HSV-1 UL15 exon-Ⅱ的原核表达及抗体制备

Prokaryotic expression and antibody preparation of HSV-1 UL15 exon-Ⅱ

目的构建pQE-HSV-1 UL15 exonⅡ原核表达载体,在大肠埃希菌中高效表达并免疫家兔制备抗体。方法以HSV-1感染后的Vero细胞总RNA为模板,通过RT-PCR扩增HSV-1 UL15 exon-Ⅱ基因,克隆入原核表达载体pQE-31;重组质粒经酶切、测序鉴定无误后转化大肠埃希菌JM109,IPTG诱导表达;SDS-PAGE检测蛋白表达情况;表达蛋白用Ni-NTA亲和层析纯化及透析复性处理后免疫家兔制备特异性抗体,抗体的免疫原性用Western blotting进行检测。结果通过RT-PCR扩增获得了HSV-1 UL15 exon-Ⅱ全长基因;构建的pQE-HSV-1 UL15 exonⅡ重组质粒经酶切及测序鉴定无误;阳性转化菌株经IPTG诱导后SDS-PAGE电泳显示表达蛋白的相对分子质量约为43 000,与理论值一致,蛋白表达率约为35%～40%;表达蛋白经纯化及透析复性后免疫家兔获得的抗血清经Western blotting鉴定具有较好的特异性和敏感性。结论成功构建了pQE-HSV-1 UL15exonⅡ原核表达载体,诱导蛋白表达并进一步用表达的蛋白制备了特异性抗体,为HSV-1末端酶全长基因表达情况的鉴定及最终构建抗病毒组装药物的分子筛选模型奠定了前期实验基础。

Objective To construct pQE-HSV-1 UL15 exon Ⅱ recombinant plasmid and express UL15 exon Ⅱ in E coli,and to prepare specific antibody by immunizing rabbit with the obtained prokaryotic protein.Methods Using the total RNA of HSV-1-infected Vero cell as template,full length HSV-1 UL15 exon-Ⅱ gene was amplified by RT-PCR and then was cloned into pQE-31 vector.The correct recombinant plasmid pQE-HSV-1 UL15 exon-Ⅱ was transformed into E coli JM109 and induced with IPTG for protein expression.The expression protein...