含有RSV G蛋白片段和CTL表位融合蛋白的表达及纯化

Expression and purification of fusion protein containing segment of glycoprotein G and CTL epitope of protein M2 from RSV

目的:构建表达呼吸道合胞病毒(RSV)G蛋白片段(G126,来自100～225氨基酸)和来自RSVM2蛋白的CTL表位的原核表达质粒,探索适宜的表达和纯化条件,获得融合表达产物,为进行体内外实验检测奠定基础。方法:利用PCR技术,从质粒pUC18(G10)、pUC18(CTL)中扩增G126和CTL基因,通过DNA重组技术构建含DsbA G126Linker CTL(DsbA GLC)融合基因的表达载体pET(GLC)。转化宿主菌E.coliBL21(DE3)plysS进行诱导表达,并采用亲和层析纯化目的蛋白。结果:DsbA GLC在E.coliBL21(DE3)plysS中可获得高效表达,表达量可达菌体总蛋白的21%。通过可溶性测试,DsbA GLC能以可溶性形式表达。纯化后目的蛋白的纯度可达到95%以上。结论:实现RSVG126和M2蛋白CTL表位的融合表达,所获得的重组蛋白可作为抗原用于RSV重组蛋白疫苗的筛选。

Objective: To construct a prokaryotic expression system for the amino acid region 100-225(G126) of glycoprotein G and CTL epitope of protein M2 from the respiratory syncytial virus (RSV), and search an appropriate inducing and purification condition to gain soluble fusion protein. Methods: G126 gene and CTL gene were amplified by PCR from plasmid pUC18(G) and pUC18(CTL), respectively. The recombinant expression plasmid pET(GLC) containing DsbA-G126-Linker-CTL(DsbA-GLC) fusion gene was constructed by recombinant DNA technique and then transformed into Escherichia coli BL21(DE3)plysS. The expression product was purified by affinity chromatography. Results: The expression level of DsbA-GLC fusion protein was about 21% of total cellular protein. The DsbA-GLC fusion protein existed in both suspension and precipitation of the lysis solution of BL21(DE3)plysS〔pET(GLC)〕. After purification, the purity of fusion protein in elution was more than 95%. Conclusion: The fusion and expression of DsbA-GLC is achieved. The fusion protein could be used as an antigen for screening recombinant protein vaccine against RSV.