登革病毒3型E蛋白的原核表达及抗体的制备  

Prokaryotic expression and antibody preparation of E protein of Dengue virus type 3

作　　者：林华[1,2,3] 杨会强[1] 李竹石[1] 杨健[1,4] 赵宇[1] 刘俐[1] 葛永红[1] 曾献武[1] 曹毅[2] 乔代蓉[2] 李玉华[5] 

机构地区：[1]成都生物制品研究所有限责任公司,610023 [2]四川大学生命科学学院 [3]四川出入境检验检疫局 [4]川北医学院 [5]中国食品药品检定研究院

出　　处：《中华微生物学和免疫学杂志》2013年第5期371-374,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家高技术研究发展计划（SS2012AA020901）

摘　　要：目的原核表达并纯化登革病毒3型（DENV-3）E蛋白，并用于制备其抗体。方法采用PCR技术克隆DENV-3E蛋白基因，插入原核表达质粒载体pET-32a（＋），转化大肠杆菌RoseRa（DE3），IPTG诱导表达。表达产物经纯化后，免疫家兔，制备DENV-3E蛋白抗血清，并用于Westernblot鉴定。同时，采用ELISA测定其抗体效价，并初步应用于DENV-3的检测。结果重组表达载体经双酶切及测序证实构建正确；表达的重组蛋白相对分子质量约为65×103，主要以包涵体形式表达；用纯化的包涵体蛋白免疫家兔后，获得的特异性抗血清效价为1：20480，该血清可与DENV-3发生特异性反应。结论本研究原核表达并纯化了DENV-3E蛋白，制备了高滴度的特异性兔抗血清。制备获得的DENV-3E蛋白抗体可用于登革病毒的鉴定，为登革病毒相关研究奠定了基础。Objective To prepare the antibody against E protein of Dengue virus type 3 with puri- fied E protein of DENV-3 expressed in prokaryotic cells. Methods The gene encoding E protein of DENV- 3 was amplified by polymerase chain reaction （ PCR）, and then inserted into prokaryotie expression vector pET-32a （＋） to construct the recombinant plasmid. The expression plasmid was then transformed into E. coli Rosetta （ DE3 ） to induce the expression of protein by IPTG. The antiserum of E protein of DENV-3 was pre- pared by immunizing rabbits with the purified recombinant protein and identified by Western blot. Antibody titer was determined by ELISA . The prepared polyclonal antibody was used to identify DENV-3 . Results The recombinant plasmid was successfully constructed as confirmed by restriction enzyme analysis and gene sequencing. The expressed target protein, with a relative molecular mass of about 65×103 , was mainly exis- ted in form of inclusion body. The prepared polyclonal antibody, with a tier of 1 ： 20 480, showed high spe- cificity. Indirect immunofluorescent assay showed that the prepared polyelonal antibody recognized the whole virus antigen of DENV-3 strain. Conclusion High titer polyclonal antibody against E protein of DENV-3 strain was successfully prepared, which laid a foundation for further studies on E protein and rapid detection of DENV-3 virus in clinic.

关 键 词：登革病毒 E蛋白 原核表达 多克隆抗体 

分 类 号：R3[医药卫生—基础医学]