I型单纯疱疹病毒糖蛋白D成熟肽基因毕赤酵母表达载体的构建及序列分析

Construction and Sequence Analysis of Recombinant Pichia Pastoris Containing Glycoprotein D Mature Peptide Gene of Herpes Simplex Virus Type 1 Stocker Strain

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 type 1 envelope glycoprotein To construct pichia pastoris expression vector of herpes simplex virus D （gD） mature peptide gene, and sequence the gene expressing the recombinant gD. Methods The herpes simplex virus type 1 glycoprotein D mature peptide gene containing dominant antigenic determinants was amplified by PCR and cloned into the pGEM-T vector. Through transformation and conformation, the chining vector and the expressing vector were digested by restriction enzymes, then ligated, and transferred into E. Coli DHSα. The recombinant vector was confirmed by PCR, enzyme digestion and sequencing. The physical and chemical properties as well as antigenicity of the HSVI-gD were predicted through sequence analysis. Results The recombinant yeast expression vector pPIC9K gD was constructed. Sequence analysis confirmed that the cloning fragment was gD gene mature peptide, and it was highly conserved. The molecule weight was 40.52 ku and pI was 7.67 through prediction by using bio-software. It contained over six antigenic determinants of whole mature peptide. Conclusion The recombinant pichia pastoris expression vector of HSVI-gD mature peptide gene was successfully constructed.