摘要
目的应用简化的两步法细菌内同源重组高效制备TGF-β1基因重组腺病毒载体,为下一步的基因治疗奠定基础。方法应用PCR技术扩增TGF-β1-pcDNA3.1质粒中的目的基因片段,将TGF-β1目的基因片段定向克隆至穿梭质粒pAdTrack-CMV中,转化DH5a感受态细胞,用卡那霉素平板筛选阳性克隆,提取质粒,将酶切鉴定正确的质粒用PmeI线性化后采用两步法同源重组:先将pAdEasy-1转化入BJ5183细菌中,制备pAdEasy-1-BJ5183感受态细胞,再将PmeI线性化的pAdTrack-CMV-TGF-β1转入其中,进行同源重组,用卡那霉素平板筛选小的阳性克隆,提取质粒,酶切或PCR鉴定。结果经酶切,PCR鉴定成功构建了TGF-β1重组腺病毒载体。结论运用简化的两步法细菌内同源重组可以在大肠杆菌中快速高效地构建重组腺病毒载体。
Objective To construct recombinant replication-defective human adenovirus serotype 5 vector carrying TGF-β1 by simplified two-step homologous recombination protocol in bacteria,and to establish the foundation of continuous gene therapy. Methods TGF-β1 was firstly subcloned into a transfer vector pAdTrack-CMV and the positive recombinant pAdTrack-CMV-TGF-β1 was linearized by Pme I. Transformed the adenoviral backbone plasmid pAdEasy-1 into E. coli BJ 5183. In BJ 5183, homologous recombination was occurred and recombinant adenoviral plasmid pAdEasy-1-TGF-β1 was generated. Results After confirmed by restriction enzyme digesting and PCR,recombinant adenoviral vector carrying TGF-β1 was successfully constructed. Conclusion The results indicate that the recombinant adenoviral vector may be constructed quickly and efficiently by simplified two-step homologous recombination protocol.
出处
《临床消化病杂志》
2007年第3期150-151,共2页
Chinese Journal of Clinical Gastroenterology
