伪狂犬病病毒gE基因主要抗原表位区的原核表达及其在疫苗接种和自然感染鉴别诊断中的应用

根据伪狂犬病病毒（PRV）Min-A株gE基因序列，利用PCR方法扩增了PRV-gE基因不含信号肽、胞内区和跨膜区的主要抗原表位区，并克隆到原核表达载体pGEX-6p-1中，获得的重组质粒命名为pGEX-tgE。经SDSPAGE电泳分析证实克隆的部分gE基因获得了表达，融合表达产物大小约为63kD，并在终浓度为0.6mmol/L的IPTG诱导下，3.5h其表达量达到高峰。通过改变诱导条件，有效抑制了包涵体形成，提高了重组蛋白的溶解性。Western blot分析证实表达的重组gE蛋白具有抗原反应活性。将表达产物利用亲和层析法纯化后作为ELISA抗原，通过对其特异性、敏感性及工作条件的优化试验，和对48份PRV阴性血清样品的检测结果的统计学分析，建立了猪伪狂犬病tgE-ELISA鉴别诊断方法。通过对400份送检血清样品的检测结果分析，表明其与PRV全病毒ELISA试验的符合率高达95%以上，与基于抗gE蛋白单抗竞争性ELISA的符合率达94%。此方法可用于gE基因缺失PRV疫苗免疫动物和PRV自然感染动物的鉴别诊断。

Expression of Truncated gE Gene of Pseudorabies Virus(PRV) and Primary Application in Differential Diagnosis of PRV Vaccination and Infection

With the application of gE gene deleted pseudorabies virus(PRV) vaccine worldwide, a corresponding differential diagnosis based on gE glycoprotein is needed in the project of PRV eradication. In this study, PRV gE gene without signal and transmembrane region was amplified by PCR and cloned into pGEX 6P 1, generated pGEX gE. After transformation of BL21 with pGEX gE, an expressed fusion protein(about 63kD) was identified by SDS PAGE. The recombinant proteins are produced as inclusion bodies. By changing the inductive conditions, the formation of inclusion bodies was inhibited and tended to increase the percentage of soluble recombinant protein. The antigenic reactivity of the recombinant protein was confirmed by Western blotting with polyclonal antibodies against PRV. Using purified recombinant tgE as antigen, an ELISA was developed to detect sera of PRV infected pigs and sera of pigs immunized with gE deleted PRV vaccine. The total of 400 serum samples collected from field were comparatively tested with the tgE ELISA and a commercial competitive ELISA based on monoclonal antibody against gE, the results indicated that the coincidental rate between the two tests is about 94%.