HSV2多功能蛋白ICP27真核表达载体构建及其在Vero细胞中的表达

Construction of HSV2 eukaryotic expression plasmid pCDNA3.0ICP27 and expression in Vero cells

摘要:

目的构建单纯疱疹病毒2型（HSV2） 多功能蛋白感染细胞多肽27（ICP27）真核表达载体pCDNA3.0ICP27，并检测其在非洲绿猴肾细胞（Vero）中的表达。方法提取病毒株HSV2333 DNA，用高保真DNA聚合酶对多功能蛋白ICP27基因进行高保真扩增，用双酶切连接至真核表达载体pCDNA3.0中。pCDNA3.0ICP27经双酶切、测序验证，并通过脂质体介导质粒瞬时转染Vero 细胞，经RTPCR和Western blot检测ICP27蛋白的表达。结果pCDNA3.0ICP27经双酶切可切出目的片段，测序结果经比对，与基因库中的序列完全一致。转染后经RTPCR 和Western blot检测，证实转染重组质粒组有ICP27蛋白表达，而转染空质粒组及未转染组没有检测到ICP27的表达。结论成功构建了HSV2 多功能蛋白ICP27真核表达载体pCDNA3.0ICP27，并能在Vero 细胞中表达。

Abstract:

ObjectiveTo construct herpes simplex virus type 2（HSV2） eukaryotic expression plasmid pCDNA3.0ICP27 and to evaluate its expression in Vero cells．MethodsThe target sequence of ICP27 gene was obtained and highfidelity amplified from HSV2 DNA. The ICP27 gene was cloned into a eukaryote plasmid pCDNA3.0 after restrictive endonucleases digestion．The pCDNA3.0ICP27 was checked and verified by double digestion and DNA sequence analysis. Vero cells were transiently transfected with pCDNA3.0ICP27 by lipofectamine 2 000 in vitro. RTPCR and Western blot analysis were employed to detect the expression of ICP27. ResultsThe 1741bp DNA fragment was obtained by DNA and cloned into pCDNA3.0. The recombinant plasmid pCDNA3.0ICP27 was subjected to sequence analysis which indicated all nucleotides were identical to the HSV2 ICP27 sequence provided by Genbank. Being transfected by lipofectamine 2 000, the expression of ICP27 in Vero cells was detected.ConclusionRecombinant plasmid pCDNA3.0ICP27 was constructed and expressed successfully in Vero cells．