摘要
目的构建KLF2过表达BGC823和KLF2敲减MGC803胃癌稳转细胞株。方法克隆人源KLF2基因,连接到Age I/Age I酶切后GV358载体上,构建GV358-KLF2重组质粒,转染293T细胞,包装慢病毒;构建KLF2shRNA,连接到Bam HI和EcoRI双酶切后的PHBLV-U6-ZSGreen-Puro载体上,经鉴定后转染293T细胞。采用RT-PCR及Western印迹法检测稳转细胞株中KLF2的表达。结果利用慢病毒介导,重组质粒G V358-K L F2和K L F2-shRN A转导入B G C823细胞、M G C803细胞并稳定表达。RT-PC R和W estern印迹法结果均显示过表达稳转株KLF2表达量明显升高(P<0.05),敲减稳转株KLF2表达量明显下降(P<0.05)。结论成功构建了K L F2过表达B G C823细胞株和K L F2敲减M G C803细胞株,为后续K L F2功能实验奠定了基础。
ObjectiveTo establish gastric cancer cell line with over expressed KLF2and knockdown KLF2.MethodsThe recombinant plasmid GV358KLF2was constructed by cloning human KLF2gene into AgeI/AgeI enzyme digested GV358vector,then the GV358KLF2plasmid was transfected into293T cells to pack lentivirus.Three pairs of synthesized shRNAs were inserted into BamHI and EcoRI enzyme digested PHBLV U6ZSGreen Puro vector,and packed into lentivirus.The expression of KLF2was detected by RT PCR and Western blot.ResultsRecombinant plasmids GV358KLF2and KLF2shRNA were successfully transfected into BGC823and MGC803respectively,and expressed stably.The results of RT PCR and Western blotting showed that the expression of KLF2was increased in KLF2overexpressed BGC823cells(P<005),and decreased in KLF2knockdown MGC803cells(P<005).ConclusionKLF2overexpression BGC823cell line and KLF2knockdown MGC803cell line have been successfully established,which may be used for the study of KLF2function.
作者
王春梅
陈金联
WANG Chun-mei;CHEN Jin-lian(Dept, of Gastroenterology, East Hospital, Tongji University, Shanghai 200120, China;Dept, of Gastroenterology,South Campus of Sixth Peopled Hospital, Shanghai Jiaotong University, Shanghai 201499, China)
出处
《同济大学学报（医学版）》
CAS
2017年第5期18-23,共6页
Journal of Tongji University(Medical Science)
基金
上海市自然科学基金(16ZR1429100)
