呼吸道合胞病毒重组蛋白DsbA-G1F/M2的制备方法及免疫原性研究  

Preparation of Recombinant Protein DsbA-G1F/M2 of Respiratory Syncytial Virus and Its Immunogenicity

作　　者：曾瑞红[1] 龚伟[1] 梅兴国[1] 刁志花[1] 郝丽梅[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：《中国药学杂志》2007年第2期90-93,共4页Chinese Pharmaceutical Journal

摘　　要：目的建立呼吸道合胞病毒(RSV)重组蛋白DsbA-G1F/M2的制备方法,并研究其免疫原性。方法PCR扩增G1和F/M2基因片段,经酶切后插入原核表达载体pET-DsbA中,构建重组质粒pET-DsbA-G1F/M2,转化大肠杆菌(Escherichiacoli)BL21(DE3),IPTG诱导表达,采用Ni+螯合亲和色谱法纯化尿素变性的包涵体溶液,梯度透析复性,Western-blot鉴定重组蛋白的RSV特异性,免疫BALB/c小鼠,用ELISA测定抗体滴度,MTT法测定细胞毒性T细胞活性(CTL)。结果构建的重组质粒在E.coli中表达了RSV特异性的重组蛋白,经变性、纯化并复性后获得了高纯度的DsbA-G1F/M2(>90%);该蛋白诱导了高滴度的RSV特异性抗体和CTL活性。结论建立了重组蛋白DsbA-G1F/M2的制备方法,该蛋白具有良好的免疫原性,为进一步研究开发RSV重组亚单位疫苗奠定了基础。OBJECTIVE To establish a method for preparing recombinant protein DsbA-G1 F/M2 of respiratory syncytial virus and to investigate its immunogenicity. METHODS The G1 and F/M2 gene fragments were amplified by PCR method and then ligated into the prokaryotic expression vector pET-DsbA after being digested by corresponding endonucleases. The recombinant plasmid pETDsbA-G1 F/M2 was transferred into E. coli BL21 （DE3） and the expression of the molecule was induced by IPTG. The inclusion degenerated in urea solution was purified by affinity chromatography, renatured by gradient dialysis, and characterized by Westemblot. DsbA-G1F/M2 was used to immune BALB/c mice. Anti-RSV antibody was measured by ELISA and RSV-spocific CTL responses by MTT. RESULTS The recombinant expression plasmid successfully expressed RSV specific fusion protein in E. col BL21 （ DE3 ）. The purity of the fusion protein was over 90% after being purified by affinity chromatography and renatured. The protein induced high level anti-RSV antibody titers and RSV-spocific CTL responses. CONCLUSION The processing above can be a scheme to obtain the high purity and RSV specific recombinant protein DsbA-G1 F/M2.

关 键 词：呼吸道合胞病毒 重组蛋白DsbA—G1F/M2 原核表达 纯化 免疫原性 

分 类 号：R963[医药卫生—微生物与生化药学]