中文摘要：

目的构建大鼠IP3R1miRNA慢病毒表达载体,并利用PC12细胞株观察其对IP3R1基因的沉默效应。方法根据已公布的IP3R1基因序列（GenBankNo.NM_001007235）,设计并合成4对miRNA的OligoDNA（miRNA1、miRNA2、miRNA3和miRNA4）,退火形成双链DNA后,与载体pcDNATM6.2-GW/EmGFP-miR连接,构建pcDNATM6.2-GW/EmGFP-miR-IP3R1表达载体;用Gateway技术将该表达载体先后与中间载体pDONRTM221、慢病毒载体pLenti6/V5-DEST连接,构建慢病毒表达载体pLenti6/V5-DEST-IP3R1,包装产生慢病毒,收集上清,感染PC12细胞株,用Real-timePCR和Western blotting技术检测慢病毒表达载体对PC12细胞内IP3R1表达的沉默效应。结果测序结果表明,4对pcDNATM6.2-GW/EmGFP-miR-IP3R1表达载体序列与参考序列一致,将重组获得的慢病毒表达载体pLenti6/V5-DEST-IP3R1转染至PC12细胞株,48h后IP3R1的mRNA和蛋白表达下调,以miR-NA2和miRNA3的沉默效应最佳。结论成功构建了大鼠IP3R1慢病毒表达载体pLenti6/V5-DEST-IP3R1,其可使大鼠PC12细胞株IP3R1表达下调,为利用RNA干扰技术进一步研究细胞内钙释放通道的功能奠定了基础。

英文摘要：

Objective To construct lentiviral expression vector of rat IP3R1 gene,and identify its silencing effect by using PC12 cell lines. Methods Oligo DNA sequences of 4 pairs of miRNA,named as miRNA1,miRNA2,miRNA3 and miRNA4,were designed according to IP3R1 gene sequence （GenBank：NM_001007235）. The single strand of oligo DNA was annealed to form double strand DNA,and then connected with the empty plasmid pcDNA TM 6.2-GW/EmGFP-miR. By using gateway technology,the expression vector pcDNA TM 6.2-GW/EmGFP-miR-IP3R1 was linked into lentiviral destination vector pLenti6/V5-DEST to form the lentiviral expression vector pLenti6/V5-DEST-IP3R1,then it was transformed into infectious lentiviral particles and to infect PC12 cell lines. Silencing effect of gene IP3R1 was detected by Real-time PCR and Western blot. Results The sequence of expression vector pcDNA TM 6.2-GW/EmGFP-miR-IP3R1 was proved correct using sequencing method. After the transfection of letivirus vector pLenti6/V5-DEST-IP3R1 into PC12 cell lines,the IP3R1 mRNA and protein level were down-regulated 48h later,of which miRNA2 and miRNA3 sequence showed the best silencing effect,and the expression of IP3R1 in the blank control and negative control showed no significant changes. Conclusions Lentiviral expression vector pLenti6/V5-DEST-IP3R1 was constructed successfully. pLenti6/V5-DEST-IP3R1 may render the IP3R1 expression in PC12 cell lines down-regulated,and it provides a foundation for studying the function of calcium release channel IP3R1.