| 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1 143 bp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51 ku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1 143 bp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。 |
| 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 |
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| 基金项目:西部地区高发人兽共患传染性疾病防治项目(2013-179) |
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| Cloning and prokaryotic expression of invA gene from Salmonella pullorum |
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LIU Zhike,ZHANG Qiuyu,YANG Ningning,et al
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| Abstract: |
| 【Objective】Cloning of Salmonella pullorum invasion protein A (invA) gene and prokaryotic expression, analysis of the antigenicity of recombinant protein invA,in order to lay a theoretical foundation for further research of the Salmonella pullorum rapid diagnosis test strips and new epitope vaccine research.【Method】 Salmonella pullorum strains as a test strain,the invA gene was cloned and the bioinformatics analysis that the invA gene was carried out.The invA gene was subcloned into the prokaryotic expression vector pET-30a,strucure pronucleus recombinant plasmid pET-30a-invA.The recombinant plasmid 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.【Result】Obtained a complete length of invA gene was about 1 143 bp successfully, which could encode 381 amino acids residues.Bioinformatics results showed that the protein encoded by invA gene had 17 antigenic determinants,and its transmembrane region was obvious,the protein had a domain which corresponding to the typical domain of low complexity between the 92－105 amino acids.The recombinant plasmid pET-30a-invA was successfully constructed and expressed as about 51 ku of fusion protein invA in E.coli BL21(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 information of the protein was confirmed,and the recombinant protein has good immunogenicity. |
| Key words: Salmonella pullorum invA gene prokaryotic expression bioinformatics analysis immunogenicity |
