Construction and transfection efficiency analysis of 5/11 chimeric adenovirus harboring RGD-4C / 第二军医大学学报

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 3X10 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.

Adenovirus vVector, a Most Potential System for Cardiovascular Disease？ / 中国医科大学学报

Cardiovascular disease is a major cause of morbidity and mortality in the contemporary society. Cardiac gene therapy has already been investigated in clinical studies profoundly. Adenovirus vector could transfer the gene efficiently, but low organ specificity and immuno- genie properties limited its application, With adenovims vector improved in the last few years, the third adenovims vector is probably the most potential vector systems for cardiovascular disease. This review will give a broad overview of the molecular basis of the adenovims vectors, their advantages, the development of increasingly efficient gene transfer approach, and experimental and clinical studies targeting the heart, application barriers and modified strategies.

Recombinant Adenovirus Vector Mediated Expression of HCMV UL49 Gene in Mice / 中国生物工程杂志

Human cytomegalovirus (HCMV) is extremely species specific and does not replicate in experimental animal tissues.To overcome the problem and establish suitable animal models for studying antiviral strategies,the expression of HCMV UL49 gene was explored in mice.UL49-GFP gene was subcloned into the adenovirus shuttle plasmid pDC316,the products(pDC316-UL49-GFP)were co-transfected with helper plasmid pBHGloxE1,3Cre into HEK293 cell lines by liposome reagent,recombinant adenovirus(Ad-UL49-GFP) was generated and confirmed by PCR and Western blot.Ad-UL49-GFP was propagated in 293 cells and purified.The titer of viral stocks was determined by end-point dilution assay.The purified adenoviruses were delivered into mice via the tail vein injection.Fluorescence quantitative PCR and Western blot experiments were used to examine the tissue distribution and duration of UL49 gene expression.The results showed that the recombinant adenovirus were present in vivo.The expression level in tissues arranged in descending order was liver,spleen,kidney,heart and lung.3 days after injection,the liver,spleen,kidney,heart and lung expressed protein UL49 in high lever and then declined gradually.14 days after injection,UL49 protein expression was disappear in some organs except liver and spleen.In conclusion,transgene animal model carrying UL49 gene was successfully established.Therefore,the system may be suitable for selecting anti-HCMV drugs targeting UL49 gene.