EB病毒融合基因重组腺病毒表达载体的构建  

Construction of Ebv Selfusien Gene Z2A Recombinant Adenovirus Vector

作　　者：朱伟[1] 山长武[1] 陈廷[1] 罗兵[2] 

机构地区：[1]济宁医学院微生物学教研室 [2]青岛大学医学院微生物学教研室

出　　处：《济宁医学院学报》2007年第1期29-32,共4页Journal of Jining Medical University

摘　　要：目的构建EB病毒(EBV)潜伏膜蛋白2A(LMP2A)编码基因和即刻早期基因(BZLF1)融合基因的重组腺病毒表达载体。方法逆转录-聚合酶链反应分别获得LMP2A和BZLF1编码序列的cD-NA,采用剪接式重叠延伸(spliced overlap exetension,SOE)技术将两段基因通过多肽接头(Gly4Ser)3的DNA序列进行连接,构建融合基因Z2A。将融合基因Z2A定向亚克隆到pAdTrack-CMV质粒上,在原核细胞E.coli BJ5183中完成穿梭质粒与骨架质粒pAdEasy-1的同源重组,构建融合基因Z2A真核表达载体pAd-Z2A。经抗生素培养板筛选重组体,然后转染293细胞,获得复制缺陷型重组腺病毒vAd-Z2A。结果重组腺病毒载体经限制性核酸内切酶酶切,电泳后可观察到长31kb和4.5kb两条DNA条带,测序鉴定结果表明序列正确;从感染重组腺病毒vAd-Z2A的293细胞中检测到融合基因Z2A的表达。结论本研究成功地构建了EBV LMP2A和BZLF1融合基因Z2A重组腺病毒表达载体,为进一步研究Z2A的功能提供了实验基础。Objective To construct Z2A recombinant adenovirus vector and study the effect of Z2A expression on EBV -associated tumor. Methods Both LMP2A and BZLF1 cDNA were cloned from B95 - 8 cell line by RT- PCR, and the fusion ! gene（LMP2A - linker - BZLF1） was constructed by using a polypeptide linker （Gly4Ser）3. Z2A vector pad - Z2A was constructed by inserting the Z2A fusion gene into pad- easyl eukaryot- ie expressing plasmid. 293 cells were transfected with pad - Z2A. Results The electrophoresis of EcoRV and Bgl Ⅱ digested products showed two DNA fragments which was 31 kb and 4.5kb, respectively,The result of DNA sequencing demonstrated that Z2A has inserted in the expected site and the insertion sequence was exactly correct. The results showed that Z2A positive recombinant adenovirus could express Z2A. Conclusion Z2A recombinant adenovirus vector has been constructed and can be used for further research.

关 键 词：LMP2A BZLF1 融合基因 腺病毒 表达载体 

分 类 号：R394.8[医药卫生—医学遗传学]