OC-STAMP基因过表达载体及稳转细胞系的构建  

Construction of the OC-STAMP over-expressed vector and its stable expression cell strain

作　　者：袁慧敏[1] 何茳萍[2] 张光亚[2] 张丹丹[2] 陈凤玲[2] 

机构地区：[1]蚌埠医学院临床学院,安徽蚌埠233030 [2]上海交通大学医学院附属第九人民医院内分泌科,上海201900

出　　处：《遵义医学院学报》2017年第2期143-149,共7页Journal of Zunyi Medical University

基　　金：上海市宝山区科学技术委员会科研项目(NO:14-E-3);上海交通大学医学院博士创新基金资助项目(NO:BXJ201638)

摘　　要：目的构建破骨细胞多次跨膜蛋白(OC-STAMP)基因过表达质粒,体外转染RAW264.7细胞构建稳转细胞系,为进一步研究OC-STAMP的功能提供工具。方法根据NCBI数据库中OC-STAMP基因信息,设计引物,采用PCR扩增其基因片段;运用基因重组方法双酶切目的基因,并将其克隆至含有绿色荧光蛋白(GFP)的p CDH-CMV慢病毒载体,经酶切测序对重组质粒进行鉴定;转染成功构建的质粒至293T细胞中Western blotting验证蛋白过表达情况;采用ps PAX2和p MD2.G两种包装质粒包装p CDH-CMV-oc-stamp重组质粒获得病毒液,病毒感染RAW264.7细胞获得OC-STAMP过表达稳转细胞系,Q-PCR和Western blotting验证蛋白过表达情况。结果酶切和测序鉴定表明成功构建p CDH-CMV-oc-stamp的基因重组质粒;将成功构建的p CDH-CMV-oc-stamp质粒转染至293T,可观察到大量绿色荧光表达;将成功包装获得的病毒液感染RAW264.7细胞后q-PCR及Western blotting结果证实OC-STAMP成功过表达。结论成功构建p CDH-CMV-oc-stamp重组质粒及OC-STAMP过表达的稳转细胞系。Objective To construct the Osteoclast stimulatory transmembrane protein （OC-STAMP） over-expressed plasmid and transfect RAW264.7 cell in vitro to construct stable expression cell strain.Methods Primers were designed according to the gene information on OC-STAMP in National Center for Biotechnology Information,and the OC-STAMP gene was amplified by Polymerase Chain Reaction （PCR）.Using the gene recombination technology,the double enzyme-digested OC-STAMP gene was cloned into the pCDH-CMV-MCS-EF1-Puro vector with GFP.Recombined plasmids were identified with enzyme digestion and sequencing and transfected into 293T cell,.Western blotting was used to detect its expression.Lentiviruses were harvested using pLenti-CMV-OC-STAMP co-transfected with psPAX2 and pMD2.G into HEK293T cells,then RAW264.7 cells were transduced with lentiviruses.Quantitative real-time RT-PCR and western blotting were used to confirm its over-expression.Results The results of enzyme digestion and sequencing revealed that the recombinant plasmids pLenti-CMV-OC-STAMP was successfully constructed.After transfecting 293T cells with the successfully constructed plasmid pLenti-CMV-OC-STAMP,a largeamount of green fluorescence was observed.After transfecting RAW264.7 cells with lentiviruses,the results of quantitative real-time RT-PCR and western blotting showed that the successful over-expression of pLenti-CMV-OC-STAMP.Conclusion The recombinant plasmids pLenti-CMV-OC-STAMP is successfully constructed and the stable expression cell strain is also constructed successfully.

关 键 词：破骨细胞多次跨膜蛋白 质粒构建 病毒转染 RAW264.7细胞 

分 类 号：R589.9[医药卫生—内分泌]