人肽脱甲酰基酶基因真核表达载体的构建及在NIH3T3细胞中的表达  

Construction of eukaryotic expression vector carrying HsPDF gene and its expression in NIH3T3 cells

作　　者：何睿[1] 黄恺飞[1] 罗学刚[1] 邢莹莹[1] 奚涛[1] 

机构地区：[1]中国药科大学生物技术研究中心,南京210009

出　　处：《中国药科大学学报》2008年第6期575-579,共5页Journal of China Pharmaceutical University

摘　　要：目的:构建人肽脱甲酰基酶(HsPDF)基因的真核表达载体并转染NIH3T3细胞,研究HsPDF基因在真核细胞中的表达及功能。方法:通过RT-PCR从人宫颈癌细胞系HeLa中扩增获得HsPDF基因,克隆到真核表达载体pcDNA3.1(-),利用阳离子脂质体转染NIH3T3细胞,G418筛选建立稳定表达细胞系;RT-PCR检测转染后HsPDF基因的mRNA转录水平;MTT法测定转染后细胞增殖能力的变化;苏木精-伊红(HE)染色观察HsPDF对NIH3T3细胞形态的影响。结果:成功构建重组表达载体pcDNA3.1(-)-HsPDF。该载体被转染入NIH3T3细胞后,外源HsPDF基因获得了有效的转录与稳定表达。HsPDF显著促进NIH3T3细胞的增殖,并使细胞呈现出一定的恶变特征。结论:HsPDF基因在NIH3T3细胞中获得了稳定表达并表现出明显的生物学功能,为深入研究奠定了基础。Aim： To construct an eukaryotic expressing vector carrying Homo sapiens peptide deformylase （ HsPDF） gene and to investigate the expression and the function of HsPDF in NIH3T3 cells. Methods： The HsPDF gene was cloned from HeLa cells by RT-PCR and then inserted into pcDNA3.1 （-） eukaryotic expressing vector. The recombinant plasmid was transfected into NIH3T3 cells using positive ion liposome method before stable cell lines were established by screening with G418. The transcription of HsPDF in the transfected cells was examined by RT-PCR. MTT assay was used to evaluate the effects of HsPDF on the proliferation. Cytomorphological analysis was performed by hematoxylin-easin （HE） staining. Results： The HsPDF gene containing eukaryotic expressing vector was successfully constructed. The transcription and expression of HsPDF in the transfected cells were confirmed. High growth rate of NIH3T3 cells transfected with pcDANA3. 1 （ - ） -HsPDF was shown and several phenotypes of transformed cells were obtained. Conclusion： The HsPDF gene with its functions was expressed in NIH3T3 cells.

关 键 词：人肽脱甲酰基酶 载体 构建 NIH3T3细胞 表达 

分 类 号：Q78[生物学—分子生物学]