伪狂犬病病毒变异株gE蛋白单克隆抗体的制备与鉴定

GENERATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST GE PROTEIN OF PSEUDORABIES VIRUS VARIANT STRAIN

为制备伪狂犬病病毒变异株gE蛋白的单克隆抗体,PCR扩增伪狂犬病病毒gE蛋白主要抗原表位区的基因片段,并将其克隆至原核表达载体p Cold-TF。经1 mmol/L IPTG低温诱导,表达了约90 k Da的gE融合蛋白。融合蛋白用镍离子金属亲和层析柱纯化后,免疫6周龄BALB/c雌性小鼠,经细胞融合和筛选,获得了2株稳定分泌抗gE蛋白单克隆抗体的杂交瘤细胞(5B2和6E6)。间接免疫荧光试验(indirect immunofl uorescence assay,IFA)显示2株单克隆抗体均能与PRV变异株(JS-2012)反应,但与gE缺失的疫苗毒Bartha-K61株无反应。Western blot结果显示5B2和6E6与gE蛋白不反应,提示2株单克隆抗体针对的可能是gE蛋白的构象表位。本研究获得的gE蛋白单克隆抗体为伪狂犬病病毒变异株的鉴定与功能研究以及野毒株和疫苗株的鉴别诊断提供了良好的工具。

The gene encoding the key antigenic epitope regions of gE of pseudorabies virus （PRV） was amplified in PCR and inserted into the pCold-TF expression vector. Expression of the recombinant fusion protein about 90 kDa was observed under lmmol/L IPTG environment. The recombinant gE protein purified with nickel ion metal affinity chromatography was used to immunize 6-week-old BALB/c female mice for the preparation of monoelonal antibodies （McAbs）. Two hybridoma cell lines （5B2 and 6E6） were obtained for stably producing anti-gE protein McAb. These two MeAbs reacted with PRV variant strain （JS-2012） but not with the gE deleted vaccine virus Bartha-K61 strain in indirect immunofluorescence assay （IFA）. Western blot results showed that 5B2 and 6E6 did not react with PRV（JS-2012）. The results indicated that 5B2 and 6E6 might be against confomational epitope of gE. These specific monoclonal antibodies might provide a good tool for the development of PRV diagnostic method.