构建携带人Sox2和EGFP基因慢病毒表达载体  

Construction of A lentiviral expression vector harboring human Sox2 and EGFP genes

作　　者：赵宗霞[1] 李芬霞[1] 王治荣[2] 常丽花 陈庆[3] ZHAO Zong-xia LI Fen- xia WANG Zhi-rong CHANG Li-hua CHEN Qing(Department of Obstetrics and Gynaecology, The Second Hospitial Affiliated to Xi＇an Medical University, Xi＇an, Shanxi, 710038, CHINA Department of Gynaecology, The Hospitial of Northwestern Polytechnical University, Xi＇ an, Shanxi, 710072, CHINA Department of Obstetrics and Gynaecology, The Second Hospitial Affiliated to Xi＇ an Jiaotong University, Xi＇ an, Shanxi, 710005, CHINA)

机构地区：[1]西安医学院第二附属医院妇产科,陕西西安710038 [2]西北工业大学医院妇科,陕西西安710072 [3]西安交通大学第二附属医院妇产科,陕西西安710005

出　　处：《中国优生与遗传杂志》2016年第12期27-28,44,F0002,共4页Chinese Journal of Birth Health & Heredity

基　　金：陕西省教育厅自然科学基金资助项目(2013JK0797)

摘　　要：目的为了研究宫颈癌的发病机制,构建携带人Sox2基因和EGFP基因的慢病毒表达载体。方法使用限制性内切酶Sac II酶切p Lenti-EF1a-Sox2-IRES-EGFP质粒,回收线性片段并补平Sac II切口,然后Xba I酶切该线性片段,回收3.432kb目的片段便获连接用Sox2-IRES-EGFP片段;Eco RI线性化p FUGW,然后用BKL试剂盒将酶切产物补平,然后继续用Xba I酶切已线性化的p FUGW(Eco R I酶切位点已补平),获得9.122kb和819bp片段,回收9.122kb目的片段以获得连接用载体片段。最后使用DNA连接试剂盒(Ta Ka Ra)中的Solution I将连接用Sox2一IRES-EGFP(3.432kb)和连接用载体片段(9.122kb)连接,连接产物转化,次日挑选单菌落,提取质粒行酶切鉴定。所构建新载体命名为p FUSGW。获p FUSGW载体后,按Invitrogen公司所推荐的标准程序进行慢病毒包装,然后确认慢病毒是否成功生产;使用携带Sox2和EGFP基因的慢病毒感染293FT细胞和宫颈癌细胞株CC-2、CC-3以建立相应的病毒感染体系。结果经酶切鉴定证实成功构建p FUSGW,按标准程序生产的携带Sox2和EGFP基因的慢病毒上清可高效率感染宫颈癌细胞株CC-2、CC-3。结论成功构建了携带人Sox2和EGFP基因的慢病毒表达载体p FUSGW,为宫颈癌发病机制的相关后续研究奠定良好的基础。Objective：In order to study the pathogenesis of cervical cancer,we construct lentiviral expression vectors harboring human Sox2 gene and EGFP gene. Methods：Sox2-IRES-EGFP was obtained from p Lenti-EF1a-Sox2-IRES-EGFP by digestion with Sac II,blunted at Sac II site,and then by digestion with Xba I,and subsequently directionally cloned into the restriction sites Eco RI（blunted at Eco RI site）and Xba I of the lentiviral vector p FUGW after EGFP gene was released from p FUGW by digestion with Eco RI,blunted at Eco RI site,then by digestion with Xba I,designated p FUSGW,as verified by enzyme digestion.According to the standard protocol from Invitrogen,lentiviruses were produced,followed by confirmation using EGFP assay under fluorescent microscope after infecting 293 FT cells,Lentiviruses harboring Sox2 and EGFP genes were employed to infect human cervical carcinoma cells（CC-2,CC-3）. Results：p FUSGW was successfully generated.Lentivirus supernatant harboring Sox2 and EGFP genes efficiently infected CC-2 and CC-3.Conclusion：The lentiviral expression vector harboring human Sox2 gene and EGFP gene was successfully constructed,it lay a good foundation for further research the pathogenesis of cervical cancer.

关 键 词：SOX2 EGFP 慢病毒载体 

分 类 号：R737.33[医药卫生—肿瘤]