hTERT启动表达VacA基因的重组杆状病毒载体的构建  

Construction of recombinant baculovirus bectors carrying VacA gene regulated by hTERT promoter

作　　者：罗彩凤[1] 涂晶晶[1] 张嘉文[1] 邵世和[1] 朱虹[1] LUO Cai-feng TU Jing-jing ZHANG Jia-wen SHAO Shi-he ZHU Hong(Medical College of Jiangsu University, Zhenjiang 212013, China)

机构地区：[1]江苏大学医学院,江苏镇江212013

出　　处：《中国地方病防治》2016年第9期966-969,共4页Chinese Journal of Control of Endemic Diseases

基　　金：江苏省自然科学基金项目(KB20161345);江苏省2014年度研究生课题(JGLX14_088)

摘　　要：目的构建人端粒酶逆转录酶启动子(hTERT)调控的、携带幽门螺杆菌细胞空泡毒素A(Vacuolatingcytotoxin A,VacA)基因的选择性表达重组杆状病毒。方法应用酶切、连接方法将hTERT连接于杆状病毒穿梭质粒pFastBac^(TM)DUAL载体P10启动子序列之后,和SV40分别调控目的基因VacA的表达和终止;将重组穿梭质粒电转入DH10Bac^(TM)E.coli感受态细胞,经转座重组获得重组杆粒Bacmid-VacA。利用脂质体Cellfectin~将重组杆粒转染Sf9昆虫细胞,包装重组杆状病毒。结果应用PCR验证、双酶切分析、序列测定等方法验证重组质粒基因片段连接正确无突变,通过转座重组获得携带有VacA基因片段的重组杆粒。结论携带VacA基因的选择性复制重组杆粒成功构建。Objective Constructing the baculovirus of selective replication carrying Vacuolatingcytotoxin A gene controlled by the human telomerase promoter（hTERT）.Methods Applied the method of cleavage and enzyme linked to connect the h TERT gene after P10 promoter of p Fast BacTMDUAL shuttle vector in order to control VacA protein expression.HTERT promoter and SV40 respectively controlled VacA gene expression and termination.Recombinant baculovirus shuttle vectors were transfected into DH10 BacTME.coli competent cells by electroporation.Recombinant Bacmid- VacA were obtained by transpositional recombination.Bacmid- VacA were transfected into Sf9 cells by Cellfectin~ in order to obtain selective replication baculovirus.Results PCR validation,restriction enzyme double- digest analysis,DNA sequencing were applied to validate the recombinant plasmid,we obtained recombinant bacmid carrying VacA.Conclusion Selective replication bacmid carrying VacA gene was successfully constructed.

关 键 词：杆状病毒 空泡毒素A 人端粒酶逆转录酶启动子 

分 类 号：R532.21[医药卫生—内科学]