非洲猪瘟病毒CD2v蛋白单链抗体的制备及鉴定

Preparation and identification of scFv against CD2v protein of African swine fever virus

为了获得特异性针对非洲猪瘟病毒(African swine fever virus,ASFV)CD2v蛋白单链抗体(scFv),本研究以CD2v阳性单克隆抗体杂交瘤细胞为模板,通过PCR扩增得到抗体重链可变区(VH)和轻链可变区(VL)基因,利用Overlap-PCR在VH和VL基因间引入短肽(Gly4Ser)2连接形成scFv。将其插入原核表达载体p ET28a(+)中,并转化至大肠杆菌BL21(DE3),经1 mmol/L IPTG于25℃条件下诱导表达,通过ELISA对复性scFv蛋白进行反应活性评价。结果显示,通过CD2v蛋白的阻断ELISA筛选出分泌阻断效果良好的单抗杂交瘤细胞株4B8,并成功扩增获得单克隆抗体4B8的重链可变区(VH,399 bp)、轻链可变区(VL,369 bp)及由GS Linker连接而成的4B8-scFv基因(744 bp);SDS-PAGE结果显示,4B8-scFv在大肠杆菌BL21(DE3)中以包涵体形式表达,通过尿素变性及透析复性,得到纯度较高的4B8-scFv(相对分子质量约为34 ku);间接ELISA结果表明,4B8-scFv与CD2v蛋白具有良好的反应活性。

To obtain specific single-chain variable fragment(sc Fv)antibodies against the CD2v protein of African swine fever virus(ASFV),CD2v-positive monoclonal antibody hybridoma cells were used as templates,and its variable regions of the heavy(VH)and light(VL)chains were amplified by PCR,respectively,in this study.The scFv genes were constructed through insertion of short peptide linker(Gly4Ser)2between VH and VL by Overlap-PCR.The sc Fv genes were inserted into the prokaryotic expression vector p ET28a(+),which was transformed into Escherichia coli BL21(DE3).The sc Fv protein expression was induced with 1 mmol/L IPTG at 25℃,then sc Fv reactivity was evaluated by ELISA.Results showed that the antibodies hybridoma secreted by cell line 4B8 could effectively block antibodies from pig sera against CD2v protein in ELISA.And 4B8-sc Fv gene(744 bp)was successfully constructed by joining VH(399 bp)and VL(369 bp)of a monoclonal antibody(m Ab)via a GS linker.SDS-PAGE result revealed that 4B8-sc Fv was expressed as inclusion bodies in E.coli BL21(DE3).High-purity 4B8-sc Fv(34 ku)was produced in inclusion bodies,which were further solubilized in urea denaturation with urea and refolding.Indirect ELISA results demonstrated good reactivity of 4B8-sc Fv with the CD2v protein.