携带RGD多肽的嵌合型腺病毒载体AD11-RGD-4C-EGFP的构建及其感染效率的研究

Construction and transfection efficiency analysis of 5/11 chimeric adenovirus harboring RGD-4C

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 5/11 chimeric adenovirus harboring RGD-4C,and observe its transfection efficiency after transfecting it into hepatocarcinoma cell lines HepG2,BEL-7404 and BJ cells.Methods RGD-4C was inserted into the HI loop region of 5/11 chimeric adenovirus fiber gene and the insertion outcome was confirmed by enzyme digestion and PCR analysis.The confirmed plasmid was co-transfected into E.coli BJ5183 together with adenoviral backbone plasmid pPE3 to produce recombinant plasmid by homologous recombination.Recombinants were selected and co-transfected into 293 cell line with PDC328-EF1-EGFP to produce recombinant adenovirus.The recombinant adenovirus production was confirmed by PCR analysis and was amplified and purified.The virus titer of recombinant adenovirus was determined.AD11-RGD-4C-EGFP was used to infect HepG2,BEL-7404,and BJ cell lines,and their transduction efficiency was determined by fluorescence microscope.Results A 1 123 bp target gene fragments was obtained by PCR from the recombinant adenovirus;meanwhile,we prepared high titer recombinant adenovirus,with the titer being 1.3×10^10pfu/ml after purification.At 10 MOI,the infection efficiency of recombinant adenovirus was much higher than control adenovirus（AD11-EGFP） after 48 h infection.Conclusion We have successfully constructed a chimeric adenovirus harboring RGD-4C,which paves a way for enhancing the infection efficiency of adenovirus in bio-therapy.