Construction Of Adenovirus Vector Expressing Angiopoietin-1 And Its Expression In Kideny Of Diabetic Rats

Background and objective:Angiopoietin-1(Ang-1),a new vascular-specific growth factor, has been found as ligands for the Tie-2 to promote vascular maturation,stabilization,and antipermeability,etc.play an important role in physiological and pathological angiogenesis.The development of diabetic nephropathy was to a large extent attributed to renal microvascular injury and disorder of angiogenetic regulatory factors.This study was designed to construct a recombinant adenovirus vector expressing Ang-1 gene,and observe Ang-1/Tie-2 expression in the kidney of diabetic rats.The purpose of this study was to provide experimental evidence for the further interventional investigation of renal microvasculopathy in diabetic rats.Methods:(1)Primer was designed and synthesized to amplify Ang-1 gene from plasmid pMD18-Ang-lwith PCR.The product and plasmid pDC315-GFP were digested with EcoR I,and then the products digested were connected with In-Fusio exchange enzyme,pDC315-GFP-Ang-1 was transformed into E.coliDH5a. Recombinant E.coliDH5a was,and identified by colony PCR and DNA sequencing.(2) HEK293 was cotransfected with shuttle plasmid pDC315-GFP-Ang-1 and auxiliary packaging plasmid pBHG loxâ–³E1,3 Cre.Target protein was observed by GFP fluorescence and Western Blot.Titer of recombinant adenovirus was detected with end-point dilution method after amplification and purification.(3) Diabetic rats induced by Streptozotocin was injected with adenovirus carrying Ang-1 in the tail vein.Ang1 and Tie-2 expression in renal tissue of diabetic rats were analyzed by immunofluorescence,immunohistochemistry,and RT-PCR..Results:(1) Ang-1 gene,which was amplified from plasmid pMD18- Ang-1,was 1558 base pair, and accord with the result in the Genebank through DNA sequencing.(2) After HEK293 cells transfected with recombinant adenovirus(pDC315-GFP-Ang-1),the GFP-Ang-1 fluorescence was observed,the band corresponding to molecular weight of of fusion protein was further confirmed.The titer of recombinant adenovirus was 1×10¹¹ pfu / ml.(3) The expression levels of Ang-1/Tie-2 mRNA and protein in renal tissue of diabetic rats were higher than that in normal rats, with peak levels from 20 to 28 weeks.Conclusion:The constructed adenovirus vector expressing ang-1 has a high titer,and may successfully express target protein,and upregulate expression levels of Ang-1/Tie-2 mRNA and protein in renal tissue of diabetic rats.