鸡白痢沙门氏菌invA基因的克隆与原核表达  

Cloning and prokaryotic expression of invA gene from Salmonella pullorum

作　　者：刘志科[1] 张秋雨[2] 杨宁宁 徐锦凤[3] 徐明国 荆明龙 吴文星 曹旭东[4] 任艳[4] 石峰[3] 陈创夫[1] LIU Zhike;ZHANG Qiuyu;YANG Ningningla;XU Jinfenglb;XU Mingguola;JING Minglong;WU Wenxingla;CAO Xudong;REN Yanlc;SHI Fenglb;CHEN Chuangfula(a College of Animal Science and Technology;College of Life Sciences;School of Medicine,Shihezi University,Shihezi,Xinj iang 832003,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]河南科技学院动物科学学院,河南新乡453003 [3]石河子大学生命科学学院,新疆石河子832003 [4]石河子大学医学院,新疆石河子832003

出　　处：《西北农林科技大学学报（自然科学版）》2018年第8期9-15,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：西部地区高发人兽共患传染性疾病防治项目(2013-179)

摘　　要：【目的】克隆鸡白痢沙门氏菌侵袭蛋白A(invA)基因并进行原核表达,分析重组蛋白invA的抗原性,为沙门氏菌快速诊断试纸条和新型表位疫苗的研发提供理论依据。【方法】以鸡白痢沙门氏菌野毒株为供试菌,克隆其invA基因,对invA基因编码的蛋白进行生物信息学分析。将invA基因克隆到pET-30a原核表达载体上,构建原核重组表达质粒pET-30a-invA,将其转化到大肠杆菌BL21(DE3)中进行诱导表达,对表达产物进行SDS-PAGE和Western-blot分析,检测其目的蛋白的表达情况和免疫反应特性。【结果】成功获得了1 143bp的完整的invA基因,编码381个氨基酸。生物信息学分析结果表明,invA基因编码的蛋白有17个抗原决定簇,其跨膜区域明显,92-105位氨基酸为low complexity典型结构域。构建获得了原核重组表达质粒pET-30a-invA,在大肠杆菌BL21(DE3)中成功表达了约51ku的invA融合蛋白;Western-blot检测结果显示,该融合蛋白具有良好的免疫反应性。【结论】成功克隆出了1 143bp大小invA基因,明确了其编码蛋白的生物信息,其诱导表达后获得免疫反应性良好的重组蛋白。[Objective] Cloning of Salmonella pullorum invasion protein A （invA） gene and prokaryot- ic expression, analysis of the antigenicity of recombinant protein invA,in order to lay a theoretical founda- tion for further research of the Salmonella pullorum rapid diagnosis test strips and new epitope vaccine re- search. [Method] Salmonella pullorum strains as a test strain,the invA gene was cloned and the bioinfor- matics analysis that the invA gene was carried out. The invA gene was subcloned into the prokaryotic ex- pression vector pET-30a, strucure pronucleus recombinant plasmid pET-30a-invA. The recombinant plas- mid was transformed into E. coli BL21 （DE3）. The expressed protein was identified by SDS-PAGE and Western-blotting;and detection of its target protein expression and immune response characteristics. [Re- sult] Obtained a complete length of invA gene was about 1 143 bp successfully, which could encode 381 a- mino acids residues. Bioinformatics results showed that the protein encoded by invA gene had 17 antigenicBL21（DE3）. Western blotting showed that the fusion protein had good immunoreactivity. [Conclusion] Cloned the invA gene successfully,which the length of invA gene was 1 143 bp size,and the biological in formation of the protein was confirmed,and the recombinant protein has good immunogenicity.

关 键 词：鸡白痢沙门氏菌 invA基因 原核表达 生物信息学分析 免疫反应性 

分 类 号：S858.31[农业科学—临床兽医学]