人轮状病毒结构蛋白VP7基因毕赤酵母表达质粒的构建  

CONSTRUCTION OF HUMAN ROTAVIRUS STRUCTURAL PROTEIN VP7 GENE IN PICHIA PASTORIS EXPRESSION VECTOR

作　　者：卢颖[1] 吴学敏[1] 单颖[1] 佟伟[1] 张逸博[1] 李会[1] 

机构地区：[1]辽宁医学院免疫与病原生物学教研室锦州.121001

出　　处：《现代预防医学》2009年第6期1127-1129,共3页Modern Preventive Medicine

摘　　要：[目的]构建人轮状病毒结构蛋白VP7基因重组酵母表达质粒VP7-pPICZαA。[方法]从VP7-pET32a质粒中PCR扩增轮状病毒VP7基因,EcoRⅠ、XbaⅠ双酶切VP7基因产物和酵母表达载体pPICZαA,T4DNA连接酶连接目的基因片段和pPICZαA,转化到大肠杆菌Top10中,Zeocin筛选转化子并进行PCR、酶切和测序鉴定。[结果]阳性克隆菌经酶切和PCR和测序鉴定,结果与预期相符,目的基因正确插入pPICZαA中。[结论]成功构建轮状病毒结构蛋白VP7基因重组酵母表达质粒VP7-pPICZαA,为轮状病毒结构蛋白VP7基因的酵母表达奠定了重要的实验基础。[Objective] To construct human rolavirus structural protein VP7 gene inlo recombinant Pichia pastoris expression vector VP7-pPICZαtA. [ Methods] VP7 gene of rotavirus was amplified from VP7-pET32a by PCtl. VP7 gene product fragments and Piehia pastoris expression vector pPICZc（A were double-digested by EcoR I and Xba I and connected by T4 DNA ligase. Ligation mixtures were transformed into competent Topl0 cells, and positive clones were screened with Zeoein. Transformants were identified by enzyme digesting, PCR. and sequencing analysis. [Results] It was proved that VP7 gene segments were inserted into pPICZαA correctly after identification with PCR enzyme cutting and sequencing analysis. [Conclusion] Eukaryotic expression vector VP7-pP1CZαA was successfully constructed, which provided a solid experimental basis for further studies on the VP7 expression in Pichia pastoris.

关 键 词：轮状病毒 表达载体 VP7基因 毕赤酵母 

分 类 号：R737.9[医药卫生—肿瘤] R373.21[医药卫生—临床医学]