EBV-LMP1 cDNA的克隆测序与原核表达质粒构建  

Cloning and Sequence Analysis of EBV-LMP1 cDNA and the Constuction of Prokaryotic Expression plasmid

作　　者：林勇平[1,2] 吴淑华[3] 杨英[1] 汤郡[1] 马桂璋[1] 

机构地区：[1]广州医学院微生物与免疫教研室 [2]广州医学院医学检验系510182 [3]病毒基因工程国家重点实验室

出　　处：《热带医学杂志》2002年第4期337-340,共4页Journal of Tropical Medicine

基　　金：国家自然科学基金 (No .39970 2 92 );卫生部科学研究基金 (No .98 2 346 );广东省自然科学基金研究项目资助 (No.990 183)

摘　　要：　目的　克隆EBV LMP1cDNA ,构建EBV LMP1基因的原核表达重组质粒。方法　通过RT PCR技术从B95 8细胞中扩增EBV LMP1cDNA ,克隆至pGEM Teasy载体上并测序 ;利用引物上的BamHⅠ与HindⅢ酶切位点将LMP1基因插入载体pET 32a,转化JM10 9菌。提取质粒 ,限制性内切酶消化 ,琼脂糖凝胶电泳鉴定。结果　扩增的LMP1cDNA长度为 115 8bp ,测序结果与已知序列吻合 ,重组原核表达质粒经酶切鉴定表明获得正确重组子。结论　成功克隆了EBV LMP1cDNA ,并构建了pET 32a LMP1原核表达载体 。Objective To clone EBV LMP1 cDNA and construct a prokaryotic expression plasmid containing EBV LMP1 gene.Methods Amplified EBV LMP1 cDNA from B95 8 cell by RT PCR technique was cloned into pGEM T easy vector and sequenced.LMP1 gene was then subcloned into pET 32a vector.The constructed recombinant plasmid was transferred into E.coli JM109.The tramformatans were screened and indentified by restriction analysis and eletrophrasis. Results The size of amplified LMP1 cDNA was 1158bp,The DNA sequence of the cloned gene was the same as the published sequence.The recombinant prokaryotic expression plasmid pET 32a LMP1 was isolated and confirmed by enzyme cleavage analysis and elotrophrasis. Conclusions The LMP1 gene was sucessfully amplfied and cloned into pET 32a prokaryotic expression vector.The expression of LMP1 in E.coli provided the basic material for studying the oncogenic role of LMP1 and vaccination against EBV.

关 键 词：LMP1 CDNA 克隆 测序 原核表达 EB病毒 

分 类 号：R373.11[医药卫生—病原生物学]