人乳头瘤病毒58型(HPV58)主要衣壳蛋白L1的可溶性表达及应用  

The soluble expression and application of human papillomavirus type 58 (HPV58) major capsid protein L1

作　　者：陈玉梅 栗宁 薛明岩 周景明 祁艳华 殷佳佳 张雨晴 李昱雅 王爱萍 CHEN Yumei;LI Ning;XUE Mingyan;ZHOU Jingming;QI Yanhua;YIN Jiajia;ZHANG Yuqing;LI Yuya;WANG Aiping(School of Life Science,Zhengzhou University,Zhengzhou 450001,China)

机构地区：[1]郑州大学生命科学学院,河南郑州450001

出　　处：《细胞与分子免疫学杂志》2022年第6期553-558,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：河南省高校科技创新团队计划(19IRTSTHN006)。

摘　　要：目的原核表达获得人乳头瘤病毒58型主要衣壳蛋白L1(HPV58 L1)并进行鉴定。方法将HPV58 L1蛋白的重组表达菌pE⁃SUMO⁃58 L1(BL21)经IPTG进行诱导表达,SDS⁃PAGE及Western blot法鉴定重组SUMO⁃58 L1蛋白的表达情况;经Ni柱纯化获得重组SUMO⁃58 L1蛋白,经泛素样蛋白酶1(ULP1)酶切去除SUMO标签获得重组HPV58 L1蛋白,红细胞凝集试验(HA)验证HPV58 L1蛋白活性;再将重组HPV58 L1蛋白免疫BALB/c小鼠,ELISA测定免疫血清效价,间接免疫荧光试验(IFA)检测免疫血清与HEK293T细胞瞬时表达的HPV58 L1蛋白的反应情况。结果重组蛋白SUMO⁃58 L1在大肠杆菌中可溶性表达量较高,其相对分子质量(M_(r))约72000。经Ni⁃NTA亲和纯化后,再用ULP1去除SUMO标签最终得到M_(r)约58000的HPV58 L1蛋白,该重组蛋白具有类似于HPV病毒的凝集小鼠红细胞的血凝活性,其血凝价为1∶16。ELISA测定HPV58 L1蛋白免疫小鼠血清效价最高可达1∶10240,且免疫血清可与HEK293T细胞瞬时表达的HPV58 L1蛋白发生特异反应。结论经大肠杆菌表达系统成功获得了具有血凝活性的重组HPV58 L1蛋白,其免疫血清可用于免疫细胞化学检测真核细胞表达的HPV58 L1蛋白。Objective The major capsid protein L1 of human papillomavirus type 58(HPV58 L1)was obtained and identified by prokaryotic expression.Methods The recombinant expression strain pE⁃SUMO⁃58 L1(BL21)was induced by IPTG.The recombinant protein SUMO⁃58 L1 was expressed in E.coli and identified by SDS⁃PAGE and Western blot analysis.Then the recombinant protein SUMO⁃58 L1 was purified by Ni⁃column and the SUMO⁃tag was removed by ubiquitin⁃like protease 1(ULP1)digestion.Subsequently,the bioactivity of recombinant protein HPV58 L1 was verified by hemagglutination assay(HA).BALB/c mice were immunized with HPV58 L1,and the antibody titers in sera of the immunized mice were detected by ELISA.And then the reaction between the immune serum and the HPV58 L1 protein transiently expressed by HEK293T cells was detected by indirect immunofluorescence assay(IFA).Results The soluble expression of the recombinant protein SUMO⁃58 L1 was identified by SDS⁃PAGE and Western blot analysis,with yields of soluble protein SUMO⁃58 L1 being about 50%of total soluble bacterial proteins.The relative molecular mass(M_(r))of SUMO⁃58 L1 was about 72000.After Ni⁃NTA affinity was purified and the SUMO⁃tag was removed by ULP1 digestion,M_(r) of recombinant protein HPV58 L1 reached about 58000.The recombinant protein HPV58 L1 showed hemagglutination activity similar to that of natural HPV,with hemagglutination value of 1∶16.After immunizing BALB/c mice,the titer of immune serum observed was about 1∶10240 by ELISA;and the sera of the immunized mice reacted specifically with HPV58 L1 proteins which were transiently expressed in HEK293T cells by IFA.Conclusion The recombinant protein HPV58 L1 also has hemagglutination activity,which can be successfully obtained from E.coli.The sera of the HPV58 L1 protein immunized mice can be used for immunocytochemical detection of HPV58 L1 protein expressed in eukaryotic cells.

关 键 词：人乳头瘤病58型(HPV58) L1蛋白 原核表达 间接免疫荧光技术(IFA) 

分 类 号：R711.74[医药卫生—妇产科学] R737.33[医药卫生—临床医学] Q816[生物学—生物工程]