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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 exonⅡ prokaryotic expression vector and express it in Escherichia coli and immunize rabbits to prepare antibodies. Methods HSV-1 UL15 exon-Ⅱ gene was amplified by RT-PCR from the total RNA of Vero cells infected with HSV-1 and cloned into prokaryotic expression vector pQE-31. The recombinant plasmids were confirmed by restriction enzyme digestion and sequencing Escherichia coli JM109, induced by IPTG; SDS-PAGE detection of protein expression; expression protein using Ni-NTA affinity chromatography purification and dialysis refolding immune rabbit preparation of specific antibodies, antibody immunogenicity with Western blotting detection. Results The full-length HSV-1 UL15 exon-Ⅱ gene was amplified by RT-PCR. The constructed recombinant plasmid pQE-HSV-1 UL15 exon Ⅱ was identified by enzyme digestion and sequencing. The positive transformed strain was induced by IPTG and then analyzed by SDS-PAGE The relative molecular mass of the expressed protein was about 43 000, consistent with the theoretical value, the protein expression rate was about 35% -40%. The antiserum obtained from the immunized rabbit after purification and dialysis refolding was identified by Western blotting Good specificity and sensitivity. Conclusion The recombinant prokaryotic expression vector pQE-HSV-1 UL15exonⅡ was successfully constructed, and the expression of the protein was induced. The specific antibody was also prepared by using the expressed protein. The expression of full-length HSV-1 gene was identified and the final construction of antiviral drug Molecular screening model laid the foundation for the previous experiment.