mold material.The results showed that the degradation rate of different components to mould was different.The supernatant of bacterial liquid had the strongest degradation ability to mould,the degradation ability of bacterial liquid fermentation liquid was the second,and the degradation ability of intracellular liquid was the worst.Therefore,it was preliminarily determined that the active ingredient of the antibacterial substance of Bacillus amyloliquefaciens SSY2 was an extracellular secretion,which was mainly present in the supernatant of the bacterial liquid.After 30%,60%,80% and 100% saturated ammonium sulfate precipitation,the optimum ammonium sulfate saturation concentration was determined to be 60%;After ultrafiltration purification,an antibacterial active substances with molecular mass greater than 3 ku was obtained.The results of the physical and chemical property test showed that,the active substance was produced early (24 h) in the logarithmic phase,and it had good passage stability.It had protease activity and resistance to pepsin,but it had lower resistance to protease K,alkaline and neutral protease;The antibacterial activity at 60 to 121℃ was stable,which proved that it was not sensitive to heat;There was no significant difference in the antibacterial activity of the supernatant against molds in pH 6.0 to 8.0 environment,it was proved that antimicrobial substances had strong acid resistance;Antimicrobial substances had good passage stability,and it could be stored at 4℃ for a long time.The results could provide basis for further research and development of fungal biological control agents.

In order to obtain porcine epidemic diarrhea virus (PEDV) isolate and study its pathogenicity,virus isolation was conducted in Vero cells through inoculating diarrhea sample collected from a pig farm in Shandong province.The strain was identified by cytopathogenic effect (CPE),IFA,electron microscope observation and RT-PCR methods,and the S gene sequence of virus was analyzed.The 3-day-old piglets were inoculated orally with 10^3.5 TCID₅₀/mL virus and observed for clinical symptoms and pathological changes.The 3-day-old piglets were inoculated orally with 3 mL different titers of virus.The mortality of piglets in each group was calculated to determine the minimum lethal dose.The results indicated that a strain of PEDV was successfully isolated in the cell culture and designated PEDV SD201604.The strain replicated well in Vero cells and could cause obvious CPE.The infectious titer of the 6th passage of PEDV SD201604 was 10^3.5 TCID₅₀/mL.The strain was detected to be PEDV positive by IFA and RT-PCR methods.Electron microscope observation showed that the size of the virus particle was about 100 nm in diameter,with an obvious envelope and spikes,which was the typical morphological feature of PEDV.Then the virus was identified as a prevalent PEDV variant of China through the analysis of S gene sequence.In the animal pathogenicity experiment,five 3-day-old piglets inoculated orally with 10^3.5 TCID₅₀/mL of PEDV SD201604 strain all showed typical PEDV clinical symptoms and pathological lesions,and 3 of them died in the test.The minimum lethal dose (MLD) experiment showed that piglets all died when they were orally inoculated with 3 mL of virus with a virus content of 10^4.5 TCID₅₀/mL.Together,the experimental results provided references for PEDV isolation,identification and biological characteristics research.

This study was aimed to obtain a tandem fusion protein of three copies of CPA C-terminal (CPA_C),and evaluate its immunogenicity.Based on the known CPA sequence of Clostridium perfringens type A(GenBank accession No.:AY823400.1),CPA_C gene was optimized,three copies of CPA_C genes (GCPA_C3) were tandemly linked in the same direction with the flexible amino acid linker (GGGS) gene and synthesized.In roder to get the recombinant protein of three copies of CPA_C(rGCPA_C3),GCPA_C3 was cloned into prokaryotic expression vector pET-30a(+) for expression and purification.Reactivity of rGCPA_C3 with antiserum of Clostridium perfringens type A was detected by Western blotting.Four rabbits were immunized with rGCPA_C3 emulsified with oil adjuvant of ISA 201 to prepare antiserum and detect the neutralizing titer.The results showed that rGCPA_C3was presented predominantly in an insoluble form (inclusion bodies),and it could react with the antiserum of Clostridium perfringens type A.After the first immunization,sera from rabbits immunized with rGCPA_C3could neutralize 30 to 50 MLD/mL Clostridium perfringens type A toxin,and 70 to 100 MLD/mL after twice immunization.Moreover,rabbits in rGCPA_C3immunized group fully survived at the dose of 1 rabbit MLD of Clostridium perfringens type A toxin challenge,whereas all of the rabbits died (100%,4/4) in control groups.These data suggested that rGCPA_C3was a potential vaccine candidate for genetic engineering subunit vaccine of Clostridium perfringens type A.

In order to abtain a high activity antimicrobial peptide,an expression vector was constructed with a tandem of Melittin and Mytilin-B.According to the partiality codon of Pichia pastoris,the Melittin(3-14)-Mytilin-B(13-27) hybrid antimicrobial peptide,Mel-MytB (MEM) gene was synthesized by SOE method,and cloned into pPICZa-A to construct the recombinant expression vector pPICZa-A-MEM.pPICZα-A-MEM was linearized by Sac Ⅰ and electroporated into Pichia pastoris X-33.The results showed that under the control of the promoter alcohol oxidase (AOX),an approximately 3.0 ku Mel-MytB protein was expressed,which had thermal stability and acid stability.The antibacterial activity was basically unchanged after boiling for 40 min and pH 2.0-10.0.Antibacterial assays demonstrated that Mel-MytB had broad-spectrum antibacterial activity.The MIC to Escherichia coli ATCC25922,Staphylococcus aureus ATCC25923,Staphylococcus aureus ATCC6538,Salmonella enterica ATCC13076,Salmonella Typhimurium ATCC14028,Vibrio parahaemolyticus ATCC17802,Vibrio vulnificus ATCC27562 and Bacillus subtilis ATCC6633 were 5.1,2.2,2.0,4.6,7.6,9.4,13.2 and 59.4 μg/mL,respectively.Therefore,the recombinant antimicrobial peptide Mel-MytB had a good application prospect in disease prevention and animal feed additives.

The two-component signal transduction system is widely distributed in various prokaryotes.Its basic structure consist of a histidine protein kinase (HK) and a response-regulatory protein (RR).The system is essential for the virulence and growth of bacteria which can perceive various external stimuli and react,so that bacteria can adapt to various adverse conditions and enhance the survival ability in the host.In this paper,12 two-component systems of Mycobacterium tuberculosis including DosS/DosT-DosR,MprA-MprB,PrrA-PrrB and PhoP-PhoR are briefly reviewed.The structure and function of PhoP-PhoR are highlighted.PhoP-PhoR is one of the most basic and most important two component systems of Mycobacterium tuberculosis,composed of receptor PhoR and effector PhoP,in which PhoR can receive external stimuli such as Mg²⁺,Cl^- or H⁺,and then drive PhoP to regulate the transcriptional expression of target genes.As an effector protein,PhoP can be found through the analysis of crystal structure,which can be used to regulate the target gene by combining with DNA,including the control of cell wall composition,the regulation of lipid metabolism and pH,and the regulation of the virulence network of Mycobacterium tuberculosis.In addition,the advantages of PhoP mutant strain as a vaccine candidate is also introduced in the article,including the weakening of PhoP mutant strain in the mouse model,and immune protection in immunized Ganges River monkeys and guinea pigs,indicating that the PhoP mutant has a good potential for vaccine development.In this paper,the structure and function of PhoP-PhoR two-component system and the research of PhoP mutant as vaccine candidate are reviewed to provide a theoretical basis for research of the virulence mechanism of Mycobacterium tuberculosis and human tuberculosis vaccine.

This experiment was aimed to study the biological characteristics of Streptococcus agalactiae (S.agalactiae)and provide a theoretical basis for the prevention and treatment of cow mastitis caused by S.agalactiae.S.agalactiae was separated and identified based on bacterial molecular biology;according to GenBank-registered S.agalactiae 16S rRNA,genus-specific cfb (CAMP factor),virulence gene and drug resistance gene sequences,14 pairs of primers were designed using Oligo 6.0 and Primer Premier 5.0 softwares,then,a rapid PCR detection method was established;The resistance tests of 20 common antibiotic drugs was carried out.The results showed that 17 strains of S.agalactiae were successfully identified,and the virulence genes were highly homologous to the corresponding sequences of S.agalactiae reported on NCBI,which were ≥ 99%;A total of 6 drug resistance genes were detected;The resistance rates of the isolates to penicillin,erythromycin,lincomycin,clindamycin,vancomycin,ampicillin,novobiocin and sulfisoxazole were higher,and the drug resistance rates were 100%,94.1%,94.1%,94.1%,94.1%,82.3%,82.3% and 47.1%,respectively,severely resistant to penicillin;And the resistance rates to aminoglycosides,tetracyclines,cephalosporins and quinolones were low,which were 15.7%,14.7%,7.7% and 3.9%,respectively.This study results indicated that the established PCR rapid detection method was sensitive and reliable,and there was a multi-drug resistance of some β-lactams,macrolides,sulfonamides and other antibiotics in Yunnan.

Veterinary medicine is of great significance in the prevention and control of diseases and healthy breeding in animal husbandry production.However,it is limited in clinical use due to poor palatability,large irritation to gastric mucosa,poor stability,and poor targeting of infection sites of certain drugs.Coating technology has significant advantages in improving drug palatability,improving drug stability and preparing controlled release and targeted dosage forms.This article summarizes the research progress on coating technology at home and abroad to improve drug palatability,improve drug stability,preparation of control and sustained-release formulations and efficient targeted drug delivery,analyzes the principle of the formation of coating film,the advantages and disadvantages of several common coating technologies and the challenges faced by their industrialization and looks forward to the application of coating technology in the field of veterinary medicine,it is respected to provide new ideas for the development of veterinary drug formulations based on coating technologies.

Urine is a liquid excretion of human and vertebrate through the urinary system and urinary tract in order to meet the needs of metabolism.In recent years,camel urine attracts public attention due to its extensive medical effects which is mainly used to adjunctive treatment of diseases in the Middle East,including fever,cold,or even cancer.In recent years,studies have shown that camel urine contains some unique biochemical components,such as concanaline,which has cytotoxic and antitumor activity in different tumors.It has been proved that camel urine plays an important role in antibacterial,liver protecting,anti-tumor and antiplatelet activities.This paper introduced the effective biochemical components in the urine of camel,and elaborated the medical effects of camel urine in various diseases aiming to provide sufficient and detailed scientific support for further researches on camel urine.

To reveal the sulfadimidine (SM₂) residue in main animal-source products in Guangxi,4 637 samples were collected from 2013 to 2017,and were detected by the method of SM₂-CLEIA.A total of 122 samples,involving milk,pork,aquatic product and swine urine,were detected to have SM₂ residue above the national standards,of which 92 were swine urine,with contents ranging from 100.51 to 860.44 μg/kg.Antibiotic residues in swine urine could contaminate water and soil,and thus enter the food chain,and their enrichment effects through animals and vegetables would be harmful to human health,which deserved due attention.Samples of buffalo milk,chicken livers,duck livers and duck meat all met standard in the tests.A survey of sources of the 122 samples displayed that the average amount of SM₂ residue was decreasing annually,and that the qualified rates of the samples from intensive livestock farms were higher than those from individual farms,and those from supermakets were higher than those from farm's markets.The content of SM₂ residue in samples taken in 2014 was the highest over the five years.IFS was employed to evaluate the safety of animal-derived foods in Guangxi,and the results were far less than 1,showing that the potential risk of SM₂ residues to the human body was low in Guangxi.

Currently,parasitic disease is one of the more serious zoonoses,and avermectins are the main drugs to control them in animal husbandry.Avermectins are macrolide antibiotics and all have powerful insect-resistant efficacy.However,they also have neurotoxicity and developmental toxins,and are highly fat-soluble.They are widely distributed in animals and have a long metabolic cycle.They are highly prone to cause drug residues in animal-derived foods and endanger human health through the food chain.It is important to detect the residue of avermectins in animal original samples.At present,the methods for analyzing avermectin residues in animal source samples include liquid chromatography-ultraviolet detection,liquid chromatography-fluorescence detection,enzyme-linked immunosorbent assay,liquid chromatography-tandem mass spectrometry,and so on.Here,the authors reviewed various methods for the detection of avermectins in animal original samples published in recent years,aiming at exploring the advantages and disadvantages of these methods and providing a reasonable basis for the more accurate and effective detection of the residues of avermectins in animal source food.