布氏杆菌 Ⅳ 型分泌系统相关蛋白基因RicA的克隆、原核表达及免疫原性分析
Cloning and Prokaryotic Expression of Related Proteins Gene RicA in Brucella Type Ⅳ Secretion System and Its Immunogenicity Analysis

为克隆并原核表达布氏杆菌（Brucella melitensis）疫苗菌株 M5 Ⅳ型分泌系统相关蛋白基因 RicA，并进行表达蛋白的免疫原性分析，应用 PCR 技术从布氏杆菌疫苗菌株 M5 全基因组扩增 RicA 基因片段，亚克隆至 pMD18-T 载体后测序分析，构建重组表达载体 pET-28a(+)-RicA，转化 E.coli BL21(DE3) 表达菌，以不同浓度 IPTG 诱导表达不同时间，SDS-PAGE 鉴定表达效果，表达蛋白经 AKTA 蛋白纯化系统纯化，用 Antheprot 5.0 软件分析其理化特性及抗原性，Western Blot 检测其免疫原性。结果表明：RicA 基因全长528 bp，编码175个氨基酸，与基因库中已知菌株的核苷酸同源性和氨基酸同源性均达到99%以上。SDS-PAGE 表明，RicA 融合蛋白在大约23 ku 处出现条带，纯化后条带单一。软件分析发现 RicA 蛋白融合抗原性较强，Western Blot检测证明其有较好的免疫原性。说明，成功克隆并表达布氏杆菌疫苗菌株 M5 RicA 基因，为进一步研究其与布氏杆菌 Ⅳ 型分泌系统及布氏杆菌致病机制之间的关系奠定了基础。
For cloning and expressing prokaryotic gene RicA of Brucella melitensis vaccine strain M5, which is related with Brucella type Ⅳ secretion system ，and for analyzing immunogenicity of the expressed protein, RicA gene was amplified from Brucella melitensis vaccine strain M5 by PCR technology, and TA cloned to pMD18-T vector for sequence analysis. Recombinant expression vector pET-28a(+)-RicA was constructed and transformed to expression stain E.coli BL21.Different concentrations of IPTG and different times were used to induce expression product. RicA protein was identified by SDS-PAGE and purified by protein purification system AKTA. Software Antheprot 5.0 was applied to analyze its physicochemical properties and antigenicity. Western blot was employed to test its immunogenicity.Results showed that full length of RicA gene was 528 bp, encoding 175 amino acids. Compared with those known strains in Genbank,both its nucleotide homology and amino acid homology achieved 99%. SDS-PAGE showed that RicA fusion protein appeared in the approximately 23 ku position and became a single band after it was purified. The software analysis and western blot indicated that RicA fusion protein possessed stronger antigenicity and better immunogenicity respectively. We successfully cloned RicA gene and expressed RicA protein of the Brucella melitensis vaccine strain M5, and laid a foundation of further research on relationships between RicA gene and Brucella type Ⅳ secretion system, as well as Brucella pathogenic mechanism.