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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 Pichia pastoris expression vector of herpes simplex virus type 2 (HSV-2) glycoprotein D (gD), which lays the foundation for the further development of recombinant antigen diagnostic reagents and research subunit vaccines. Methods: gD2 gene was amplified from HSV-2 genome by PCR, and then two restriction enzyme sites XhoⅠ and XbaⅠ were added to both ends of the gene by PCR. After double digestion, the PCR products were correctly read The box was cloned into pichia pastoris expression vector pPICZαA; transformed into E. coli competent cells TOP10F ’, the antibiotic was screened transformants. Results: The homology of gD2 fragment amplified by PCR with the HSV-2G strain in GeneBank was 98.4% and the amino acid homology was 95.7%. Four expression vectors were constructed during the experiment, which were identified by double-enzyme digestion after agarose electrophoresis. Conclusion: Four gD2 Pichia expression vectors were constructed, two of which contained the CTL epitope sequence of HSV-2gB.