猪附红细胞体MSG1基因人工合成、克隆、表达及免疫原性分析

Synthesizing,cloning and expression of Mycoplasma suis MSG1 gene using PCR-based accurate synthesis and overlap-extension PCR,and its immunogenicity analysis

目的利用引物重叠扩增法人工合成猪附红细胞体粘附蛋白基因MSG1,并检测其免疫原性。方法将人工合成的猪附红细胞体粘附蛋白基因MSG1产物插入到pMD18-T载体中,亚克隆至原核表达载体pET28c,构建pET28c_msg1表达载体,转入E.coli BL21,IPTG诱导表达。用SDS-PAGE,Western-blot方法分析鉴定,表达产物具有很好的抗原性。纯化后的重组蛋白接种昆明小白鼠后,用重组蛋白作为抗原包被酶标板,以检测接种昆明小白鼠血清抗体的产生。结果结果表明MSG1基因在大肠杆菌中能够得到高效的表达,并能够刺激小鼠产生较好的抗体。结论人工成功的合成了猪附红细胞体粘附蛋白基因MSG1,其基因可在大肠杆菌中高效表达,并可刺激小鼠产生较好的抗体。

The adhesion protein MSG1 of Mycoplasma suis was synthesized by overlap PCR and inserted into vector pMD18-T,and then subcloned to prokaryotic expression vector pET28c.The recombinant plasmid pET28c_msg1 was transformed into E.coli BL21 for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot,suggested that the recombinant protein had good antigenicity.The purified recombinant protein was adopted to vaccinate Kunming mice,and the recombinant protein as antigen-coated ELISA plates was employed to detect antibodies in Kunming mouse serum.The results showed that MSG1 gene could be efficiently expressed in E.coli,and could produce better antibodies in mice.This experiment is significance to analyze the function of MSG1 gene in future research.