呼吸道合胞病毒重组蛋白候选疫苗的质粒构建、表达及免疫原性和保护性研究  

Plasmid Construction, Expression, Immunogenicity and Protective Efficacy of Recombinant Protein Candidate Vaccine of Respiratory Syncytial Virus

作　　者：曾瑞红[1] 龚伟[1] 方学平[1] 张振亚[1] 梅兴国[1] 

机构地区：[1]军事医学科学院毒物药物研究所,北京100850

出　　处：《生物工程学报》2005年第4期534-539,共6页Chinese Journal of Biotechnology

摘　　要：PCR扩增呼吸道合胞病毒(respiratory syncytial virus,RSV)M2蛋白的CD8+T细胞表位F/M2:81-95和RSV-G蛋白的B细胞表位片段G:125～225(简称G1),以一个Linker连接,插入质粒pET-DsbA中构建原核表达重组质粒,转染E.coli BL21(DE3)后成功表达了融合蛋白DsbA-G1-Linker-F-M2:81-95(简称D-G1LF/M2),Western-blot结果表明该融合蛋白是RSV特异性的,采用Ni+螯合亲和层析法纯化变性的包涵体溶液,经梯度透析法复性,用该蛋白免疫BALB/c小鼠,结果表明被免疫小鼠肺部及血清中产生了高滴度的抗D-G1LF/M2及抗RSV IgG抗体和中和抗体,同时还诱导产生了RSV特异性的CTL应答;IgG的亚型IgG1/IgG2a的比值为2.66;用RSV攻击免疫后的小鼠,病毒滴定法检测肺部RSV滴度,结果表明D-G1LF/M2对小鼠肺部具有保护作用.To construct plasmid of recombinant protein candidate vaccine of respiratory syncytial virus, express it in E.coli, and to investigate its immunogenicity and protective efficacy. A CD8~ +T cell epitope from respiratory syncytial virus(RSV) M2 protein F/M2:81-95 and the G:125～225 (G1) gene fragments from RSV-G protein containing B cell epitopes were amplified by PCR method and then inserted into the prokaryotic expression vector pET-DsbA after bonding to a linker. The fusion protein DsbA-G1-Linker-F/M2:81-95 (D-G1LF/M2) was expressed successfully in E.coli BL21(DE3). The product was proved to be RSV-specific by Western-blot. After purified by affinity chromatography on Ni~ + Sepharose and renatured by gradient dialysis.D-G1LF/M2 was used to immune BALB/c mice.D-G1LF/M2 induced high anti-D-G1LF/M2 IgG, anti-RSV IgG and neutralizing antibody titers in serum and lung of BALB/c mice, and elicied RSV-specific CTL responses. The IgG subclass distribution revealed that IgG1/IgG2a ratio was 2.66. Viral titration indicated that D-G1LF/M2 could protect BALB/c mice against RSV challenge in lung.

关 键 词：呼吸道合胞病毒(RSV) 融合蛋白D—G1LF/M2 中和抗体 CTL 

分 类 号：R392.11[医药卫生—免疫学]