人巨细胞病毒重组基因表达质粒的构建

Construction of expression vector for human cytomegalovirus recombinant gene

目的 用基因工程技术克隆人巨细胞病毒 (Humancytomegalovirus ,HCMV)中抗原性较强的蛋白片段—gp5 2C末端和pp15 0C末端的DNA序列 ,插入pPIC9K质粒 ,构建适于酵母表达系统的重组表达质粒。方法 根据人巨细胞病毒糖蛋白gp5 2和磷酸蛋白pp15 0C末端的cDNA序列 ,设计出 2对引物。利用PCR的方法 ,以病毒培养液上清为模板 ,进行二轮PCR扩增 ,把两个目的基因串联在一起 ,将所得的DNA片段经EcoRⅠ和NotⅠ双酶切后用T4连接酶与pPIC9K质粒进行连接 ,然后导入大肠杆菌DH5α ,筛选出阳性转化子 ,并用PCR和双酶切鉴定阳性重组子。结果 从人巨细胞病毒培养液上清中扩增出目的基因 ,PCR反应和双酶切鉴定结果与预期相符。

Objective To construct a recombinant plasmid for expressing recombinant peptides of human cytomegalovirus (HCMV) in Yeast Pichia Pastoris expression system. Methods Two pairs of primers were designed based on the DNA fragments of c terminal of gp52 and pp150 of HCMV since the product peptides of these 2 fragments showing strong antigenicity. The 2 fragments were obtained in series by over lapping PCR with HCMV culture as template, PCR amplification and digestion with EcoR Ⅰ and Not Ⅰ. The DNA fragment was linked to plasmid pPIC9K with T4 DNA ligase, and the recombinant product was transformed into competent E. coli DH5α. Positive clone was screened and identified with PCR and EcoRⅠ and NotⅠ digestion. Results The target genes were amplified from human cytomegalovirus culture, and proved to be consistent with the objective sequences. Conclusion The recombinant plasmid containing c terminal gp52 and pp150 genes of HCMV is constructed in success.