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目的构建单纯疱疹病毒I型包膜糖蛋白D成熟肽基因毕赤酵母表达载体,并对序列进行分析,为进行高抗原性的真核表达重组gD蛋白奠定基础。方法 PCR扩增HSV1-gD成熟肽基因,将该段基因克隆于pGEM-T克隆载体,转化鉴定后,与巴斯德毕赤酵母表达载体(pPIC9K)酶切连接,转化大肠杆菌DH5α,筛选测序确定构建了pPIC9K-gD的真核表达载体,对克隆的序列进行分析,预测表达产物的理化特性及抗原性。结果获得了重组的酵母表达载体pPIC9K-gD。测序结果证实为HSVI-gD成熟肽基因,序列分析其高度保守,预测蛋白分子量为40．52 ku,pI为7．67,包含完整成熟肽分值达1．7的多个抗原决定簇。结论成功构建了HSV1-SD成熟肽基因的毕赤酵母表达载体。
Objective To construct the Pichia pastoris expression vector of herpes simplex virus type I glycoprotein D mature gene and analyze the sequence, which will lay the foundation for the highly antigenic eukaryotic expression of recombinant gD protein. Methods The HSV1-gD mature peptide gene was amplified by PCR and cloned into pGEM-T cloning vector. After transformed and identified, it was ligated with the pPIC9K vector and transformed into E. coli DH5α. The eukaryotic expression vector pPIC9K-gD was constructed and the cloned sequence was analyzed to predict the physicochemical properties and antigenicity of the expressed product. As a result, a recombinant yeast expression vector pPIC9K-gD was obtained. The sequence of HSVI-gD mature peptide was confirmed by sequence analysis. The sequence of the gene was highly conserved. The predicted protein molecular weight was 40.52 ku and the pI was 7.67. It contained multiple antigenic determinants with intact mature peptide score of 1.7. Conclusion The Pichia pastoris expression vector of HSV1-SD mature peptide gene was successfully constructed.