人TRB3过表达载体的构建及检测-东南大学学报医学版

人TRB3过表达载体的构建及检测

单位：1. 南京医科大学上海一院临床医学院消化科, 上海 200080;
2. 上海交通大学附属第一人民医院消化科, 上海 200080;
3. 上海交通大学附属第九人民医院消化科, 上海 200030

分类号：R734.7

出版年·卷·期（页码）：2017·36·第一期（17-20）

摘要：

目的：构建TRB3真核表达重组质粒，并用肝癌细胞MHCC-97H检测其表达。方法：从肝癌组织中提取并扩增TRB3基因的编码区，将其克隆到真核表达载体pcdh-CMV-MCS-GFP中，酶切并检定，将克隆成功的质粒转染至MHCC-97H细胞中，用qPCR和Western Blot检测TRB3的表达水平。结果与结论：成功扩增了TRB3的编码区，并克隆至真核表达载体中；转染后72、96h，MHCC-97H细胞的TRB3 mRNA和蛋白表达显著增高；成功构建的TRB3过表达载体可用于后续实验。

Objective:To construct human TRB3 gene overexpression vector and identify its expression in hepatocellular carcinoma cells MHCC-97H.Methods:TRB3 gene was extracted from liver cancer tissue of human and amplified it by reverse transcription.Then it was cloned into pcdh-CMV-MCS-GFP vector for constructing eukaryotic expression vector.The successful cloned vector was transfected into hepatocellular carcinoma cells MHCC-97H,and TRB3 expression was determined by qPCR and Western blot.Results and Conclusion:The coding region of human TRB3 gene is successfully amplified,and cloned into the vector;the expression of TRB3 mRNA and protein are upregulated in MHCC-97H cells after transfected;TRB3 overexpression vector is successfully constructed,which could be used into the post-study.

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