盖塔病毒6K蛋白单克隆抗体的制备与鉴定

为用于猪群中盖塔病毒(Getah virus,GETV)的流行病学调查,并研究其6K蛋白功能,制备了该蛋白的单克隆抗体。将6K基因克隆至原核表达载体pCold-TF,并转化大肠杆菌BL21(DE3),进行IPTG低温诱导表达。将诱导的重组菌超声裂解,离心后收集上清,经过镍柱亲和和切胶纯化后,免疫BALB/c小鼠,取免疫小鼠脾细胞和SP2/0骨髓瘤细胞进行融合。使用间接免疫荧光(IFA)法筛选阳性杂交瘤细胞,获得1株GETV 6K单克隆抗体,命名为25C5。将其注射小鼠腹腔,制备6K单抗腹水。IFA检测结果显示,单抗25C5可以结合GETV蛋白并产生特异性荧光。在Western-blot和免疫沉淀试验中,单抗25C5可以结合GETV感染的ST-R细胞样品,其特异性条带大小约6 ku。以上结果表明,单抗25C5免疫学反应特性良好,可用于检测GETV,为该病毒的分离鉴定、致病机制和蛋白功能等研究奠定了基础。

Preparation and identification of monoclonal antibody against 6K protein of porcine Getah virus

To help the epidemiological survey of Getah virus(GETV)in pigs and study the function of its 6 K protein,a monoclonal antibody against GETV 6 K protein was prepared.6 K gene was cloned into the prokaryotic expression vector p Cold-TF,and transformed into Escherichia coli BL21(DE3)which was then induced by IPTG at low temperature.The induced recombinant bacteria were lysed by ultrasound to obtain the supernatant,which was purified by Ni2+affinity chromatography and gel-slicing,and then used to immunize BALB/c mice.The spleen cells of immunized mice were collected and fused with SP2/0 myeloma cells.The positive hybridoma cells were screened by indirect immunofluorescence assay(IFA)method,and then a GETV 6 K monoclonal antibody was obtained,named as 25 C5.The positive hybridoma cells were injected into mice intraperitoneally to prepare 6 K monoclonal antibody ascites.The results showed that 25 C5 can bind to the GETV antigen in the IFA,Western-blot and immunoprecipitation assays.The size of specific band was about 6 ku.Taken together,25 C5 obtained here can be used as a powerful tool to detect GETV,and lays the foundation for further studies on such as isolation,identification,pathogenic mechanism and protein functions of GETV.