仙台病毒F基因的克隆及原核表达

Expression of Fusion Gene of Sendai Virus in Prokaryotic System

目的利用原核表达系统表达仙台病毒(Sendai Virus)F蛋白主要抗原片段FP(S),并对表达产物进行免疫学初步研究。方法根据GenBank公布的仙台病毒F蛋白(gi:9627219)的基因序列设计特异性引物,通过RT-PCR扩增出F基因的主要抗原片段FP(S),插入pMD-18-T载体中,鉴定正确后克隆入pQE31原核表达载体中,将鉴定正确的pQE31-FP(S)转化大肠埃希菌M15,IPTG诱导表达,对大肠埃希菌裂解物进行SDS-PAGE和Western-blot验证。结果大肠埃希菌表达的FP(S)相对分子质量约26×103,与预期相符;能与SeV阳性血清发生特异性反应,出现单一条带。结论原核表达的FP(S)蛋白有良好的抗原性,为检测仙台病毒抗体的ELISA检测方法的研究奠定了基础。

Objective To express fusion gdne of Sendai virus in prokaryotic system and analyze its immunogenicity. Methods PCR primers were designed according to fusion protein of Sendai virus （gi：9627219）. FP（S） cDNA specific for the primary antigen form of fusion gene of Sendai virus was produced by RT-PCR and was cloned into pMD18-T vector for sequence analysis. The pMD18-T-FP（S） was digested by restriction enzymes and then subcloned into the prokaryotic expression vector pQE31. The FP（S） was expressed in E. coli M15 by IPTG induction and purified, which was confirmed by SDS-PAGE and Western blot. Results The recombinant protein FP（S） was about 26 × 10^3 and could react with mice-anti Sendai virus serum. Conclusion The recombinant protein FP（S） has a good immunogenicity and the results of the present study provided a good basis for detection of SeV-specific antibodies by ELISA assay.