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目的构建含有人三叶因子3(trefoil factor family 3,TFF3)基因的腺病毒载体,并观察其对胆管上皮细胞迁移的调控作用。方法利用PCR技术对目的基因TFF3全长进行扩增,测序后亚克隆至腺病毒穿梭质粒构建载体pAdTrack-CMV-TFF3,转化至含腺病毒骨架质粒pAd-Easy的大肠杆菌株BJ5183感受态细胞中进行同源重组,筛选重组子并经内切酶PacⅠ线性化,转染293细胞进行病毒包装及扩增,并进行滴度测定。构建的TFF3腺病毒载体感染人胆管上皮细胞后用RT-PCR及Western blot检测目的基因表达水平,并进行划痕实验观察细胞迁移能力的变化。结果成功扩增了大小约为458 bp的人TFF3基因,测序报告显示目的基因序列与NCBI报道一致;成功构建了TFF3的同源重组腺病毒穿梭载体,经293细胞包装及扩增的病毒滴度为1.3×109CPU/mL。RT-PCR及Western blot结果显示转染人胆管上皮细胞后其TFF3表达明显增强,细胞迁移能力也明显高于对照组。结论成功构建含TFF3基因的腺病毒载体,为进一步研究TFF3在胆管上皮损伤修复中的作用奠定了基础。
Objective To construct an adenovirus vector containing human trefoil factor family 3 (TFF3) gene and observe its regulation on the migration of bile duct epithelial cells. Methods The full-length TFF3 gene was amplified by PCR and subcloned into shuttle vector pAdTrack-CMV-TFF3. The recombinant plasmid was transformed into E. coli strain BJ5183 competent cells containing the adenovirus backbone plasmid pAd-Easy Homologous recombination was carried out. The recombinant was screened and linearized with restriction endonuclease Pac I. The 293 cells were transfected into 293 cells for virus packaging and amplification, and the titer was determined. The constructed TFF3 adenovirus vector was used to infect human cholangiocarcinoma cells. RT-PCR and Western blot were used to detect the expression of the target gene. The scratch test was performed to observe the changes of cell migration. Results The human TFF3 gene with a size of about 458 bp was successfully amplified. Sequencing results showed that the sequence of the target gene was consistent with that of NCBI. The homologous recombinant adenovirus shuttle vector of TFF3 was constructed successfully. The virus titer 1.3 × 109CPU / mL. The results of RT-PCR and Western blot showed that the expression of TFF3 in transfected human bile duct epithelial cells was significantly increased and the cell migration ability was also significantly higher than that in the control group. Conclusion The successful construction of adenoviral vector containing TFF3 gene lays the foundation for further study on the role of TFF3 in the repair of bile duct epithelial injury.