Experimental Method on Amplification and Purification of Adenovirus Mediated Sarcoplasmicreticulum Ca~(2+) ATPase

Objective:This study provided stable and efficient methods for sarcoplasmicreticulum Ca2+ ATPase(SERCA2a) gene bank and established the basis of transferring gene experiments by amplification and purification of recombinant adenovirus(rAd) mediated SERCA2a gene.Method:Human embryonic kidney(HEK) 293 cells were transfected by adenovirus mediated SERCA2a gene with 100μL at virus concentration of 1.9 × 1012 pfu/mL,when the cells appeared cytopathic effect(CPE),collected and destroyed cell wall with freeze-thaw steps to deliver virus,got the Ad-SERCA2a-GFP products by 2×CsCl-gradient purification and measured DNA plasmids by the ultraviolet spectrophotometer.Result: Green fluorescence were successfully expressed in HEK293 cells.The infectious titer,the number of virus particle and the ratio of infectious titer to virus particle for the product were 1.3±0.58×1012 pfu/mL,The OD260/OD280 ratio was 1.57±0.49(n=50).Conclusion:We established the reliably experimental method for amplification of rAd.SERCA2a by HEK 293 cells,and provided high titer rAd.SERCA2a for gene therapy research of heart failure,as well as had certainly referred values for the amplification and purification of recombination adenovirus carrying other genes.