穿膜肽HIV-TAT与呼吸道合胞病毒G蛋白片段G1及CTL表位融合蛋白的表达及纯化  

Expression and Purification of Fusion Protein Containing Cell-penetrating Peptide HIV-TAT as well as Glycoprotein G1 and CTL Epitope of Respiratory Syncytial Virus

作　　者：张竞[1] 龚伟[2] 梅兴国[2] 阎浩林[1] 

机构地区：[1]沈阳药科大学生命科学与生物制药学院,沈阳110016 [2]军事医学科学院毒物药物研究所,北京100850

出　　处：《中国生物制品学杂志》2008年第11期951-953,共3页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(30671923)

摘　　要：目的在大肠杆菌中表达含穿膜肽HIV-TAT与呼吸道合胞病毒(RSV)G蛋白片段G1及CTL表位的融合蛋白,并进行纯化。方法以质粒pET-G1F/M2为模板,扩增含TAT、RSVG蛋白片段G1和CTL表位F/M2的融合基因片段,与原核表达质粒pET-His连接,构建融合表达质粒pET-TAT-G1F/M2,转化E.coli BL21(DE3)plysS进行诱导表达,采用Ni2+螯合亲和层析法纯化经尿素变性的包涵体,梯度透析复性纯化的目的蛋白,并进行Westernblot鉴定。结果在E.coli中可高效表达重组蛋白TAT-G1F/M2,表达量占菌体总蛋白的30%以上,目的蛋白主要存在于包涵体中。经变性、纯化、复性可获得高纯度(>95%)特异性的TAT-G1F/M2蛋白。结论已在大肠杆菌中成功表达了融合蛋白TAT-G1F/M2,为进一步进行RSV体内体外免疫学研究奠定了基础。Objective To express the fusion protein containing Cell-penetrating peptide HIV-TAT as well as the glycoprotein G1 and CTL epitope of respiratory syncytial virus （RSV） in E. coli and purify the expressed product. Methods Amplify the fusion gene encoding HIV-TAT as well as G1 protein and CTL epitope F/M2 of RSV using plasmid pET-G1F/M2 as a template and insert into prokaryotic expression vector pET-His. Transform the constructed recombinant plasmid pET-TAT-G1F/M2 to E. coli BL21 （DE3）plysS for expression under induction of IPTG. The expressed product in a form of inclusion body was denaturalized with urea, purified by nickel ion chelate affinity chromatography, renaturalized by gradient dialysis and identified by Western blot. Results The recombinant fusion protein TAT-G1F/M2 expressed in E. coli mainly existed in inclusion body and contained more than 30% of total somatic protein. After denaturalization, purification and renaturalization, the expressed protein reached a purity of more than 95%. Conclusion Fusion protein TAT-G1F/M2 was successfully expressed in E. coli, which laid a foundation of in vitro and in vivo immunological study on RSV.

关 键 词：呼吸道合胞病毒 G蛋白 CTL表位 穿膜肽 融合蛋白 表达 纯化 

分 类 号：Q786[生物学—分子生物学]