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

摘要/Abstract

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。

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

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

中图分类号:

S852.61⁺2

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.

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.

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