表达Ⅰ型耐热肠毒素的重组大肠杆菌对7日龄仔猪小肠功能的影响

doi:10.16431/j.cnki.1671-7236.2019.02.012

摘要/Abstract

摘要：

试验旨在研究表达Ⅰ型耐热肠毒素（STa）的重组大肠杆菌对7日龄仔猪小肠功能的影响。选取24头7日龄仔猪，随机分为4个处理组，分别为对照组、STa组、LMG194组和K88组。整个试验期间均以人工乳饲喂，预饲期4 d，试验第5天进行攻毒，STa组、LMG194组及K88组分别在早晚两次口服灌喂2×10⁹ CFU大肠杆菌LMG194-pBAD-STa、LMG194和K88，对照组灌服等量生理盐水，试验第7天早上各组灌服D-木糖并采血，屠宰并取空肠、回肠组织样品，测量仔猪血常规指标、血浆D-木糖含量及二胺氧化酶（DAO）活力，以及吸收与转运相关基因与蛋白表达量。结果显示，与对照组相比，STa组仔猪血液中中性粒细胞、中性粒细胞比率显著降低（P<0.05），淋巴细胞比率显著升高（P<0.05），LMG194组与K88组仔猪血液中中性粒细胞比率显著降低（P<0.05），淋巴细胞比率显著升高（P<0.05）；STa组与K88组仔猪血浆D-木糖含量显著降低（P<0.05）；STa组、LMG194组及K88组血浆DAO活力显著升高（P<0.05）；STa组与LMG194组空肠AQP8、AQP10、KCNJ13和b^0，+AT基因表达量均显著降低（P<0.05），K88组空肠AQP8、AQP10基因表达量显著升高（P<0.05）；STa组回肠AQP8基因表达量显著降低（P<0.05），AQP10、SGLT-1基因表达量显著升高（P<0.05），LMG194组、K88组AQP8基因表达量显著升高（P<0.05），K88组AQP10、KCNJ13、b^0，+AT、SGLT-1基因表达量显著降低（P<0.05）；STa组、LMG194组及K88组空肠HSP70蛋白的表达量显著升高（P<0.05）。以上结果表明，表达Ⅰ型耐热肠毒素的重组大肠杆菌可导致7日龄仔猪小肠产生炎症，吸收与转运能力下降。

关键词: 耐热肠毒素(STa); 重组大肠杆菌; 仔猪; 肠道

Abstract:

This experiment was aimed to investigate the effects of recombinant E.coli expressing heat-stable enterotoxin(STa) on small intestinal function in 7 days old piglets.Twenty-four 7 days old piglets were randomly divided into four treatments of control,STa,LMG194 and K88 groups,which were fed with artificial milk throughout the experiment.Pre-feeding period was 4 d.STa,LMG194 and K88 groups were orally administered with 2×10⁹ CFU E.coli LMG194-pBAD-STa,LMG194 and K88 twice in the morning and evening on the 5th day,respectively.The control group was given the same amount of normal saline.On the 7th morning of the experiment,each group was given D-xylose and blood was collected.Then they were slaughtered and the samples of jejunum and ileum tissue were collected,blood routine indicators,the content of plasma D-xylose,the activity of diamine oxidase (DAO),and the expression of absorption and transport related genes and protein were measured.The results showed that compared with control group,the neutrophils and neutrophil ratio were significantly decreased in blood,the lymphocyte ratio was significantly increased in STa group (P<0.05),the neutrophil ratio was significantly decreased in blood,the lymphocyte ratio was significantly increased in LMG194 and K88 groups (P<0.05);The content of D-xylose in plasma was significantly decreased in STa and K88 groups (P<0.05);The activity of DAO in plasma was significantly increased in STa,LMG194 and K88 groups (P<0.05).In jejunum,compared with control group,the expression levels of AQP8, AQP10,KCNJ13 and b^0,+AT genes were significantly reduced in STa and LMG194 groups (P<0.05),the expression levels of AQP8 and AQP10 genes were significantly increased in K88 group (P<0.05).In ileum,the expression level of AQP8 gene was significantly reduced,the expression levels of AQP10 and SGLT-1 genes were significantly increased in STa group (P<0.05).The expression level of AQP8 gene was significantly increased in LMG194 and K88 groups (P<0.05),the expression levels of AQP10,KCNJ13,b^0,+AT and SGLT-1 genes were significantly reduced in K88 group (P<0.05).The relative expression level of HSP70 was significantly increased in STa,LMG194 and K88 groups (P<0.05).These results suggested that recombinant E.coli expressing STa could lead to inflammation in small intestinal,absorption and transfer capacity were decreased in 7 days old piglets.

Key words: heat-stable enterotoxin (STa); recombinant E.coli; piglets; intestinal

中图分类号:

S828

吴梦郡, 李思源, 纪昌正, 余魁, 董毅, 赵广宇, 赵迪, 吴涛, 侯永清. 表达Ⅰ型耐热肠毒素的重组大肠杆菌对7日龄仔猪小肠功能的影响[J]. 《中国畜牧兽医》, 2019, 46(2): 424-432.

WU Mengjun, LI Siyuan, JI Changzheng, YU Kui, DONG Yi, ZHAO Guangyu, ZHAO Di, WU Tao, HOU Yongqing. Effects of Recombinant E.coli Expressing Heat-stable Enterotoxin on Small Intestinal Function in 7 Days Old Piglets[J]. , 2019, 46(2): 424-432.

参考文献

[1] 陈培富,朱兴全.大肠杆菌耐热肠毒素的结构与特性[J].中国预防兽医学报,2012,34(3):244-246. CHEN P F,ZHU X Q.Structure and properties of heat-stable enterotoxin from Escherichia coli[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(3):244-246.(in Chinese)
[2] 周海慧,周继恩,章跃炎.一起由产肠毒素大肠埃希氏菌引起的食源性疾病[J].中国卫生检验杂志,2013,23(7):1809-1810. ZHOU H H,ZHOU J E,ZHANG Y Y.Identification of a foodborne disease due to enterotoxigenic Escherichia coli[J].Chinese Journal of Health Laboratory Technology,2013,23(7):1809-1810.(in Chinese)
[3] NAGY B,FEKETE P Z. Enterotoxigenic Escherichia coli (ETEC) in farm animals[J].Veterinary Research,1999,30:259-284.
[4] OKAMOTO K.Properties and actions of heat-stable enterotoxin of Escherichia coli[J].Journal of Natural Toxins,2000,9(3):213-229.
[5] ZHANG W P,BERBEROV E M,FREELING J,et al.Significance of heat-stable and heat-labile enterotoxins in porcine colibacillosis in an additive model for pathogenicity studies[J].Infection and Immunity,2006,74(6):3107-3114.
[6] ERUME J,BERBEROV E M,KACHMAN S D,et al.Comparison of the contributions of heat-labile enterotoxin and heat-stable enterotoxin b to the virulence of enterotoxigenic Escherichia coli in F4ac receptor-positive young pigs[J].Infection and Immunity,2008,76(7):3141-3149.
[7] YAMAMOTO T,NAKAZAWA M.Detection and sequences of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene in enterotoxigenic E.coli strains isolated from piglets and calves with diarrhea[J].Journal of Clinical Microbiology,1997,35(1):223-227.
[8] YAMAMOTO T,TANEIKE I.The sequences of enterohemorrhagic Escherichia coli and Yersinia pestis that are homologous to the enteroaggregative E.coli heat-stable enterotoxin gene:Cross-species transfer in evolution[J].FEBS Letters,2000,472(1):22-26.
[9] MÉNARD L P,LUSSIER J G,LÉPINE F,et al.Expression,purification,and biochemical characterization of enteroaggregative Escherichia coli heat-stable enterotoxin 1[J].Protein Expression and Purification,2004,33(2):223-231.
[10] COTÉ G A,BUCHMAN A L.Antibiotic-associated diarrhea[J].Expert Opinion on Drug Safety,2006,5:361-372.
[11] 王磊.表达单一肠毒素重组大肠杆菌的构建及仔猪腹泻模型的建立[D].武汉:武汉轻工大学,2016. WANG L.Construction of recombinant Escherichia coli expressing single enterotoxin and establishing of piglet diarrhre model[D].Wuhan:Wuhan Polytechnic University,2016.(in Chinese)
[12] FU W J,STROMBERG A J,VIELE K,et al.Statistics and bioinformatics in nutritional sciences:Analysis of complex date in the era of systems biology[J].Journal of Nutritional Biochemistry,2010,21(7):561-572.
[13] HOU Y Q,WANG L,YI D,et al.Dietary supplementation with tributyrin alleviates intestinal injury in piglets challenged with intrarectal administration of acetic acid[J].British Journal of Nutrition,2014,111(10):1748-1758.
[14] KIM H M,HAN S B,OH G T,et al.Stimulation of humoral and cell mediated immunity by polysaccharide from mushroom Phellinus linteus[J].International Journal of Immunopharmacology,1996,18(5):295-303.
[15] 丁厚康,马卫明,刘莲莲,等.大肠杆菌对兔血液生理生化指标和实质器官组织结构的影响[J].江西农业大学学报,2013,35(3):556-562. DING H K,MA W M,LIU L L,et al.Effect of colibacillosis on blood physiobiochemical indices and histoogical structure of parenchymal organs in rabbits[J].Acta Agriculturae Universitatis Jiangxiensis,2013,35(3):556-562.(in Chinese)
[16] 刘放南,罗楠,谭力,等.高效液相色谱法测定成人肠吸收试验中D-木糖的正常值[J].肠外与肠内营养,2004,11(3):23-25. LIU F N,LUO N,TAN L,et al.The measurement of D-xylose normal range of blood and urine of healthy volunteers using HPLC after D-xylose absorption testing[J].Parenteral and Enteral Nutrition,2004,11(3):23-25.(in Chinese)
[17] 王猛,侯永清,丁斌鹰.精胺对断奶仔猪生长性能和肠道吸收功能的影响[A].动物营养与饲料研究——第五届全国饲料营养学术研讨会论文集[C].2006. WANG M,HOU Y Q,DING B Y.Effect of spermine on growth performance and intestinal maturation in weaned piglet[A].Animal Nutrition and Feed Research-Proceedings of the 5th National Symposium on Feed Nutrition[C].2006.(in Chinese)
[18] 马玉敏,王恬.初乳中生长因子对肠道生长发育的影响[J].猪业科学,1999,3:14-16. MA Y M,WANG T.Effect of growth factors in colostrum on intestinal growth and development[J].Swine Industry Science,1999,3:14-16.(in Chinese)
[19] 陈英.腹内高压对肠黏膜屏障功能损伤的实验研究[D].南宁:广西医科大学,2013. CHEN Y.Experimental studies on the injury of intestinal barrier function induced by intra-abdolminal high press[D].Nanning:Guangxi Medical University,2013.(in Chinese)
[20] 赵凯科,郑立,毛智斌,等.水通道蛋白与胃肠道疾病关系研究进展[J].动物医学进展,2016,37(1):77-80. ZHAO K K,ZHENG L,MAO Z B,et al.Progress on relationship between aquaporins and gastrointestinal disease[J].Progress in Veterinary Medicine,2016,37(1):77-80.(in Chinese)
[21] YAMAMOTO T,KURAMOTO H,KADOWAKI M.Downregulation in aquaporin 4 and aquaporin 8 expression of the colon associated with the induction of allergic diarrhea in a mouse model of food allergy[J].Life Sciences,2007,81(2):115-120.
[22] 职爱民,左建军,周响艳,等.不同浓度赖氨酸对猪肠黏膜上皮细胞碱性氨基酸转运载体mRNA表达的影响[J].中国农学通报,2010,26(21):6-11. ZHI A M,ZHUO J J,ZHOU X Y,et al.The influence of different lysine concentration on the cationic amino acid transporter mRNA expression of porcine IEC[J].Chinese Agricultural Science Bulletin,2010,26(21):6-11.(in Chinese)
[23] 张璇,耿仙,陈兴娟,等.蛋白激酶C对内向整流钾离子通道的调节机制[J].第二军医大学学报,2009,30(8):956-960. ZHANG X,GENG X,CHEN X J,et al.Modulation mechanism of PKC upon inwardly rectifying potassium channels[J].Academic Journal of Second Military Medical University,2009,30(8):956-960.(in Chinese)
[24] GOULD G W,HOLMAN G D.The glucose transporter family:Structure,function and tissue-specific expression[J].Biochemical Journal,1993,295(2):329-341.
[25] LOOS M,GEENS M,SCHAUVLIEGE S,et al.Role of heat-stable enterotoxins in the induction of early immune responses in piglets after infection with enterotoxigenic Escherichia coli[J].PLoS One,2012,7(7):398.
[26] NIEWOLD T A,VAN DER MEULEN J,KERSTENS H H D,et al.Transcriptomics of enterotoxigenic Escherichia coli infection.Individual variation in intestinal gene expression correlates with intestinal function[J].Veterinary Micicrobioly,2010,141(1):110-114.
[27] DZAMAN-SERAFIN S,TELATY?SKA-MIESZEK B,CIECHANOWSKI K.Heat shock proteins and their characteristics[J].Polski Merkuriusz Lekarski,2005,19(110):215.
[28] KREGEL K C.Heat shock proteins:Modifying factors in physiological stress responses and acquired thermotolerance[J].Journal of Applied Physiology,2002,92(5):2177-2186.
[29] 郭辉,徐月敏,叶章群,等.细胞因子及热休克蛋白在慢性前列腺炎患者精浆中的含量及其临床意义[J].中华男科学杂志,2012,18(12):1088-1092. GUO H,XU Y M,YE Z Q,et al.Levels of cytokines and heat-shock protein 70 in the seminal plasma of patients with chronic bacterial prostatitis and chronic prostatitis/chronic pelvic pain syndrome[J].National Journal of Andrology,2012,18(12):1088-1092.(in Chinese)