新疆出血热病毒多表位基因的原核表达及间接ELISA方法的建立

Prokaryotic expression of a multi-epitope gene of Xinjiang hemorrhagic fever virus and development of an indirect ELISA for virus detection

目的：原核表达纯化新疆出血热病毒（ XHFV） YL04057株的多表位肽,以其为抗原建立检测动物血清抗XHFV抗体的间接ELISA方法。方法依照对XHFV YL04057株核蛋白和糖蛋白氨基酸序列的亲水性和抗原决定簇位点分析,设计包含有保守性强的共6个优势B细胞表位的多表位基因片段,经串联重复成为双拷贝多表位基因 MEPX2,采用化学法合成后构建原核表达质粒pET-32a（＋）-MEPX2,经诱导表达和镍柱亲和层析纯化获得重组rMEPX2多表位肽,Western blot检测其抗原性,以其作为包被抗原采用方阵滴定法建立间接ELISA方法检测动物血清,并与商品化试剂盒检测结果进行比较。结果经双酶切鉴定和测序证实构建的重组表达质粒pET-32a（＋）-MEPX2正确,经SDS-PAGE分析和Western blot鉴定证实rMEPX2表达正确,相对分子质量约为49＆#215;103,能被His-tag单抗及天然感染XHFV的羊血清特异性识别。以纯化的rMEPX2为抗原建立了检测XHFV IgG抗体的间接ELISA方法,用此法对108份绵羊血清样品进行检测,并与双抗原夹心法商品化试剂盒检测比较,结果显示基于rMEPX2建立的检测方法具有较高的特异性。结论成功获得抗原性强的重组多表位肽rMEPX2,以其为抗原建立的间接ELISA方法可用于XHFV特异性抗体的检测。

Objective To express and purify the multi-epitope peptide of Xinjiang hemorrhagic fe-ver virus （ XHFV） in a prokaryotic expression system and to further use it as diagnostic antigen to establish an indirect ELISA for the detection of XHFV in animal sera. Methods A double copy multi-epitope gene （ MEPX2 ） segment with repeated tandem including six highly conservative and dominant B cell epitopes was designed based on the analysis of hydrophilicity and antigenic determinant sites in amino acid sequences of nucleoprotein and glycoprotein from XHFV strain YL04057. The multi-epitope gene MEPX2 was synthesized by chemical method and then inserted into the prokaryotic expression vector pET-32a （＋） to construct the prokaryotic expression plasmid pET-32a （＋）-MEPX2 . The recombinant expression plasmid was transformed into E. coli BL21 （DE3） to induce the expression of rMEPX2 by IPTG. The expressed products were purified by Ni-NTA purification system. The antigenicity of rMEPX2 was identified by Western blot assay. The puri-fied rMEPX2 was used as coating antigen to establish an indirect ELISA by checkerboard for the detection of＆amp;nbsp;XHFV in animal serum samples. The results of virus detection were compared with those by using commer-cial ELISA kit. Results The recombinant expression plasmid pET-32a （＋）-MEPX2 was constructed suc-cessfully as confirmed by restriction enzyme digestion and DNA sequencing analysis. Results of the SDS-PAGE and Western blot assay confirmed that the rMEPX2 was expressed correctly with a relative molecular mass （Mr） of about 49＆#215;103. The rMEPX2 was recognized specifically by His-tag mouse monoclonal antibody and by positive serum samples of sheep naturally infected with XHFV. The indirect ELISA for the detection of XHFV IgG antibody was established by using the purified rMEPX2 as antigen. Compared with the commer-cial ELISA kit, the established indirect ELISA showed good specificity for the detection of XHFV in 108 sheep serum samples. Conclusion The recombinant multi-epitope protein rMEPX2 with good antigenicity was obtained successfully. The established indirect ELISA based on the rMEPX2 could be used for the detec-tion of specific antibodies against XHFV.