汉滩病毒84FLi株L片段全长cDNA克隆的构建及鉴定  

Cloning of the Full-length Complementary DNA of L Gene of Hantaan Virus 84FLi Strain

作　　者：张野[1] 李新红[1] 黄长形[1] 王平忠[1] 姜泓[1] 白雪帆[1] 

机构地区：[1]第四军医大学唐都医院全军感染病诊疗中心,陕西西安710038

出　　处：《生物技术通讯》2007年第6期905-908,共4页Letters in Biotechnology

基　　金：国家自然科学基金项目(30200010)

摘　　要：目的:克隆汉滩病毒84FLi株L片段的cDNA。方法:提取病毒总RNA,反转录为cDNA模板,用高保真的Taq酶扩增出含有L片段部分克隆的片段84L1(1-1875)、84L2(1725-3352)、84L3(2921-4961)和84L4(4727-6533),将这4个片段分别与T载体pGEM-T-Easy连接,转化感受态大肠杆菌后获得4个克隆pGEM-L1、pGEM-L2、pGEM-L3和pGEM-L4。利用酶切连接方法获得pGEM-L12和pGEM-L34,2段产物覆盖全长的L片段,并且于2921-3352 nt区域内相互重叠;再利用酶切连接的方法获得了含有L片段全序列的cDNA克隆。结果:获得6个含有L片段部分片段的克隆,进而酶切组装为含有L片段全序列的cDNA克隆。结论:应用分段克隆和逐次拼接法成功构建了84FLi株L片段全序列的cDNA克隆。Objective： To clone the full-length complementary DNA of L gene of Hantaan virus 84FLi strain. Methods： Total viral RNA was extracted and complementary DNA was synthesized by reverse transcription as template for PCR. Four PCR products, 84L1 （1-1875）, 84L2 （1725-3352）, 84L3 （2921-4961）and 84L4 （4727-6533）, containing parts of L gene of 84FLi, respectively, were inserted into pGEM-T-Easy vector. Recombinants pGEM-L1, pGEM-L2, pGEM-L3 and pGEM-L4 were then transformed into competent E.coli DH5a. Recombinants pGEM-L12 and pGEM-L34, which overlapped in 2921-3352 nt region, were obtained by enzyme digestion and ligation, eDNA clone containing the full-length complementary DNA of L gene was obtained by enzyme digestion and ligation. PCR and restriction analysis were performed to identify the positive clone. Results： Six eDNA clones containing parts of L gene of 84FLi were constructed, and eDNA clone of the full-length L gene was obtained. Conclusion： The full-length complementary DNA of L gene of Hantaan virus 84FLi strain was cloned successfully.

关 键 词：汉坦病毒 依赖RNA的RNA聚合酶 基因克隆 

分 类 号：Q78[生物学—分子生物学]