Construction Of Adenovirus Vectors Of Rat Glucocorticoid Receptor Gene And Expression In Rat Mesangial Cell

Objective: To construct adenovirus vectors of rat glucocorticoidreceptor and to transfect them into rat mesangial cells and to detect theexpression of GR protein by Western Blot.Methods: The rat glucocorticoid receptor gene from pcDNA1.Neo—Rat GR was amplified by polymerase chain reaction (PCR). Theamplified rat GR cDNA was inserted into the downstream of CMVpromoter, hence the shuttle plasmid pShuttle-CMV-Rat GR wasestablished by ligation. The linearized shuttle plasmid wasco-transformed into BJ5183 bacteria with backbone vector AdEasy-1 toobtain the recombinant adenoviral plasmid pAd—Rat GR by homologousrecombination. The recombined adenovirus DNA was transfected intoHEK293A cells for packing and the amplification product ofreplication-deficient Ad—Rat GR virus was purified by CsCl densitygradient centrifugation. The expression of rat GR was verified by WesternBlot in rat mesangial cell after infected with Ad—Rat GR.Results: The recombinant plasmid pAd—rat GR was established byhomologous recombination and confirmed by restriction endonucleasedigestion and sequencing. EGFP expression was observed 48 hours after packing of the linearized pAd—rat GR in HEK293A cells and 1.0×10~9PFU/ml titer of Ad—rat GR was obtained by CsCl gradient purification.The rat mesangial cell was infected with this titer of Ad—rat GR viruses,and then total protein in the rat mesangial cells was extracted. WesternBlot showed that rat GR protein was expressed obviously.Conclusion: The recombinant adenoviruses of rat GR gene areconstructed successfully and can up-regulate the expression of rat GRprotein in the rat mesangial cells.