HSP70基因的克隆与表达.pdfVIP

21 3 2005 5 IMMUNOLOGICAL JOURNAL Vol. 21 No. 3 May 2005 201 [ ]2005) HSP70 a, c a a c b 金欢胜 , 吴雄飞 , 金锡御 , 杨青浩, 倪 兵 ( 第三军医大学a. 西南医院肾科; b. 基础部全军免疫学 研 所, 重庆400038; c. 新桥医院泌尿外科, 重庆400037) [ ] HSP70 HSP70 HepG2, RTPCR HSP70cDNA, T , HSP70 pAAVMCS NIH3T3 , Wester blot HSP70pAAVMCS RTPCR HSP70 HSP70 NIH3T3 , ECLWester blottimg HSP70pAAV MCS, HSP70 [ ] HSP70; ; ; [ ] R692. 5 [ ] A Cloning and expression of HSP70 gene a, c a a c b JIN Hua she g , WU Xio gfei , JIN Xiyu , YANG Qi ghao , NI Bi g ( a. Department of Kidney , Southwestern Hospital, b. Institute fo Immunology , PLA , c. Department of Urinary Surgery , XiqiaoHospital, hird Military Medical University , Chongqing 400038, China) [Abstract] Objective To co struct HSP70 eukaryotic expressi g plasmids for studyi g its role a d mecha ism i ischemia preco di tio i g ( IP) of kid ey. Methods Fullle gth HSP70 ge e was amplified by reverse tra scriptio polymerase chai reactio ( RTPCR) method from cell li e HepG2 of liver ca cer, a d the clo ed i to eukaryotic expressi g vector pAAVMCS by usi g the TA vector as i termediary. Followi g the recombi a t plasmids tra sformed i to NIH3T3 cells, the expressio of HSP70 protei i the host cells was verified by usi g ECL Wester blotti g. Results Fullle gth HSP70 ge e was amplified by RTPCR method correctly. The correct clo i g of HSP70 ge e i pAAV MCS was co firmed by restrictive e zyme digestio a d seque ci g. The results of ECL Wester blotti g i dicated that the target ge e could ex press i the mammalia cell li e NIH3T3. Conclusion Recombi a t plasmid HSP70pAAVMCS has bee clo ed successfully, which will provide the base for i vestigati g the role a d the mecha ism of HSP70 i the ischemia preco ditio of kid ey. [Ke words] HSP70; Ischemia preco ditio ; Eukaryotic