PRL-3基因RNA干扰慢病毒载体的构建及稳定感染HepG2单克隆细胞株的建立  

Construction of a lentiviral vector for RNA interference of PRL-3 gene and establishment of monoclone of stably infected HepG2 cell lines

作　　者：黄德好[1] 苏树英[1] 陈规划[2] 

机构地区：[1]佛山市第一人民医院胆道外科,广东佛山528000 [2]中山大学附属第三医院肝移植科,广州510630

出　　处：《中国临床药理学杂志》2011年第11期862-866,共5页The Chinese Journal of Clinical Pharmacology

基　　金：国家自然科学基金资助项目(30571769);国家973重点基础研究发展规划基金资助项目(2009CB522404)

摘　　要：目的构建PRL-3基因RNA干扰慢病毒载体并建立被稳定感染的HepG2单克隆细胞株。方法设计PRL-3基因特异性RNAi靶序列,合成靶序列的DNA oligo,退火形成双链DNA,与含绿色荧光蛋白(GFP)编码基因的pGCL-GFP线性化载体连接,产生重组慢病毒质粒LV-PRL3-siRNA,转化感受态细胞,PCR筛选阳性克隆,并测序鉴定。用Lipofectamine 2000将LV-PRL3-siRNA、pHelper 1.0和pHelper 2.0这3种质粒共转染293T细胞,包装产生慢病毒,经孔稀释法,测定病毒滴度。重组慢病毒感染肝癌HepG2细胞,进行RealTime-PCR和Western-Blot验证PRL-3基因的沉默效果,筛选出最佳干扰序列组及阴性对照组的单克隆细胞株。结果成功构建PRL-3基因RNA干扰慢病毒载体并获得相应的慢病毒,浓缩病毒悬液的滴度为6E+8 Tu.mL-1。重组慢病毒对HepG2细胞PRL-3基因表达有明显沉默作用,其中第一干扰序列效果最明显。筛选出最佳干扰序列组及阴性对照组的单克隆肝癌细胞株。结论成功构建人PRL-3基因特异性的慢病毒干扰载体,并筛选出最佳干扰序列组及阴性对照组的单克隆肝癌细胞株。Objective To construct a lentiviral vector for PRL-3 gene RNA interference（RNAi） and establish monoclone of stably infected HepG2 cell lines.Methods The effective sequence of siRNA targeting PRL-3 gene was confirmed.Both sense and anti-sense DNA-oligo of the targeting sequence were designed,synthesized and cloned into the pGCL-GFP vector,which contained coding gene of green fluorescent protein（GFP）.The resulting lentiviral vector containing PRL-3 siRNA was called LV-PRL3-siRNA,and it was confirmed by PCR and DNA sequencing.293T cells were co-transfected with LV-PRL3-siRNA,pHelper 1.0 and pHelper 2.0 and the lentivirus was produced.The titer of virus was tested according to the expression level of GFP.HepG2 cell lines were infected with recombinant lentivirus and the silencing effect of PRL-3 gene was assessed by Real-Time PCR and Western-Blot.Monoclone cell lines of infected HepG2 which exhibited the most significant PRL-3 gene knock-down were screened.Results Recombinant lentiviral vector expressing siRNAs targeting PRL-3 gene was successfully established and confirmed by DNA sequencing.The recombinant lentivirus were harvested from 293T cells with a viral titer of 6E＋8 Tu·mL-1. Results of Real-Time PCR and Western-Blot showed that expression level of PRL-3 gene was down regulated in infected HepG2 cell lines.Monoclone cell lines of infected HepG2 with the most significant PRL-3 gene knock-down was successfully screened.Conclusion Lentiviral vector for PRL-3 gene RNA interference was successfully constructed and monoclone cell lines of infected HepG2 with the most significant PRL-3 gene knock-down was successfully screened,which lays a foundation for further investigating functions of PRL-3 in hepatocellular carcinoma.

关 键 词：RNA干扰 慢病毒 PRL-3基因 肝细胞癌 

分 类 号：R735.7[医药卫生—肿瘤] R965.2[医药卫生—临床医学]