Construction Of Recombinant Adenovirus Vector Of Human STGFβRⅡ Gene

Background and ObjectiveDiabetic nephropathy(DN) is one of the microvascular complications of diabetes mellitus and the leading cause of end-stage renal disease. Disordered glomerular mesangial cell synthesis and catabolism of the components of the extracellular (ECM) under high glucose conditions are the immediate cause of DN. Many research show that overexpression of transforming growth factor-β (TGF-β) mediates the effects of high glucose concentration, and that TGF-β is probably a determinant in the development of DN.Therapeutic strategy to down-regulate TGF-β level under high glucose conditions provide an approach to inhibit the progression of diabetic nephropathy.Soluble TGFβ receptor II (sTβRII) is the extracellular segment of TGFβRII which is combined with TGF-β competently and thus suppresses the TGF-β activity and improves renal function.To reveal the role of sTβRII inhibiting the activity of TGF-β, renal tubular interstitial cell proliferation , ECM accumulation and tissue fibrosis under high glucose conditions, and investigate the possibility of sTβRII gene therapy for DN, we constructed the recombinantadenovirus vector of sTβRII gene. MethodsWith the template provided by researcher Yao, sTβRII gene was amplified by the use of a platinum Taq polymerase. Then the sTβRII segment was cloned into TA plasmid(pMD19-T simple) and identified by the way of PCR .restriction digestion and sequence analysis.TA-sTβRII and pShuttle-IRES-hrGFP-1 (PSI) were digested by two restriction endonucleases—Sal I and Xho I .Then sTβRII segment was cloned into PSI plasmid. The clones were identified by PCR and restriction digestion. PSI- sTβRII was linearized with Pme I enzyme and electro-porated into BJ5183-AD-1 electroporation competent cells. And the plates of transformations were incubated overnight at 37℃. After that, several smallest and isolated clones were picked from the plates and identified by the way of PCR and restriction digestion.Then the recombinants were cutted with Pac I restriction enzyme. And the restricted recombinants were transfected into the AD-293 cells by MBS mammalian transfection kit. The cells which appeared cyto-pathetic effection(CPE) were harvested and subjected to three rouds of freezing/thawing by alternating the tubes between -70℃ and 37℃,vortexing and microcentrifugation. Ultimately, the primary viral supernatant was preserved in -70℃ , named primary virals, and used to infect AD-293 cells to gain the second generation virals and evaluate them primarily.Results1. Amplification of sTβRII gene and TA- sTβRII plasmid.With a platinum Taq polymerase and template the sTβRII segment gene was amplified and then cloned into TA plasmid. PCR, restriction digestion and sequence analysis all provided the evidence that it was the right segment.2. Construction and examination of PSI- sTβRHPSI- sTβRII was obtained by combination of digested PSI and sT βRII . Then it was identified by the way of PCR and restriction digestion .Agrose gel electrophoresis ensured that there was a band of about 500bp.3. Construction and examination of AD-sTβRIIBy the way of electroporating PSI-sTβRII into BJ5183-AD-1, we gained AD-sTβRII vector and identified them.4. Package of recombinant adenovirus plasmids in AD-293 cells and infecting AD-293 cells.At the time of 13 days after transfection of recombinants, 90% AD-293 cells were found to appear CPE. The primary virals were harvested and succeeded to infect AD-293 cells. It was confirmed that recombinant adenvirus of sTβRII gene were active.ConclusionsWe succeed to construct and identify human sTβRII -recombinant adenovirus plasmid. And we identify that the expressed sTβRII is able to restrain TGFβ1 from inhibiting MvlLu cells proliferation.