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目的:构建靶向肝脏的小凹蛋白-1 caveolin-1基因重组腺病毒并对该表达体系进行鉴定。方法:根据GeneBanks中caveolin-1的序列设计引物,以大鼠肝组织提取总RNA为模板,PCR扩增caveolin-1,并将caveolin-1序列克隆至pAdTrack-CMV质粒中,再与pAd-easy质粒进行同源重组构建重组腺病毒载体,利用脂质体法于293细胞中进行包装重组腺病毒,RT-PCR检测caveolin-1基因的转录,Westernblotting检测caveolin-1蛋白的表达情况。结果:PCR及Western blotting检测结果表明成功构建出caveolin-1基因重组腺病毒。结论:成功构建出caveolin-1基因重组腺病毒,为进一步研究caveolin-1基因在肝纤维化及肝硬化疾病中的作用机制打下实验基础。
Objective: To construct a recombinant adenovirus targeting caveolin-1 gene of liver and to identify the expression system. Methods: Primers were designed according to the sequence of caveolin-1 in GeneBanks. The total RNA of rat liver tissue was used as a template to amplify caveolin-1 by PCR. The caveolin-1 sequence was cloned into pAdTrack-CMV plasmid and ligated with pAd-easy The recombinant adenovirus vector was constructed by homologous recombination. The recombinant adenovirus was packaged in 293 cells by lipofectamine. The caveolin-1 gene transcription was detected by RT-PCR. The expression of caveolin-1 protein was detected by Western blotting. Results: PCR and Western blotting results showed that caveolin-1 gene was successfully constructed. CONCLUSION: The caveolin-1 gene recombinant adenovirus was successfully constructed and laid the experimental foundation for further study of the mechanism of caveolin-1 gene in liver fibrosis and cirrhosis.