人TRB3过表达载体的构建及检测

Construction and identification of human TRB3 gene overexpression vector

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q PCR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、96 h,MHCC-97H细胞的TRB3 m RNA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 hepato- cellular 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 up- regulated in MHCC-97H cells after transfected;TRB3 overexpression vector is successfully constructed,which could be used into the post-study.