CYP4F2真核荧光表达载体的构建及其对HK2细胞ACE表达水平的影响

生物技术通报 ›› 2013, Vol. 0 ›› Issue (12): 194-197.

CYP4F2真核荧光表达载体的构建及其对HK2细胞ACE表达水平的影响

李晓泽^{1, 2} 石磊¹ 李晋^{1, 2} 严华成¹ 李健¹ 赵树进¹

1.广州军区广州总医院，广州 510010
2.华南理工大学生物科学与工程学院，广州 510640

收稿日期:2013-06-22 出版日期:2013-12-20 发布日期:2013-12-20
作者简介:李晓泽，硕士研究生，研究方向：药物代谢酶与心血管疾病；E-mail：xiaozeli@foxmail.com
基金资助:
国家自然科学基金面上项目（81100945）

Construction of a Specific GFP Expression Vector of Human CYP4F2 Gene and Its Influence on Expression of ACE

Li Xiaoze ^{1, 2}, Shi Lei¹, Li Jin^{1, 2}, Yan Huacheng¹, Li Jian¹, Zhao Shujin¹

1. Guangzhou General Hospital of Guangzhou Military Command，Guangzhou 510010
2. School of Biological Science and Engineering ，South China University of Technology ，Guangzhou 510640

Received:2013-06-22 Published:2013-12-20 Online:2013-12-20

摘要/Abstract

摘要：

旨在构建重组CYP4F2基因真核绿色荧光蛋白表达载体pAcGFP-CYP4F2，并转染到人肾近曲小管上皮细胞HK2中表达，为研究CYP4F2在真核细胞中的功能提供一个有效的载体；对CYP4F2过表达对HK2细胞中血管紧张素转化酶（Angiotensin converting enzyme，ACE）表达的影响进行初步研究。从肝癌细胞HepG2中提取总RNA，反转录成cDNA模板，通过PCR扩增出CYP4F2目的片段，纯化后经限制性内切酶酶切，T4连接酶连接到pAcGFP1-C1载体上，转化E. coli DH5α宿主菌，获得阳性克隆；通过PCR扩增、双酶切鉴定和测序分析，确认重组质粒完整正确；抽提重组质粒，转染HK2细胞株，荧光显微镜观察荧光蛋白表达，RT-qPCR和Western blot检测CYP4F2在细胞中mRNA表达。RT-qPCR检测ACE的表达。结果显示，CYP4F2基因目的片段成功重组，获得的重组质粒，保持了目的片段的特异性和序列完整性。荧光显微镜可观察到细胞内GFP的表达；RT-qPCR和Western blot证实CYP4F2基因在转染的细胞内表达增高。RT-qPCR结果显示，转染重组质粒后细胞ACE表达有所增高。成功构建了真核绿色荧光蛋白表达载体pAcGFP-CYP4F2，CYP4F2过表达会提高HK2细胞ACE的mRNA水平。

关键词: CYP4F2基因, 真核表达, HK2细胞, ACE

Abstract:

It was to construct an eukaryotic expression vector which is able to simultaneously express cytochrome P450, family 4, subfamily F, polypeptide 2(CYP4F2)and green fluorescent protein(GFP)in eukaryotic cells, and provide a useful tool for investigating the function of CYP4F2 gene. The full length cDNA encoding CYP4F2 was amplified by polymerase chain reaction(PCR)with cDNA of human HepG2 cell as a template, and subsequently inserted into pAcGFP1-C1 plasmid after digestion with the corresponding restriction endonuleases. The recombinant plasmid pAcGFP1-C1-CYP4F2 was confirmed by restriction endonuclease mapping, PCR amplification and DNA sequencing. HK2 cells were transfected with pAcGFP1-C1-CYP4F2 to investigate the expressions of CYP4F2 and GFP in eukaryotic cells. Fluorescence microscopy was employed to observe the expression of GFP, while RT-PCR and Western blot was utilized to analyze the expression of CYP4F2 gene in HK2 cells. Results showed that the expression vector pAcGFP1-C1-CYP4F2 was successfully constructed and resulted in simultaneously cellular expression of both CYP4F2 and GFP when transfected into HK2 cells. Fluorescence microscopy confirmed the GFP expression in the cells, RT-PCR and Western blot revealed that the expression of CYP4F2 and ACE was increased in CYP4F2 gene transfected cells.

Key words: CYP4F2, Eukaryotic expression, HK2 cells, ACE

李晓泽, 石磊, 李晋, 严华成, 李健, 赵树进. CYP4F2真核荧光表达载体的构建及其对HK2细胞ACE表达水平的影响[J]. 生物技术通报, 2013, 0(12): 194-197.

Li Xiaoze,Shi Lei, Li Jin, Yan Huacheng, Li Jian, Zhao Shujin. Construction of a Specific GFP Expression Vector of Human CYP4F2 Gene and Its Influence on Expression of ACE[J]. Biotechnology Bulletin, 2013, 0(12): 194-197.

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