【摘要】 构建单纯疱疹病毒2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。采用PCR扩增HSV2-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。结果显示,获得的重组的酵母表达载体pPIC9K-gD,测序结果证实为HSV2-gD成熟肽基因,序列分析其高度保守,预测蛋白分子量40.63kD,等电点pI为7.15,包含完整成熟肽分值达1.7的多个抗原决定簇。成功构建了HSV2-gD成熟肽基因的毕赤酵母表达载体。更多还原

【Abstract】 To construct Pichia pastoris expression vector of herpes simplex virus type 2(HSV-2)envelope glycoprotein D mature peptide gene,then analyze the gene sequences for expressing the recombinant gD.HSV-2 glycoprotein D mature peptide gene containing dominant antigenic determinants was amplified by PCR and cloned into the pGEM-T vector.Through transformation and identification,the cloning vector and the expressing vector were digested by restriction enzymes,then connected and transferred into E.coli DH5α.The recombinant was confirmed by using PCR,enzymes digestion and sequencing.The physical and chemical property and antigenicity of the HSV2-gD were predicted through sequence analysis.The recombinant yeast expression vector pPIC9K gD was constructed.Sequence analysis confirmed that the cloning fragment was gD gene of mature peptide,and it was highly conserved.The molecule weight was 40.63kD and pI was 7.15 through prediction with bio-softwares.The whole mature peptide contained more than six antigenic determinants.Finally,the recombinant Pichia pastoris expression vector containing HSV-2 gD mature peptide gene was successfully constructed.更多还原