表达肠道病毒71型中和表位融合基因的重组耻垢分枝杆菌的构建及其免疫原性初步评价  

Construction and immunogenicity of recombinant Mycobacterium smegmatis expressing neutralizing epitope fusion gene of enterovirus 71

作　　者：梁思佳 吴岳 石春薇[2] 刘洪波 LIANG Si-jia;WU Yue;SHI Chun-wei;LIU Hong-bo(Department of Clinical Laboratory,Second Affiliated Hospital,Guilin Medical College,Guilin 541199,Guangxi Zhuang Autonomous Region,China;不详)

机构地区：[1]桂林医学院第二附属医院检验科,广西桂林541199 [2]华中科技大学同济医学院基础医学院病原生物学系,湖北武汉430030

出　　处：《中国生物制品学杂志》2020年第9期974-977,982,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81460304,81660280);广西高校科学技术研究项目(KY2015LX266)。

摘　　要：目的构建表达嵌合肠道病毒71型(enterovirus 71,EV71)中和表位VP1-SP70(E1)和VP2-141(E2)融合蛋白HBc-E1/2的重组耻垢分枝杆菌(Mycobacterium smegmatis,MS),并评价其在小鼠体内的免疫原性。方法PCR法扩增目的基因HBc-E1/2,并克隆至E.coli-分枝杆菌穿梭载体pMV261,构建重组质粒pMV261-HBc-E1/2,电转化至mc2155(MS野生株ATCC 607的突变体),获得重组菌pMV261-HBc-E1/2/mc2155,将其经腹腔免疫BALB/c小鼠,间接ELISA法检测小鼠血清抗体效价。结果经双酶切及测序鉴定,重组质粒pMV261-HBc-E1/2构建正确。重组菌pMV261-HBc-E1/2/mc2155表达产物可与E1多肽、E2多肽及EV71灭活疫苗小鼠免疫血清发生特异性结合,且在相对分子质量约27500和55000处可见特异性蛋白结合条带。免疫小鼠血清抗E1及E2表位抗体效价均为1∶1600。结论成功构建了呈递HBc-E1/2的重组MS,可诱导小鼠产生EV71的特异性抗体。本实验为手足口病(hand,foot and mouth disease,HFMD)新型疫苗的研发奠定了基础。Objective To construct a recombinant Mycobacterium smegmatis(MS)for expression of a fusion gene HBcE1/2 consisting of chimeric VP1-SP70(E1)and VP2-141(E2)neutralizing epitopes of enterovirus(EV)71 and evaluate its immunogenicity in mice.Methods HBc-E1/2 gene was amplified by PCR and cloned into E.coli-Mycobacterium shuttle plasmid pMV261.The constructed recombinant plasmid pMV261-HBc-E1/2 was transformed to mc2155,a mutant of wild MS ATCC607 strain,by electroporation,and the obtained recombinant bacterial strain pMV261-HBc-E1/2/mc2155 was inoculated i.p.into BALB/c mice.The serum antibody titer of mice was determined by indirect ELISA.Results Restriction analysis and sequencing proved that recombinant plasmid pMV261-HBc-E1/2 was constructed correctly.The expressed product showed specific binding to the sera of mice immunized with E1 and E2 polypeptides and inactivated EV71 vaccine,and showed specific binding bands with relative molecular masses of about 27500 and 55000.Both the titers of anti-E1 and E2 epitope antibodies in the sera of immunized mice were 1∶1600.Conclusion A recombinant MS vaccine presenting HBc-E1/2 was successfully constructed,which induced specific antibody against EV71 in mice.It laid a foundation of the development of a novel vaccine against hand,foot and mouth disease(HFMD).

关 键 词：肠道病毒71型 中和表位 基因重组 耻垢分枝杆菌 

分 类 号：R392-33[医药卫生—免疫学]