稳定长期沉默肾小管上皮细胞Megalin基因表达的载体构建

目的：为探讨沉默Megalin基因对肾小管上皮细胞Megalin蛋白诱导表达的抑制作用,构建长期下调Megalin蛋白表达的慢病毒载体作为研究工具。方法：分别用30,50,80,100nmol/LsiRNA或对照siRNA转染人HK-2细胞,48h后用免疫荧光和RT-PCR鉴定Megalin基因表达筛选有效的序列,用有效或对照组序列的正义链和反义链,中间加loop环,两端加入XhoⅠ和hindⅢ的酶切位点作为目的序列,经过XhoⅠ和HindⅢ双酶切的慢病毒载体质粒PLL3.7作为载体,将目的序列与载体连接,转化到XL-10Gold高感受态大肠杆菌中,XhoⅠ和HindⅢ双酶切初步鉴定,测序进一步鉴定正确的序列。得到长期下调人Megalin基因表达的慢病毒载体,并包装慢病毒感染人HK-2细胞,免疫组化鉴定干扰有效,以此获取Megalin表达下调的HK-2细胞株。结果与结论：成功筛选出有效干扰人Megalin基因的siRNA,通过慢病毒感染成功获得长期稳定下调人Megalin表达的肾小管上皮细胞株。

Lentivirus Vector Construction of Long-term Silence of Megalin Protein Expression in Human Renal Tubular Epithelial Cell

Objective：To explore the down-regulation effect of megalin on human renal tubular epithelial cell （HK2） megalin receptor with the lentivirus vector of long-term silence of megalin protein expression.Methods：Eighty nmol/L or 100 nmol/L SiRNA or control siRNA was used to transfect human renal tubular epithelial cell.After 48 hours,megalin protein was detected by immunofluorescence and nucleic acid was detected by RT-PCR.The lentivirus vector containing gene for down-regulating the expression of megalin protein,two enzyme sites named Xho I and HpaI was constructed.The constructed plasmid was transformed into XL-10Gold and it was extracted.Sequencing was done to identify true clone.Lentivirus was packaged,and then human renal tubular epithelial cells were infected.At last,the HK-2 cell lines down-regulated expression of megalin protein for long term were obtained.Results and Conclusion：Compared with that in the control group,the effective SiRNA down-regulating the expression of human megalin had already been selected,and the long-term downregulation of the expression of the HK-2 cell lines megalin protein had been obtained by infected with lentivirus.