生长激素抑制素基因过表达慢病毒载体的构建及鉴定  

Construction and identification of lentiviral vector of somatostatin gene over-expression

作　　者：赵倩 夏凤艳 刘聪慧 

机构地区：[1]华北理工大学研究生院,河北唐山063000 [2]华北理工大学附属医院,河北唐山063000

出　　处：《华北理工大学学报（医学版）》2017年第5期356-360,共5页Journal of North China University of Science and Technology：Health Sciences Edition

摘　　要：(1)目的探讨构建人生长激素抑制素(Somatostatin,SST)基因过表达慢病毒载体的技术方法,为后续构建SST过表达子宫内膜癌细胞系提供基础。(2)方法通过设计SST基因引物,使用聚合酶链反应(PCR)的方法,扩增SST基因片段;使用EcoR I和BamHI两种限制性内切酶进行双酶切pLVX-mCMV-ZsGreen-PGK-Puro慢病毒载体和目的基因SST片段,将两者酶切后的片段使用T4DNA连接酶进行连接,从而使将SST基因插入pLVX-mCMV-ZsGreen-PGK-Puro慢病毒载体上,构建pLVX-SST-mCMV-ZsGreen-PGK-Puro重组载体;运用双酶切后PCR方法鉴定阳性克隆的pLVXSST-mCMV-ZsGreen-PGK-Puro载体;将重组载体转染293T细胞(人胚肾细胞),进行慢病毒包装并且观察其感染效率及测定病毒滴度。(3)结果通过PCR成功扩增SST基因并连接到pLVX-mCMV-ZsGreen-PGK-Puro慢病毒载体上,通过DNA测序鉴定,并与GenBank上的SST基因标准序列进行对比,证明pLVX-SST-mCMV-ZsGreen-PGK-Puro过表达慢病毒载体构建成功,将其转染293T细胞后可观察到绿色荧光的表达。成功包装SST过表达慢病毒载体然后测其滴度为1.0×108 TU/mL。(4)结论成功构建pLVX-SST-mCMV-ZsGreen-PGK-Puro慢病毒载体,为SST基因的后续研究提供了实验基础。Objective To construct a lentiviral vector over-expressing somatostatin （SST） gene, and to provide a basis for the subsequent construction of SST over-expressing endometrial carcinoma cell line.Methods The primers of SST gene were designed for amplification of SST gene fragments by polymerase chain reaction （PCR）.The pLVX-mCMV-ZsGreen-PGK-Puro lentiviral vector and the SST fragment of the target gene were digested with both EcoR I and BamHI restriction enzymes.The frag- ments cleaved by those were ligated using T4 DNA ligase, so that SST gene was inserted into pLVX- mCMV-ZsGreen-PGK-Puro lentiviral vector to construct pLVX-SST-mCMV-ZsGreen-PGK-Puro re- combinant vector.The candidate clones were identified by PCR after double digestion.The recombinant plasmid was transfected into 293T cells （human embryonic kidney cells）, and was packaged. The transfection efficiency was assessed under the fluorescent microscope and the virus titer was deter- mined. Results SST gene was amplified and successfully bound to the pLVX-mCMV-ZsGreen-PGK- Puro lentivirus vector. The sequences of the recombinant plasmid were confirmed successfully by DNA sequencing, and compared with the standard sequence of SST gene on GenBank. The expression ofGFP could be observed using fluorescent microscope after recombinant lentiviruses were transfected in- to 293T cells. The recombinant plasmid was packaged and its titer was 1.0x10^8TU/mL.Conclusion The pLVX-SST-mCMV-ZsGreen-PGK-Puro lentiviral vector is successfully constructed, which pro- vide the experimental basis for the next study of SST gene.

关 键 词：生长激素抑制素 慢病毒 过表达 载体构建 

分 类 号：R722[医药卫生—儿科]