森林脑炎病毒E蛋白DomainⅢ的串联表达、纯化及其多克隆抗体的制备

Tandem expression and purification of tick-borne encephalitis virus E protein DomainⅢand preparation of its polyclonal antibody

目的串联表达、纯化森林脑炎病毒(tick-borne encephalitis virus,TBEV)E蛋白DomainⅢ(EDⅢ),并制备多克隆抗体。方法Trizol法提取TBEV RNA,反转录为cDNA,以其为模板PCR扩增EDⅢ基因片段,采用重叠PCR法将2段EDⅢ基因片段通过疏水性柔性多肽(G_(4)S)_(3)连接为融合基因,与原核表达载体pET-28a(+)连接,构建重组表达质粒pET-28a-2EDⅢ,经测序鉴定后,转化至E.coli BL21(DE3)感受态细胞,IPTG诱导表达,Ni^(2+)亲和层析纯化。以复性后的重组蛋白为免疫原,免疫雌性新西兰大白兔制备多克隆抗体,间接ELISA法检测效价,Western blot法鉴定特异性。利用DNAMAN软件分析TBEV与其他黄病毒EDⅢ氨基酸序列同源性。结果重组质粒pET-28a-2EDⅢ经测序鉴定,扩增序列包含2段与GenBank上登录的TBEV“森张”株(JQ650523.1)E序列一致的基因,证明质粒构建正确。诱导表达的重组2EDⅢ蛋白相对分子质量约21000,主要以包涵体形式存在,纯度可达97.5%。兔抗2EDⅢ血清多克隆抗体效价为1∶107,可与TBEV全病毒发生特异性反应。经DNAMAN软件比对,TBEV与日本脑炎病毒(Japanese encephalitis virus,JEV)、黄热病病毒(yellow fever virus,YFV)、登革病毒(Dengue virus,DENV)EDⅢ氨基酸序列同源性分别为36.56%、9.28%、30.77%。结论成功构建了TBEV EDⅢ串联重组表达质粒pET-28a-2EDⅢ,表达的重组2EDⅢ蛋白具有良好的反应性和免疫原性,制备的多克隆抗体效价较高。

Objective To express and purify the E protein DomainⅢ(EDⅢ)of tick-borne encephalitis virus(TBEV)in tandem and prepare the corresponding polyclonal antibody.Methods The TBEV RNA was extracted by Trizol method,and then reversely transcribed into cDNA,which was used as template to amplify EDⅢgene fragment by PCR.Two EDⅢgene fragments were ligated into fusion gene by the hydrophobic flexible polypeptide(G,S),using overlapping PCR,which was then linked to prokaryotic expression vector pET-28a(+)to construct the recombinant expression plasmid pET-28a-2EDⅢ.After sequencing,pET-28a-2EDⅢwas transformed into E.coli BL21(DE3)competent cells,induced by IPTG and purified by Ni2*affinity chromatography.Female New Zealand white rabbits were immunized with the renatured recombinant protein to prepare polyclonal antibody.The antibody titer was detected by indirect ELISA and the specificity was identified by Western blot.The homology of EDⅢamino acid sequence between TBEV and other flaviviruses was analyzed by DNAMAN software.Results The recombinant plasmid pET-28a-2EDⅢwas identified by sequencing,and the amplified sequence contained two genes consistent with the E sequence of TBEV"Senzhang"strain(JQ650523.1)included on GenBank,indicating that the recombinant plasmid was constructed correctly.The recombinant 2EDⅢprotein was expressed mainly in the form of inclusion bodies,with a relative molecular mass of about 21000 and a purity of 97.5%.The titer of rabbit anti-2EDⅢserum polyclonal antibody was 1:10',which reacted specifically with TBEV whole virus.DNAMAN software alignment showed that the homology of EDⅢamino acid sequences between TBEV and Japanese encephalitis virus(JEV),yellow fever virus(YFV)and Dengue virus(DENV)was 36.56%,9.28%and 30.77%,respectively.Conclusion The TBEV envelope EDⅢtandem recombinant expression plasmid pET-28a-2EDⅢwas successfully constructed.The expressed recombinant 2EDⅢprotein had good reactivity and immunogenicity,and the prepared polyclonal antibody had high titer.