BARF1基因真核表达载体PIRES2-EGFP/BARF1的构建及鉴定  

Construction and identification of eukaryotic expression vector PIRES2-EGFP/BARF1 for BARF1 gene

作　　者：李淑英[1] 张科[1] 杜海军[2] 王湛[2] 李旭坤[1] 慈雅丽[1] 王新燕[1] 张秀军[1] 周玲[2] 

机构地区：[1]河北联合大学生物科学系,河北唐山063000 [2]中国疾病预防控制中心病毒病预防控制所肿瘤病毒室传染病预防控制国家重点实验室病毒性肿瘤组,北京100052

出　　处：《辽宁师范大学学报（自然科学版）》2011年第1期98-101,共4页Journal of Liaoning Normal University:Natural Science Edition

基　　金：传染病预防控制国家重点实验室开放课题项目(2008SKLID302)

摘　　要：提取B95-8细胞RNA,进行逆转录聚合酶链反应(Reverse transcription polymerase chain reaction,RT-PCR)扩增BARF1基因,将其克隆到pUM-T载体,转化E.coliDH5α,筛选阳性克隆,提取质粒并用EcoRI和BamHI双酶切,电泳回收BARF1片段.同时用EcoRI和BamHI双酶切真核载体PIRES2-EGFP,电泳后回收载体PIRES2-EGFP并与BARF1片段连接;转化E.coliDH5α,筛选阳性克隆.提取质粒并用EcoRI和BamHI双酶切鉴定插入方向,并进行测序分析.转染胃上皮细胞GES-1后,观察细胞形态与行为变化.得到666 bp的BARF1基因片段.经双酶切鉴定,BARF1基因已正确连接到PIRES2-EGFP真核表达载体;测序结果与B95-8细胞株序列完全一致.BARF1基因转染GES-1细胞后,细胞由梭形圆化,黏着力减弱,重叠生长.结果表明成功地构建了携带EB病毒(Epstein-Barr virus,EBV)编码BARF1基因的真核表达载体.To construct eukaryotic expression vector of BARF1 gene encoded by Epstein-Barr virus（EBV）.RNA of B95-8 cells was extracted,BARF1 gene was amplified by reverse transcription polymerase chain reaction（RT-PCR）.The PCR products of BARF1 were cloned into pUM-T vector and transformed into E.coli DH5α.The positive clones were screened,the plasmids were extracted,and digested with EcoRI and BamHI,BARF1 fragments were recovered by gel electrophoresis.At the same time,eukaryotic vectors of PIRES2-EGFP were digested with EcoRI and BamHI,the vectors of PIRES2-EGFP were recovered,and ligated with the BARF1;PIRES2-EGFP/BARF1 were transformed into E.coli DH5α,and the positive clones were screened.The plasmids were extracted,and insertion direction was identified by digestion with EcoRI and BamHI and Sequenced analysis.The changes of cell morphology and behavior were observed after BARF1 gene was transfected into gastric epithelial GES-1 cells.666 bp products of BARF1 were obtained.BARF1 gene had been correctly connected to the eukaryotic expression vector PIRES2-EGFP through double enzyme digestion;The sequencing results were completely identical with the B95-8 cells.The GES-1 cells changed after BARF1 gene was transfected into gastric epithelial GES-1 cells： spindle cell rounded,adhesion of cells reduced,overlapping cell grew.The eukaryotic expression vector PIRES2-EGFP/BARF1 was constructed successfully.

关 键 词：EB病毒 BARF1基因 真核表达载体 

分 类 号：R373.1[医药卫生—病原生物学]