全长单纯疱疹病毒Ⅱ型糖蛋白D毕赤酵母表达载体的构建  

Construction pichia pastoris plasmids toexpress complete herpes simplex virus type 2 glycoprotein D

作　　者：刘仲荣[1] 曹春来[1] 杨慧兰[1] 郭勇[2] 王捷[3] 

机构地区：[1]广州军区广州总医院皮肤科,510010 [2]广州市华南理工大学生物科学与工程学院,510640 [3]广州军区广州总医院医学实验科,510010

出　　处：《实用医学杂志》2005年第11期1117-1119,共3页The Journal of Practical Medicine

基　　金：2003年国家自然科学基金资助项目(编号:30371290)

摘　　要：目的:构建单纯疱疹病毒Ⅱ型(HSV-2)糖蛋白D(gD)的毕赤酵母表达载体,为进一步研制重组抗原诊断试剂、研究亚单位疫苗奠定基础。方法:PCR从HSV-2基因组中扩增gD2基因,再用PCR的方法在基因两端加入两个限制性内切酶切位点XhoⅠ和XbaⅠ;PCR产物经过双酶切后按照正确的读码框顺序克隆到毕赤酵母表达载体pPICZαA中;转化到大肠杆菌感受态细胞TOP10F'中,抗生素得到筛选转化子。结果:核酸序列测定PCR扩增的gD2片段与GeneBank中HSV-2G株gDDNA碱基同源性达98.4%,氨基酸同源性达95.7%。在实验过程中构建了四个表达载体,经过双酶切后琼脂糖电泳鉴定正确。结论:构建了gD2毕赤酵母表达载体四个,其中两个含有HSV-2gB的CTL表位序列。Objective To construct the recombinant plasmids for expression of the complete herpes simplex virus type 2 glycoprotein D in pichia pastoris, base for development the subunit vaccine and the diagnose reagent of herpes simplex virus type 2. Methods Cloned the complete HSV- 2 glycoprotein D gene from HSV- 2 genome, add XhoⅠ and Xba Ⅰ enzyme sites in the ends of the sequence; PCR products was inserted into the plasmid pPICZα A after digested by the XhoⅠ and Xba Ⅰ restriction enzymes according to the correct reading code frame; Transformation the recombinant vectors in Top 10' and filtration by the zeocin antibiotic. Results The vectors was confirmed by the enzymes Xba Ⅰ and Xba Ⅰ restriction digestion, and sequence analyzed compared to HSV- 2 G strain gD sequence, DNA homology 98.4% , protein homology 95.7% . Conclusion Obtained the vectors we need to express complete HSV- 2 gD in pichia pastoris. [

关 键 词：全长单纯疱疹病毒Ⅱ型 糖蛋白D 毕赤酵母 表达载体 

分 类 号：R392[医药卫生—免疫学]