论文部分内容阅读
目的构建含有人TGF-β1Ⅱ型受体胞外区及IgG1Fc段融合基因的腺病毒载体,并进行初步的功能活性鉴定。方法RT-PCR扩增人TGF-β1Ⅱ型受体胞外区及IgG1Fc段基因,通过OverlapPCR融合为目的基因hTβRⅡ-IgG1Fc;克隆至pAdTrack-CMV穿梭质粒,线性化后转染含骨架质粒的BJ5183菌,同源重组构建腺病毒质粒;将线性化的重组腺病毒质粒转染293细胞,并扩增、纯化、RT-PCR鉴定;ELISA初步检测重组腺病毒的功能活性。结果目的基因经酶切分析、测序鉴定正确;RT-PCR及ELISA表明重组腺病毒能表达hTβRⅡ-IgG1Fc,中和HLF分泌的TGF-β1。结论成功构建了含融合基因hTβRⅡ-IgG1Fc的腺病毒载体,体外实验证实表达的蛋白具有中和TGF-β1的作用,为进一步研究放射性肺纤维化的基因治疗提供实验依据。
Objective To construct an adenovirus vector containing the extracellular region of human TGF-β1 receptor and fusion gene of IgG1 Fc region, and to identify the preliminary functional activity. Methods The extracellular region of human TGF-β1 type II receptor and IgG1 Fc gene were amplified by RT-PCR and cloned into pAdTrack-CMV shuttle plasmid by Overlap PCR and cloned into pAdTrack-CMV shuttle vector. After linearization, the vector containing BJ5183 The recombinant plasmids were transfected into 293 cells. The 293 cells were amplified, purified and identified by RT-PCR. The recombinant adenoviruses were initially tested for their functional activity by ELISA. Results The target gene was identified by restriction enzyme digestion and sequencing. RT-PCR and ELISA showed that the recombinant adenovirus could express hTβRⅡ-IgG1Fc and neutralize TGF-β1 secreted by HLF. Conclusion The recombinant adenoviral vector containing fusion gene hTβRⅡ-IgG1Fc was constructed successfully. The in vitro experiments confirmed that the expressed protein has the effect of neutralizing TGF-β1 and provide experimental basis for further study of gene therapy of radiation-induced pulmonary fibrosis.