小鼠XBP1基因RNA干扰慢病毒载体的构建及筛选.docVIP

小鼠XBP1基因RNA干扰慢病毒载体的构建及筛选 钟河江，蒋建新王海燕， （第三军医大学大坪医院野战外科研究所第四研究室，全军交通医学研究所，全军交通伤重点实验室，创伤、烧伤与复合伤国家重点实验室，重庆 400042）： 构建基因RNA（RNA interference， RNAi）慢病毒载体靶序列。针对PCR 鉴定与DNA测序证实寡核苷酸链插入正确。成功构建基因RNAi 慢病毒载体。：RNA干扰；XBP1；慢病毒 Construction and of lentiviral vector of RNA interference of mouse XBP1 gene ZHONG He-jiang, JIANG Jian-xin, WANG Hai-yan, YANG Ce, ZHANG Chong, YAN Jun, LIU Qi, HUANG Su-la (State Key Laboratory of Trauma, Burns and Combined Injury, Department 4, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, 400042, China) Abstract: Objective To construct lentiviral vector of RNA interference（RNAi） of X-box binding protein 1 (XBP1) gene, and screen the effective sequence of siRNA targeting XBP1 gene. Methods According to the GenBank information of mouse XBP1 gene, four interfering sequence and a negative sequence were designed. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was synthesized, and then were formed into double-stranded DNA by annealing. The obtained products were cloned into the pGCL-GFP vector digested by the restriction enzyme of AgeⅠand EcoRⅠ to construct lentiviral vector which expressed short hairpin RNA (shRNA)，and it was identified by PCR and DNA sequencing. Tsuccessful construct vector was packaged by packaging plasmid mix. The titer of virus was tested. NIH3T3 cells were infected by lentivirus, and the interfering efficiency was determined by Real-time PCR. Results PCR identification and DNA sequencing demonstrated that insertion of oligonucleotide was correct. The titer of virus tested according to the expression level of GFP was 1?08TU/ml. Real-time PCR anaylsis confirmed XBP1-siRNA-3 had the highest interfering efficiency, and the interfering efficiency of lentivirus was more 95%. Conclusion The lentivirus RNAi vector of mouse XBP1 was constructed and screened successfully, which lays a foundation for further study of the role of XBP1 in the regulation of