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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 plasmid expressing the G protein segment of respiratory syncytial virus (RSV) (G126 from 100-225 amino acids) and the CTL epitope from RSVM2 protein, and to explore suitable expression and purification conditions to obtain the fusion expression product In vitro and in vivo experiments to establish the basis for testing. Methods: The G126 and CTL genes were amplified from the plasmids pUC18 (G10) and pUC18 (CTL) by PCR and the expression vector pET (GLC) containing the DsbA G126Linker CTL (DsbA GLC) fusion gene was constructed by DNA recombination. The transformed host strain E.coli BL21 (DE3) plysS was induced to express, and the target protein was purified by affinity chromatography. RESULTS: DsbA GLC was highly expressed in E.coli BL21 (DE3) plysS, and the expression level of DsbA GLC reached 21% of the total bacterial proteins. DsbA GLC can be expressed in soluble form by soluble testing. After purification, the purity of the target protein can reach more than 95%. Conclusion: The fusion protein of CTV epitope of RSVG126 and M2 can be expressed in E.coli. The obtained recombinant protein can be used as an antigen in the screening of RSV recombinant protein vaccine.