| 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 |
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| 引用本文： | 黄海峰,王成强,张振兴,李宝宝,郑义盈,安琪,张萌萌,章泸尹,朱姝,曹瑞勇,杨小健,聂鑫,杜丽,王凤阳.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[J].中国畜牧兽医,2018,45(12):3337-3345. |
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| 作者姓名： | 黄海峰 王成强 张振兴 李宝宝 郑义盈 安琪 张萌萌 章泸尹 朱姝 曹瑞勇 杨小健 聂鑫 杜丽 王凤阳 |
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| 作者单位： | 海南大学热带农林学院动物科技学院, 海南省热带动物繁育与疫病研究重点实验室, 海口市动物基因工程重点实验室, 海口 570228 |
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| 基金项目： | 海南省重大科技计划项目（ZDKJ2016017-01）；国家肉羊产业技术体系（CARS-38）；中央引导地方科技发展专项资金项目（ZY2017HN07） |
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| 摘 要： | 试验旨在对羊源多杀性巴氏杆菌toxA-N基因进行克隆、原核表达及纯化，并对表达蛋白toxA-N进行生物信息学分析，为探索羊源多杀性巴氏杆菌毒素基因的相关特性提供参考依据。以羊源多杀性巴氏杆菌基因组为模板，参考GenBank中公布的多杀性巴氏杆菌HN06中toxA基因序列（登录号：CP003313.1）设计引物，通过PCR技术扩增出目的片段，构建重组质粒pET28a （+）-toxA-N，转化E.coli BL21（DE3）感受态细胞，IPTG诱导表达后，经考马斯亮蓝染色及Western blotting鉴定，纯化蛋白并运用生物信息学软件对表达蛋白进行特性分析。结果显示，试验成功扩增出大小为1 515 bp的toxA-N基因片段，经BamHⅠ和NotⅠ双酶切得到大小约为5 369和1 515 bp两条片段，表明成功构建了pET28a （+）-toxA-N重组质粒，IPTG诱导表达的菌株经考马斯亮蓝染色及Western blotting鉴定后，成功表达出大小约为60 ku的toxA-N蛋白；生物信息学分析表明，toxA-N蛋白为包涵体，其分子式为C2635H4002N664O797S17，原子总个数为8 115，消光系数为84 480，不稳定指数为43.50，亲水性平均值为-0.381。在toxA-N蛋白二级结构中，α-螺旋、β-折叠、延伸链和无规则卷曲分别占53.47%、2.77%、11.28%和32.48%，与三级结构预测结果一致。本试验通过对toxA-N基因的初步研究，揭示了多杀性巴氏杆菌毒素的相关特性，对家畜的疾病预防、诊断、治疗具有重要意义。
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| 关 键 词： | 羊源多杀性巴氏杆菌 toxA-N基因 亚克隆 生物信息学分析 |
| 收稿时间： | 2018-07-31 |
Cloning,Prokaryotic Expression and Bioinformatics Analysis of toxA-N Gene of Pasteurella multocida in Goat |
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| 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].China Animal Husbandry & Veterinary Medicine,2018,45(12):3337-3345. |
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| Authors: | 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 |
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| Institution: | Key Laboratory of Animal Genetic Engineering of Haikou City, Key Laboratory of Tropical Animal Reproduction & Breeding and Epidemic Disease Research of Hainan Province, College of Animal Science and Technology, College of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China |
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| 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. |
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| Keywords: | Pasteurella multocida of goat toxA-N gene subcloning bioinformatics analysis |
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