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

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。

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

Objective To construct herpes simplex virus type 2（HSV-2） eukaryotic expression plasmid pCDNA3. 0-ICP27 and to evaluate its expression in Vero cells. Methods The 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. RTPCR and Western blot analysis were employed to detect the expression of ICP27. Results The 1741bp DNA fragment was obtained by DNA and cloned into pCDNA3. 0. The recombinant plasmid pCDNA3. 0-ICP27 was sub- jected 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. Conclusion Recombinant plasmid pCDNA 3.0-ICP27 was constructed and expressed successfully in Vero cells.