人转化生长因子β1基因重组腺病毒的构建与鉴定

背景：转化生长因子β1能促进多种细胞的生长增殖,是调控骨髓间充质干细胞向成骨方向定向分化的主要生长因子。 目的：利用AdMax系统构建人转化生长因子β1(hTGF-β1)基因的重组腺病毒表达载体。 方法：采用PCR方法克隆人转化生长因子β1基因cDNA后,插入线性化表达载体pAV-MCMV-EGFP-3FLAG中,转化E.coli DH5α感受态细胞,构建pAV-MCMV-hTGF-β1重组质粒。 结果与结论：含目的基因的重组腺病毒质粒共转染293细胞进行病毒包装、扩增后, 经PCR、Western Blot检测得到转化生长因子β1重组腺病毒,其滴度约为1.25×10¹⁰ pfu/mL。表明利用AdMax系统成功可构建人转化生长因子β1腺病毒载体,并可满足进一步的转化生长因子β1成骨作用的研究。

Construction and identification of recombinant adenovirus vectors encoding human transforming growth factor-beta 1 gene

BACKGROUND:Transforming growth factor-beta1 (TGF-β1) can promote the growth and proliferation of many cells and is one of main growth factors that regulate osteoblast differentiation of bone marrow mesenchymal stem cells. OBJECTIVE:To construct a recombinant adenoviral vector carrying the human TGF-β1 gene using the AdMax system which may be applicable in gene therapy of bone defect. METHODS:The hTGF-β1 gene sequence was amplified by PCR from cDNA template and then was inserted into shuttle plasmid pAV-MCMV-EGFP-3FLAG. The recombinant shuttle plasmid was constructed and transformed into E.coli DH5α.The recombinant pAV-MCMV-hTGF-β1 was obtained. RESULTS AND CONCLUSION:The recombinant pAV-MCMV-hTGF-β1 was packaged and amplified in 293 cells. Then the recombinant TGF-β1 adenovirus was constructed successfully. After measuring the titre of virus (1.25×10¹⁰ pfu/mL), the target gene was evaluated by PCR and Western Blot. These findings suggest that construction of hTGF-β1 adenoviral vector was successfully achieved using the AdMax system and may be available in the future study of TGF-β1 gene therapy.