肠道病毒71型多表位-mGITRL真核表达质粒的构建、表达及其免疫原性研究  

Construction,expression and immunogenicity of EV71 multiepitope-mGITRL eukaryotic plasmid

作　　者：毛镇伟[1] 刘海冰[2] 郑东[2] 彭辉勇[2] 王瑞[2] 苏兆亮[2] 陈建国[1] 

机构地区：[1]江苏大学附属人民医院检验科,江苏镇江212002 [2]江苏大学检验医学研究所,江苏镇江212013

出　　处：《细胞与分子免疫学杂志》2012年第8期797-800,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：镇江市医学重点人才项目(2010年)

摘　　要：目的:构建肠道病毒71型(EV71)多表位-mGITRL真核表达质粒并对其免疫原性进行研究。方法:串联已证实的VP1表位并合成(VP1’);PCR扩增获得mGITRL,克隆至真核共表达载体pIRES中。对重组质粒进行测序鉴定后,以脂质体介导法转染至COS7细胞,通过Western blot方法检测其在COS7细胞中的表达。将构建好的重组质粒免疫小鼠,应用ELISA法检测小鼠血清中抗VP1的抗体滴度。结果:PCR扩增出目的基因,测序证实真核共表达载体pIRES-VP1’-mGITRL构建成功,Western blot结果显示目的基因在COS7细胞和肌细胞中过表达。小鼠血清中检测出高滴度的抗VP1抗体。结论:成功扩增出目的基因并构建VP1表位的重组真核共表达载体pIRES-VP1’-mGITRL,在COS7细胞和肌细胞中过表达,并获得高滴度的抗VP1抗体。AIM： To construct an enterovirus 71（EV71） multiepitope-mGITRL eukaryotic plasmid and study its immunogenicity in BALB/c mice.METHODS： We first designed and synthesized VP1＇ epigene containing two B cells and two T cells epitopes of VP1,and amplified mGITRL gene by PCR.The VP1＇ epigene and mGITRL gene were then cloned into the expression vector pIRES to construct the recombination plasmid pIRES-VP1＇-mGITRL.The recombination plasmid was transfected into COS7 cells by liposome-mediated method.The protein expressions of VP1＇ and mGITRL were detected by Western blotting.BALB/c mice were immunized with pIRES-VP1＇-mGITRL plasmid,and its serum antibody titer was measured by ELISA.RESULTS： The recombination plasmid pIRES-VP1＇-mGITRL was successfully constructed as demonstrated by sequencing.Western blot analysis indicated that the VP1＇-mGITRL fusion protein was expressed in COS7 cells and muscle cells.After BALB/c mice were immunized with this plasmid,we detected the high titer of anti-VP1 antibody in serum.CONCLUSION： VP1＇-mGITRL fusion protein can be highly expressed in COS7 cells and muscle cells by the construction and transfection of the recombination plasmid pIRES-VP1＇-mGITRL,and it could elicit the dramatic immune response in mice.

关 键 词：EV71 VP1 mGITRL 表位 

分 类 号：R183.4[医药卫生—流行病学]