目的:构建人转化生长凶子-β_1(TGF-β_1)的真核表达载体,并检测其融合蛋白在真核细胞COS-7中的表达,为阐明TGF-B_1在肝纤维化发生、发展中的作用以及建立相应的基因沉寂治疗途径奠定基础.方法:从人的肝脏组织中提取总RNA,通过RT-PCR方法获得人TGF-β_1基凶,将其克隆到真核荧光表达载体pEGFP-N1上,构建重组质粒pEGFP-N1-TGF-β_1,转染真核表达细胞COS-7中,通过荧光显微镜检测其表达,RT-PCR和Western Blot检测TGF-β_1基因在真核细胞中的表达.结果:通过RT-PCR方法克隆人TGF-β_1基因为1173bp,与目的基因片段大小相一致;测序结果显示,于531位有1个碱基突变:T→C,为无义突变.通过RT-PCR和Western Blot 检测到TGF-β_1基因的表达,其融合蛋白分子质最约为52 ku(绿色荧光蛋白分子质量约为27 ku),说明重组质粒在COS-7细胞内正常表达.结论:成功的构建了真核表达质粒pEGFP-N1-TGF-β_1,并在真核细胞COS-7中表达,为进一步研究其在肝纤维化的基因治疗中奠定了基础.
Construction of eukaryotic expression vector encoding the human transforming growth factor-β1 and its expression in COS-7 cells
AIM:To construct the recombinant human transforming growth factor-β_1 eukaryotic expression vector and to detect its expression in COS-7, in order to lay the foundation for elucidating pathogenesis of hepatic fibrosis and constructing the correspondence genetherapy with RNA interference. METHODS:Total mRNA was extracted from human hepatic tissue and the TGFβ_1 cDNA was obtained by RT-PCR and cloned into pEGFP-N1 vector, and then the pEGFP-NI-TGF-β_1 vector was constructed,identificated and transfected into COS-7 cells. Its expression was detected by fluorescent microscopy, RT-PCR and Western Blot.RESULTS:Enzyme digestion analysis and sequencing showed that the target genes was found to be 1173 bp, and had been inserted into recombinant vector, as compared with gene reported by GenBank, O. 08% of the gene mutation(531 th:T→C)and no amino acid residues change in hTGF-β_1. RT-PCR and Western Blot demonstrated that the recombinant vector pEGFP-N1-TGF-β_1 was expressed in COS-7 ceils, and the relatived molecular mass of the GFP-TGF-β_1 fusion protein was about 52×10~3, while the relatived molecular mass of the GFP protein expressed by pBud-GFP was about 27×10~3. The Western Blot analysis indicated that the GFP-TGF-β_1 fusion protein could be recognized by anti-GFP antibody, suggesting that this fusion protein had good biological activity.CONCLUSION:The eukaryotie expression plasmid pEGFP-N1TGF-β_1 has been constructed and expressed successfully in COS-7 cells, which would lay a foundation for further study on the applications of TGF-β_1 in gene therapy of fibrosis.
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