摘要
目的构建无细胞内段基因的人转化生长因子βⅡ型受体（△TβRⅡ）的真核表达载体pEGFP／△TβRⅡ，转染L02细胞以表达融合蛋白EGFP／△TβRⅡ，并检测其生物学活性。方法以质粒H2-3FF为模板，用PCR方法扩增得到人转化生长因子βⅡ型受体（TpRⅡ）胞外段和跨膜段的cDNA，将该cDNA克隆到真核表达载体pEGFP-N2上，构建成pEGFP／△TβRⅡ重组质粒。经双酶切和核酸测序鉴定，将该重组质粒转染L02细胞，用倒置荧光显微镜观察融合蛋白EGFP的表达，并检测其生物学活性。结果L02细胞转染pEGFP／△TβRⅡ重组质粒后，在荧光显微镜下可以观察到融合蛋白EGFP的表达，所表达的蛋白能显著减轻TGF-β1对L02细胞G1～S期的阻滞作用。结论成功构建了pEGFP／△TβRⅡ重组质粒，并表达了有活性的融合蛋白EGFP／△TβRⅡ，为进一步探讨其生物学效应奠定了基础。
Objective To construct a eukaryotic expression vector pEGFP/△TβRⅡ that lacks the cytoplasmic domain of the cDNA of the human transforming growth factor-beta type Ⅱ receptor （△TβRⅡ ）, to express the fusion protein EGFP/△TβRⅡ in human embryonic hepatic cell line L02 and to determine its biological activity. Methods The cDNA of type Ⅱ truncated receptor （△TβRⅡ ） that lacks the cytoplasmic domain was amplified by PCR from the plasmid H2-3FF, and the eukaryotic expression plasmid pEGFP/△TβRⅡ was constructed by inserting the △TβRⅡ cDNA into the vector pEGFP-N2. The construction was introduced by transfection into L02 cells after the DNA sequence was confirmed by double digestion and sequencing. The fusion protein EGFP was confirmed by inverted fluorescence microscope and its biological function was determined. Results The specific protein was observed by inverted fluorescence microscope, and the protein significantly attenuated the inhibitory effect of TGFβ1 on L02 cells. Conclusion The eukaryotic expression plasmid pEGFP/△TβRⅡ was successfully constructed and the EGFP/△TβRⅡ fusion protein with biological activity obtained.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2007年第1期130-133,共4页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
湖北省自然科学基金资助项目(No.2004AA301C44)
关键词
人转化生长因子β
受体
转染
重组
transforming growth factor-beta
receptors transfection
reconstruction
