羊源多杀性巴氏杆菌toxA-N基因的克隆、原核表达及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2018.12.004

Abstract

Abstract:

This study was aimed to clone,prokaryotically express and purify toxA-N gene of Pasteurella multocida in goat,and analyze the expressed protein toxA-N by bioinformatics,which provided a reference for exploring the relevant characteristics of the Pasteurella multocida toxin gene in goat.The genome of the above-mentioned bacteria was used as a template,and the primer was designed with reference to the Pasteurella multocida HN06 toxA gene sequence published in GenBank(accession No.:CP003313.1),and the target fragment was amplified by PCR.The recombinant plasmid pET28a(+)-toxA-N was constructed and then transferred to E.coli BL21(DE3) competent cells.After inducted by IPTG,the expressed proteins were characterized by Coomassie blue staining,Western blotting,protein purification and bioinformatics softwares.The results showed that the 1 515 bp gene fragment was successfully amplified,and the two fragments of 5 369 and 1 515 bp were digested by BamH Ⅰ and Not Ⅰ,indicating that the recombinant plasmid pET28a(+)-toxA-N was successfully constructed.The Coomassie blue staining and Western blotting results showed that a size of about 60 ku toxA-N protein was successfully expressed by IPTG induction.Bioinformatics analysis results showed that toxA-N protein was an inclusion body with a molecular formula of C₂₆₃₅H₄₀₀₂N₆₆₄O₇₉₇S₁₇,the total number of atoms was 8 115,the extinction coefficient was 84 480,the instability index was 43.50,and the average hydrophilicity was -0.381.In the secondary structure of toxA-N protein,α-helix,β-turn,extended chain and random coil accounted for 53.47%,2.77%,11.28% and 32.48%,respectively,which were consistent with the prediction results of the tertiary structure.In summary,this study revealed the relevant properties of the Pasteurella multocida toxin by preliminary study of toxA-N gene,which was of great significance for disease prevention,diagnosis and treatment of livestock.

Key words: Pasteurella multocida of goat; toxA-N gene; subcloning; bioinformatics analysis

CLC Number:

S852.61⁺2

HUANG Haifeng, WANG Chengqiang, ZHANG Zhenxing, LI Baobao, ZHENG Yiying, AN Qi, ZHANG Mengmeng, ZHANG Luyin, ZHU Shu, CAO Ruiyong, YANG Xiaojian, NIE Xin, DU Li, WANG Fengyang. Cloning,Prokaryotic Expression and Bioinformatics Analysis of toxA-N Gene of Pasteurella multocida in Goat[J]. , 2018, 45(12): 3337-3345.

References

[1] 依巴代提·马木提.羊巴氏杆菌病诊疗防治[J].当代畜牧,2016,35:86. IBDATI M.The diagnosis and treatment of sheep Pasteurellosis[J].Contemporary Animal Husbandry,2016,35:86.(in Chinese)
[2] 叶尔生·吐尔逊哈孜.羊巴氏杆菌病的诊断与防控[J].当代畜牧,2016,35:90. YERSIN T.Diagnosis and prevention of Pasteurellosis[J].Contemporary Animal Husbandry,2016,35:90.(in Chinese)
[3] 徐朋朋.鸭源多杀性巴氏杆菌分离鉴定及感染雏鸭动态病理学观察[D].洛阳:河南科技大学,2011. XU P P.Isolation and identification of Pasteurella multocida and dynamic pathological observation of infected ducklings[D].Luoyang:Henan University of Science and Technology,2011.(in Chinese)
[4] 李健.猪产毒素多杀性巴氏杆菌toxA基因的功能结构域研究[D].武汉:华中农业大学,2007. LI J.Functional domain of toxA gene of porcine toxin-producing Pasteurella multocida[D].Wuhan:Huazhong Agricultural University,2007.(in Chinese)
[5] 陈品安,汪义金.羊巴氏杆菌病的流行特点及综合防治措施[J].畜牧与饲料学,2013,1:110-111. CHEN P A,WANG Y J.The epidemiological characteristics and integrated control measures of Pasteurellosis[J].Animal Husbandry & Feed Science,2013,1:110-111.(in Chinese)
[6] 唐先春,吴斌,索绪峰,等.猪多杀性巴氏杆菌的分离鉴定及生物学特性研究[J].畜牧兽医学报,2005,36(6):590-595. TANG X C,WU B,SUO X F,et al.Isolation,identification and biological characteristics of Pasteurella multocida[J].Journal of Animal Husbandry andVeterinary Medicine,2005,36(6):590-595.(in Chinese)
[7] 唐先春.猪多杀性巴氏杆菌的分离鉴定及生物学特性研究[D].武汉:华中农业大学,2004. TANG X C.Isolation,identification and biological characteristics of Pasteurella multocida[D].Wuhan:Huazhong Agricultural University,2004.(in Chinese)
[8] WANGMAN M,CHAIVISUTHANGKURA P,SRITUNYALUCKSANA K,et al.Development of monoclonalantibodies specific to ToxA and ToxB of vibrio parahaemolyticus that cause acute hepatopancreatic necrosis disease (AHPND)[J].Aquaculture,2017,474:75-81.
[9] 聂鑫,赵天靖,曹瑞勇,等.羊种布鲁氏菌LpxB基因的克隆、原核表达及其蛋白的生物信息学分析[J].中国畜牧兽医,2016,43(7):1681-1687. NIE X,ZHAO T J,CAO R Y,et al.Cloning,prokaryotic expression of LpxB gene and bioinformatics analysis of its protein[J].China Animal Husbandry & Veterinary Medicine,2016,43(7):1681-1687.(in Chinese)
[10] 曹瑞勇,聂鑫,李宝宝,等.羊源类鼻疽伯克霍尔德菌BPSS1512基因克隆、原核表达及其蛋白的生物信息学分析[J].中国畜牧兽医,2017,44(8):2234-2240. CAO R Y,NIE X,LI B B,et al.Cloning,prokaryoticexpression and bioinformatics of BPSS1512 gene from Burkholderia sinensis analysis[J].China Animal Husbandry & Veterinary Medicine,2017,44(8):2234-2240.(in Chinese)
[11] 徐开莲,朱华培,赵天靖,等.羊种布鲁氏菌Omp10基因的克隆及原核表达[J].中国畜牧兽医,2014,41(12):122-125. XU K L,ZHU H P,ZHAO T J,et al.Cloning and prokaryotic expression of Brucella Omp10 gene in sheep[J].China Animal Husbandry & Veterinary Medicine,2014,41(12):122-125.(in Chinese)
[12] 戴碧红.鸭瘟病毒UL19基因截段表达、蛋白纯化及抗体制备[D].雅安:四川农业大学,2014. DAI B H.Segmentation,protein purification and antibodypreparation of duck plague virus UL19 gene[D].Ya'an:Sichuan Agricultural University,2014.(in Chinese)
[13] HARPER M,BOYCE J D,ADLER B.The key surface components of Pasteurella multocida:Capsule and lipopolysaccharide[J].Current Topics in Microbiology and Immunology,2012,361:39-51.
[14] BAGCHI A.Structural characterization of Fis-A transcriptional regulator from pathogenic Pasteurella multocida essential for expression of virulence fac-tors[J].Gene,2015,554(2):249-253.
[15] 孙雪婧,杜晓华,杨孝朴,等.牦牛CYGB基因CDS区克隆与生物信息学分析[J].中国农业科学,2014,47(13):2690-2698. SUN X J,DU X H,YANG X P,et al.Cloning and bioinformatics analysis of CDS region of yak cattle CYGB gene[J].Chinese Agricultural Sciences,2014,47(13):2690-2698.(in Chinese)
[16] El TAYEB A B,MORISHITA T Y,ANGRICK E J.Evaluation of Pasteurella multocida isolated from rabbits by capsular typing,somatic serotyping,and restriction endonuclease analysis[J].Journal of Veterinary Diagnostic Investigation,2004,16(2):121-125.
[17] MAGNE B.Multilocus sequence analysis of Pasteurella multocida demonstrates a type species under development[J].Microbiology,2013,159(Pt3):580-590.
[18] 李卉.多杀性巴氏杆菌毒力因子及基因表达的研究进展[J].中国畜牧兽医文摘,2015,9:68-69. LI H.Research progress on virulence factors and gene expression of Pasteurella multocida[J].Chinese Journal of Animal Husbandry and Veterinary Medicine,2015,9:68-69.(in Chinese)
[19] SIEGERT P,SCHMIDT G,PAPATHEODOROU P,et al.Pasteurella multocida toxin prevents osteoblast differentiation by transactivation of the MAP-kinasecascade via the Gα(q/11)-p63RhoGEF-RhoA axis[J].PLoS Pathogens,2013,9(5):e1003385.
[20] 鲍娟.猪源多杀性巴氏杆菌分离物荚膜PCR分型与毒素研究[D].贵阳:贵州大学,2009. BAO J.PCR typing and toxin study of isolates of Pasteurella multocida isolates[D].Guiyang:Guizhou University,2009.(in Chinese)
[21] 许田亮.牦牛源多杀性巴氏杆菌分离鉴定及重组外膜蛋白OmpH、OmpA免疫效果的初步研究[D].石河子:石河子大学,2013. XU T L.Isolation and identification of Pasteurella multocida from yak and preliminary study on the immune effect of recombinant outer membrane proteins OmpH and OmpA[D].Shihezi:Shihezi University,2013.(in Chinese)
[22] 江涛,谭春萍,任灵芝,等.猪源D型产毒素多杀性巴氏杆菌毒素基因的原核表达[J].动物医学进展,2010,12:64-68. JIANG T,TAN C P,REN L Z,et al.Prokaryotic expression of toxin-producing Pasteurella multocida toxin gene from pig D-type[J].Progress in Animal Medicine,2010,12:64-68.(in Chinese)
[23] GOORMAGHTUGH E,RUYSSCHAERT J M,RAUSSENS V.Evaluation of the information content in infrared spectra for protein secondary structure determination[J].Biophysical Journal,2006,90(8):2946-2957.

Cloning,Prokaryotic Expression and Bioinformatics Analysis of toxA-N Gene of Pasteurella multocida in Goat

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LIU Jialong, ZHANG Yiwen, ZHANG Yueran, SUN Qijuan, CHEN Jian, HE Lei, JIA Yanyan, DING Ke, YU Zuhua. Cloning,Prokaryotic Expression and Bioinformatics Analysis of ELAVL1 Gene in Chicken [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1807-1817.
[2]	ZHUO Na, ZHU Zhongwu, LI Jing, HU Shixiang, WANG Jianchang, LIN Hua, CHEN Chaolin, HAN Diangang, AI Jun, CHEN Peifu. Bioinformatics Analysis and Eukaryotic Expression of ORF2 Protein of Swine Hepatitis E Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1959-1970.
[3]	FAN Bingfeng, LI Wen, LIU Lixiang, ZHAO Xiangyuan, SHAO Jing, LIN Lefeng, SUN Huimin, ZHANG Xulin, XU Baozeng. Cloning and Bioinformatics Analysis of AANAT Gene in Mink [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1307-1318.
[4]	JIA Xinyue, MA Jing, ZHANG Yanyan, MA Xun, WANG Zhengrong, BO Xinwen. Bioinformatics Analysis and Prokaryotic Expression of EGR-07243 and EGR-07243 Genes of Echinococcus granulosus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 847-858.
[5]	WEI Yirong, ZHENG Zihua, ZHANG Sanbao, SONG Ying, JIANG Haiyu, SUN Wenyue, CHENG Pengfei, LIU Yufan, ZOU Jianwei, HUANG Yanna, PAN Yan, JIANG Qinyang. Cloning,Bioinformatics Analysis and Tissue Expression of UCP1 Gene in Nubian Goats [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 440-450.
[6]	TANG Jingyu, DU Hanyu, MENG Chunchun, LIU Guangqing. Cloning,Bioinformatics Analysis and Subcellular Localization of Interferon-stimulated Gene 15 in Goats [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 2843-2854.
[7]	FU Yongzhi, MA Xiaojun, WANG Maohan, LI Zhe, CHEN Zhiyuan, QUAN Wei, ZHAO Han, SU Yongmei, WU Jianhua, ZHU Wenjin. Bioinformatics Analysis and Tissue Expression of MSTN Gene in Yangyuan Donkeys [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 2910-2919.
[8]	ZOU Jianwei, ZOU Juhong, SHEN Yujian, WEI Yi, ZHANG Sanbao, LIAN Zitong, XU Jianjian, SONG Ying, HUANG Yanna, WEI Yingming, JIANG Qinyang, ZHENG Zihua. Bioinformatics Analysis, Construction of Eukaryotic Expression Vector and Expression of LMCD1 Gene in Nubian Goats [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2033-2042.
[9]	ZHANG Limeng, LIU Aiju, LI Runting, LI Yuhua, LI Lin, NIE Xiaoning, WANG Linqing, CHEN Longxin. Cloning,Bioinformatics and Tissue Expression Analysis of CREBRF Gene in Ovis aries [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1599-1609.
[10]	LEI Xing, LI Tianfeng, LI Jiahao, MAN Xin, BAI Suying. Bioinformatics Analysis and Expression of Elovl1 Gene in Muskrat Scent Gland [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1201-1212.
[11]	SHEN Xiang, CHEN Linghui, YAO Jingjie, WANG Sanhu, BAI Yueyu, ZHANG Xiaojian. Cloning,Bioinformatics and Expression Analysis of Bovine CCR7 Gene [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1244-1252.
[12]	ZHANG Mingliang, GENG Yachun, LIAN Kaiqi, MA Lei, CHANG Ying, WANG Shuangshan, ZHANG Fuliang. Cloning,Bioinformatics Analysis and Truncated Fragment Expression for Truncated M Gene of Porcine Epidemic Diarrhea Virus [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1479-1487.
[13]	SHI Yuzhu, LUO Rutang, CHEN Chao, WU Xu, LI Ang. Cloning, Bioinformatics and Tissue Expression Analysis of GDF9 Genes in Muscovy Duck [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 817-829.
[14]	HOU Jinxing, WANG Zhanhang, ZHU Junru, JIANG Yue, LIU Shujuan, AN Xiaopeng. Cloning, Bioinformatics and Tissue Expression Profile Analysis of CSN1S1 Gene in Guanzhong Dairy Goats [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 857-865.
[15]	LI Yulong, LYU Shijie, ZHAI Yaying, ZHANG Zhijie, ZHU Xiaoting, ZHANG Zijing, CHEN Fuying, SHI Qiaoting, WANG Eryao. Cloning and Bioinformatics Analysis of POLB Gene in Jiaxian Red Cattle [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 887-896.