DPEP1基因过表达BT549细胞株的建立和鉴定  

Establishment and identification of DPEP1-overexpressing BT549 cell line

作　　者：郑美玲 魏春莉 刘晓燕 傅俊江 ZHENG Mei-ling;WEI Chun-li;LIU Xiao-yan;FU Jun-jiang(The Research Center for Preclinical Medicine,Key Laboratory of Epigenetics and Oncology of Universities,South-west Medical University,Luzhou 646000,China)

机构地区：[1]西南医科大学医学基础研究中心·四川省高校表观遗传与肿瘤重点实验室,泸州646000

出　　处：《西南医科大学学报》2021年第4期301-306,共6页Journal of Southwest Medical University

基　　金：国家自然科学基金项目(81672887);四川省教育厅项目(18ZB0647);西南医科大学科研项目(00030702)。

摘　　要：目的探讨二肽酶1(dehydropeptidase-1,DPEP1)基因表达载体的构建及DPEP1蛋白在肿瘤细胞中的过表达情况及其影响。方法将DPEP1全长基因PCR扩增,EcoR Ⅴ和Nhe Ⅰ双酶切后克隆入带有同样双酶切pcDNA5/FRT/TO表达载体,并获取pcDNA5/FRT/T0/DPEP1,筛选阳性克隆,Sanger测序确证。扩增pcDNA5/FRT/T0/DPEP1质粒并将其转染入BT549细胞系,获得DPEP1过表达,借助Western blot、CCK-8等实验技术分析其表达及影响作用。结果成功构建了DPEP1的过表达载体,转染肿瘤细胞株,并获得DPEP1过表达蛋白,目的蛋白表达明显升高,其过表达的条带灰度约为阴性对照的20倍。结论研究的成功实施为将来阐明DPEP1基因在肿瘤中的作用及机制奠定了实验基础。Objective To explore the construction of dipeptidase 1(DPEP1)gene expression vector,and to investigate the overexpression of DPEP1 protein in tumor cells and its impact. Methods Polymerase chain reaction was used to amplify the DPEP1 full-length gene,and EcoR Ⅴ and Nhe Ⅰ were used to perform the double-enzyme digestion. Then the digested products were cloned into the same double enzyme-digested pcDNA5/FRT/TO expression vector to obtain pcDNA5/FRT/T0/DPEP1. The positive clones were screened out and verified using Sanger sequencing. After that,the pcDNA5/FRT/T0/DPEP1 plasmid was amplified and transfected into cell line BT549 to obtain DPEP1 overexpression. Western blot,Cell Counting Kit-8,and other experimental techniques were used to analyze its expression and influences. Results The overexpression vector for DPEP1 was successfully constructed and transfected into the tumor cell line,and the DPEP1 overexpressed protein was obtained. The target protein had significantly up-regulated expression,and the overexpressed band had an intensity about 20 times that in the negative control. Conclusion The successful implementation of this study lays an experimental foundation for understanding the role and mechanism of action of DPEP1 gene in tumors in the future.

关 键 词：二肽酶1 载体构建肿瘤细胞系 

分 类 号：R34[医药卫生—基础医学]