组蛋白去乙酰化酶3真核表达载体的构建和鉴定

Construction and identification of eukaryotic expression plasmid pEGFP-Hdac3

目的利用pEGFP-N1真核表达载体构建组蛋白去乙酰化酶3真核表达载体pEGFP-Hdac3,并鉴定其在大鼠肝癌细胞系CBRH-7919中的表达。方法利用大鼠肝脏cDNA为模板,聚合酶链反应(PCR)获取目的基因Hdac3;用HindⅢ、BamH I双酶切真核表达载体pEGFP-N1和目的基因Hdac3,用T4 DNA连接酶连接纯化后的酶切产物;连接产物转化DH5α大肠杆菌感受态细胞并挑选阳性克隆,并通过PCR、双酶切、DNA测序鉴定构建的重组质粒。利用测序正确的重组体转染CBRH-7919细胞,检测Hdac3和PPAR-γ的mRNA表达。结果利用基因克隆技术获得组蛋白去乙酰化酶3的真核表达载体pEGFP-Hdac3重组质粒。PCR,双酶切,DNA测序证实目的基因H-dac3已成功插入pEGFP载体中。用重组质粒转染大鼠肝癌细胞系CBRH-7919后Hdac3 mRNA表达显著升高、而PPAR-γmRNA表达明显降低。结论本研究成功构建了蛋白去乙酰化酶3的真核表达载体pEGFP-Hdac3。

Objective To construct a eukaryotic expression plasmid containing rat histone deacetylase 3 （Hdac3） and to study its expression in CBRH-7919 cell line. Methods We use HindⅢ, BamHⅠ to digest pEGFP- N1 and acquired target gene Hdac3 , which was amplified by PCR with rat liver cDNA. After purification, the two were ligated together by T4 DNA ligase, and then the ligation production was used to transform DH5a competent cell. Positive clones, picked up from the agarose plate with kanamycin, were identified through PCR, digestion of HindⅢ , BamHⅠ and DNA sequence. Correct positive clones as chosen recombinant plasmlds were transfected into the CBRH-7919 cell line with LipofectamineTM 2000. Cells were observed at 48 hours after transfection using an inverted fluorescence microscope. The mRNA expression of Hdac3 and PPAR-γ were identified by real-time PCR. Results The correct constructed plasmid was confirmed by PCR, restriction digestion and DNA sequence analysis. Green fluorescence protein could be observed in the ceils after 48 hours of transfeetion under fluorescence microscope. Compared to control groups, the mRNA expression of Hdac3 increased significantly in CBRH cells transfected with pEGFP-Hdac3,while that of PPAR-γ decreased. Conclusion A eukaryotic expres- sion plasmid pEGFP-Hdac3 has been constructed successfully.