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目的在嵌合型腺病毒的fiber中插入具有整合素特异性的RGD肽,构建可提高腺病毒感染效率的腺病毒载体,观察其对人肝癌细胞株HepG2、BEL-7404以及成纤维细胞BJ的感染效率。方法将RGD-4C插入到AD5/11嵌合型腺病毒载体fiber的HI区,与腺病毒骨架质粒pPE3共转染大肠杆菌BJ5183,同源重组产生腺病毒重组质粒(pPE3-F11-RGD-4C),该重组质粒与PDC328-EF1-EGFP共转染人胚肾293细胞进行包装,产生重组腺病毒(AD11-RGD-4C-EG-FP)。用该重组腺病毒感染HepG2、BEL-7404以及BJ细胞,通过荧光显微镜观察感染效率。结果所构建的重组腺病毒PCR扩增出1 123 bp包含目的片段的基因片段。同时,制备了高滴度的重组病毒,纯化后病毒滴度为1.3×1010pfu/ml。当MOI=10,48 h时该重组病毒对HepG2、BEL-7404及BJ细胞的感染效率明显高于对照组腺病毒(AD11-EGFP)。结论成功构建了可提高腺病毒感染效率的嵌合型腺病毒(AD11-RGD-4C-EGFP),为进一步的研究奠定了基础。
OBJECTIVE: To construct a recombinant adenovirus carrying integrin-specific RGD peptide in the fiber of chimeric adenovirus and to improve the efficiency of adenovirus infection and to observe its effect on human hepatocellular carcinoma cell lines HepG2, BEL-7404 and fibroblast BJ Infection efficiency. Methods RGD-4C was inserted into the HI region of the AD5 / 11 chimeric adenovirus vector fiber and cotransfected into E. coli BJ5183 with the adenoviral backbone plasmid pPE3. The recombinant adenovirus plasmid pPE3-F11-RGD-4C ). The recombinant plasmid was cotransfected into 293 cells of human embryonic kidney with PDC328-EF1-EGFP for packaging, resulting in the recombinant adenovirus (AD11-RGD-4C-EG-FP). The recombinant adenovirus was used to infect HepG2, BEL-7404 and BJ cells, and the infection efficiency was observed by fluorescence microscopy. Results The recombinant adenovirus constructed by PCR amplified 1 123 bp gene fragment containing the target fragment. At the same time, a high titer recombinant virus was prepared and the virus titer after purification was 1.3 × 1010pfu / ml. The infection efficiency of HepG2, BEL-7404 and BJ cells was significantly higher than that of control group adenovirus (AD11-EGFP) when MOI = 10,48 h. Conclusion The chimeric adenovirus (AD11-RGD-4C-EGFP), which can improve the efficiency of adenovirus infection, was successfully constructed and laid the foundation for further research.