【摘要】 为有效表达猪流行性腹泻病毒(PEDV)的ORF3蛋白并检测表达产物的生物活性,开展了ORF3蛋白相关的生物信息分析(包括遗传进化分析、跨膜区、亲水性、抗原表位、二级结构等),以预测结果为依据,选取抗原区域进行了截短原核表达。根据CV777株的ORF3基因序列,设计了1对特异性引物,以RTPCR扩增了大小为342 bp的ORF3截短基因片段,将扩增的ORF3基因插入p ET-22b(+)载体中构建了原核表达质粒p ET-22 b(+)-ORF3。确定了最佳表达条件为诱导温度25℃、诱导时间4 h、IPTG浓度1.2mmol/L,表达的蛋白主要以包涵体形式存在,大小约18 ku。Western-blot和ELISA试验证明,该蛋白与PEDV阳性血清有良好的反应性。更多还原

【Abstract】 In order to efficiently express ORF3 protein of porcine epidemic diarrhea virus(PEDV)and detect the biological activity of the expressed protein,we first analyzed the bioinformatics of ORF3 protein,including phylogenetic analysis,transmembrane domain,hydropathicity,epitope and sec-ondary structure and so on.Based on the analytic result,a truncated nucleotide sequence of ORF3 gene was chosen to construct a prokaryotic expression system.According to the ORF3 gene of CV777 strain,a truncated ORF3 nucleotide fragment 342 bp has been amplified by RT-PCR.The amplified fragment was cloned into the prokaryotic expression vector p ET-22b(+).The result showed that 25 ℃ of induction temperature,1.2 mmol/L IPTG and 4 h of induction time were the best conditions for the protein production.The ORF3 protein,about 18 ku,was mainly existed in the form of inclusion body.Western-blotting and ELISA assay had proved it had a good reactivity with PEDV positive serum.We hope that it can provide the material for the further function study of ORF3 protein.更多还原