摘要
目的 构建人类染色体重塑复合物SWI/SNF（Switch/Sucrose NonFermentable）中重要组份ARID1A（A-T rich interaction domain）的突变过表达载体,并检测野生型ARID1A基因及突变型ARID1A基因在肝癌细胞株HepG2中的表达情况.方法 采用overlap PCR技术构建在野生型质粒pcDNA6-ARID1A基础上构建结构域缺失突变体pcDNA6-ARID1A/△ARID以及pcDNA6-ARID1A/△DUF3518;利用脂质体转染技术将野生型质粒以及构建的突变型质粒转染到肝癌细胞HepG2中进行过表达;通过Real-time PCR以及Western blot技术对野生型及突变型ARID1A在肝癌细胞中的表达情况进行鉴定.结果 经双酶切后SDS-PAGE分析以及测序验证,成功构建了真核表达载体pcDNA6-ARID1A的突变型质粒pcDNA6-ARID1A/△ARID以及pcDNA6-ARID1A/△ DUF3518;通过Real-time PCR以及Western blot的方法验证,ARID1A及ARID1 A/△ARID在肝癌细胞株HepG2中成功过表达,而ARID1A/△ DUF3518蛋白可能降解.结论 成功构建ARID1A功能域缺失型突变体,并在肝癌细胞株HepG2中稳定过表达ARID1A及ARID1A/△ARID;ARID1A/△ DUF3518蛋白的缺失暗示DUF3518结构域可能起着稳定蛋白结构的功能.
Objective To construct the mutants of ARID1A gene,which is an important component in human chromosomal remodeling complex Switch/Sucrose Non Fermentable （SWI/SNF）,and identify their overexpression in liver cancer HepG2 cells.Methods Overlap PCR was used to construct domain truncated mutantss pcDNA6-ARID1A/△ARID and pcDNA6-ARID1A/△D UF3518 based on wild type plasmids pcDNA6-ARID1A.Lipofectionmethod was used to transfect the wild type and mutants into HepG2.Real-time PCR and western blotting were used to confirm the overexpression of ARID1A and the mutants.Results SDS-PAGE and sequencingresult confirmed the successful construction of pcDNA6-ARID1A/△ARID and pcDNA6-ARID1A/△DUF3518.Real-time PCR and western blottingresult confirmed the overexpression of both mRNA and protein of wild type ARID1A and ARID1A/△ARID.The mRNA levels indicated that ARID1A/△ DUF3518 were overexpressed,but the protein levels were quite low.Conclusions Functional domain truncated mutants of ARID1A were successfully constructed.Overexpression of wild type ARID1A and ARID1A/△ARID in liver cancer HepG2 cells was successful.Loss of ARID1A/△DUF3518protein suggest that DUF3518 may contribute to the protein structure stability.
作者
肖花
廖启成
汪海燕
刘威龙
吴驰
黄健
刘嘉林
Xiao Hua Liao Qicheng Wang Haiyan Liu Weilong Wu Chi Huang Jian Liu Jialin(Shenzhen Third People＇ s Hospital, Shenzhen 518112, China （ Xiao H, Wang HY, Liu WL, Wu C, Huang J, Liu JL Gannan Medical University, Ganzhou 341000, China （ Liao QC)
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
2017年第4期343-347,共5页
Chinese Journal of Experimental and Clinical Virology
基金
深圳市肝胆疾病研究重点实验室项目（ZDSYS201504301534057）
深圳市科技计划项目（JCYJ20160427183548637）
