摘要
目的:构建针对人宫颈癌基因2(HCCR2)有效靶点的RNA干扰真核表达载体,鉴定获得干扰质粒稳定转染的人胰腺癌PANC1细胞株.方法:设计并合成多个针对HCCR2基因的RNA干扰序列,并将其插入真核表达慢病毒载体pGCsi-H1/Hygro/NEGative,通过测序和与HCCR过表达载体共转染293T细胞后行Western blot检测,筛选出有效干扰质粒;通过脂质体转染法将该有效干扰质粒pGCsi-HCCR2稳定转染至人胰腺癌细胞株PANC1.抗生素G418筛选获得稳转细胞株;Western blot检测稳转细胞株中HCCR2蛋白表达水平.结果:HCCR2干扰载体和过表达载体共转染293T细胞后Western blot检测结果表明干扰质粒3具有最佳干扰效果,选其作为最终干扰质粒.将该质粒稳定转染至胰腺癌PANC1细胞后,Western blot检测显示,干扰组的PANC1细胞株与空载体组比较,HCCR2蛋白表达水平下调,表明获得HCCR2的RNA干扰质粒稳定转染的PANC1细胞株.结论:成功构建了HCCR2的RNA干扰真核表达载体及其稳定转染的人胰腺癌PANC1细胞株.
AIM：To construct eukaryotic expression vectors expressing small interfering RNAs（siRNAs） targeting the human cervical cancer oncogene 2（HCCR2） gene and transfect them into human pancreatic cancer cell line PANC1 to obtain a cell line stably transfected with the HCCR2 siRNA plasmid.METHODS：Multiple siRNAs targeting the HCCR2 gene were designed,chemically synthesized,and cloned into the eukaryotic expression vector pGCsi-H1/Hygro/NEGative.The resulting recombinant vectors were identif ied by direct sequencing.After the recombinant pGCsi-HCCR plasmids were co-transfected with an HCCR eukaryotic expression vector into 293T cells,the protein expression of HCCR-2 was analyzed by Western blotting to identify the pGCsi-HCCR vector that had the highest gene knockdown efficiency.This recombinant vector was then transfected into PANC1 cells with LipofectamineTM 2000.G418-resistant clones were selected to obtain a stably transfected cell line.The expression of HCCR2 protein in stably transfected cell line was detected by Western blot.RESULTS：The pGCsi-HCCR-3 plasmid had the highest gene knockdown eff iciency and was used to transfect PANC1 cells.Western blotting analysis demonstrated that HCCR2 expression was signif icantly inhibited in PANC-1 cells sta-bly transfected with the pGCsi-HCCR-3 plasmid compared to cells transfected with the empty vector.CONCLUSION：Eukaryotic expression vectors expressing siRNAs targeting the HCCR2 gene were successfully constructed and a PANC-1 cell line stably transfected with the pGCsi-HCCR-3 plasmid was successfully established.
出处
《世界华人消化杂志》
CAS
北大核心
2011年第14期1463-1468,共6页
World Chinese Journal of Digestology
基金
常州市社会发展科技计划基金资助项目
No.CS20102014~~
