【摘要】 以质粒pBV222/gE为模板,经PCR扩增出750 bp的伪狂犬病病毒(PRV)gE基因片段,克隆入pET-41b质粒中并转化大肠埃希菌TG1。经PCR、酶切鉴定,筛选出阳性重组质粒pET41b/gE后,转化表达菌Balgold,IPTG诱导,目的蛋白经镍柱亲和层析纯化,SDS-PAGE凝胶电泳和Western blot分析检测。以纯化后的目的蛋白作包被原,建立间接gE-ELISA检测方法。结果表明,目的基因在30℃,0.025mmol/LIPTG诱导条件下,可在Balgold中高效表达,目的蛋白大部分以可溶性形式存在,并能被PRV阳性血清所识别,方阵滴定法确定了间接ELISA方法的抗血清最佳稀释度(800倍)和包被抗原的最佳工作浓度(1 mg/L)。更多还原

【Abstract】 A fragment of 750 bp gE gene was amplified by PCR with plasmid pBV222/gE,and cloned into the pET41b vector,and transformed into TG1.Positive clones named as pET41b/gE with interest gene were identified by restriction enzyme digestion analysis,PCR and DNA sequencing.Then the recombinant plasmid was transformed into Balgold.The interest gene was induced to express in E.coli with IPTG induction.The target protein was purified by Ni NTA affinity beads and analysed by SDS-PAGE and Western blot.And then the indirect ELISA was established with the purified recombinant protein.The results demonstrated that gE protein of PRV could be expressed efficiently in Balgold,and most of the protein expressed existed in soluble form,which could be recognized by PRV positive serum,and in the indirect ELISA assay,the ideal serum concentration was 800 dilution,and the antigen coating concentration was 1 mg/L.更多还原