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.
作者
刘志科
张秋雨
杨宁宁
徐锦凤
徐明国
荆明龙
吴文星
曹旭东
任艳
石峰
陈创夫
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)
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
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)
