人DNMT3A基因慢病毒载体构建及其乳腺癌细胞MDA-MB-231稳定表达株的建立  

Construction of human DNMT3A lentiviral vector and establishment of stale MDA-MB-231 cell line

作　　者：张慧变 王媛[1] 于林[1] 

机构地区：[1]天津医科大学,天津300070

出　　处：《山东医药》2017年第42期5-8,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81672592;81202102)

摘　　要：目的构建人类DNA甲基转移酶3A(DNMT3A)蛋白过表达慢病毒载体并建立乳腺癌细胞MDA-MB-231稳定表达株。方法从乳腺癌细胞MDA-MB-231中提取总RNA,逆转录出含DNMT3A的c DNA;以DNMT3A的c DNA为模板,根据其CDS序列设计含有EcoRⅠ和Bam HⅠ限制性内切酶位点的引物序列,采用PCR法扩增出带有双酶切位点的目的基因;采用双酶切法将目的片段连接到到慢病毒表达质粒pLVX-IRES-Hyg中,获得重组的pLVX-FLAG-DNMT3A表达载体。重组的pLVX-FLAG-DNMT3A表达载体通过与包装质粒共转染293T细胞,获得携带FLAG-DNMT3A的重组慢病毒。以慢病毒感染MDA-MB-231细胞,48 h后在培养基中加入1μg/μL的嘌呤霉素,筛选出稳定表达DNMT3A蛋白的细胞株,检测稳定株及野生型细胞DNMT3A蛋白及mRNA表达水平。结果构建的重组质粒经菌落PCR、重组质粒双酶切、质粒PCR验证及测序比对鉴定正确。用病毒感染MDA-MB-231细胞,药物筛选后获得的过表达稳定株中,DNMT3A蛋白及mRNA表达水平分别为13.2±0.48、107.10±0.46,均高于野生型及空载对照。结论成功构建了DNMT3A基因慢病毒表达载体pLVX-FLAG-DNMT3A,并在MDA-MB-231细胞中筛选出稳定表达DNMT3A的细胞株,为进一步探讨DNMT3A在乳腺癌中的作用提供了体外细胞系模型。Objective To construct the lentiviral vector of human DNA methyltransferase 3A（DNMT3A） and to establish the stable breast caner MDA-MB-231 cell line.Methods The total RNA was extracted from MDA-MB-231 cells and c DNA containing DNMT3A was inverse transcribed.DNMT3A fragment containing EcoRⅠand Bam HⅠsites was amplified by PCR and was inserted into p LVX-IRES-Hyg lentiviral vector.Then the recombinant expression vector p LVXFLAG-DNMT3A was co-transfected into 293T cell line with packaging plasmids,and the culture supermatant containing the lentiviral particles was collected to infect MDA-MB-231 cells.After 48 h,1 μg/μ puromycin was used to screen the infected cells,so as to obtain a cell line with stable expression.The protein and mRNA expression levels of DNMT3A were detected by Western blotting and PCR.Results Bacteria colonies PCR,double restriction enzyme digestion,plasmid PCR identification,and DNA sequencing demonstrated the lentiviral vector p LVX-FLAG-DNMT3A was constructed.MDA-MB-231 cells were infected by the lentivirus,and the protein and mRNA expression of DNMT3A was significantly up-regulated following screening with puromycin.Conclusions The DNMT3A gene lentiviral vector p LVX-FLAG-DNMT3A is constructed successfully.The cell line stably expressing DNMT3A is screened in MDA-MB-231 cells,which provides an in vitro model for further study of molecular function and mechanism of DNMT3A in human breast cancer.

关 键 词：DNMT3A基因 慢病毒载体 MDA-MB-231细胞 乳腺癌 

分 类 号：Q591.2[生物学—生物化学] Q784