摘要
目的:通过扩增和纯化rAd.SERCA2a,为转SERCA2a基因研究提供实验基础,并为建立基因库提供稳定可靠的实验方法。方法:用100μL 1.9×1012 pfu/ml rAd.SERCA2a感染HEK293细胞,出现细胞病变效应时收获细胞,经物理反复冻融方法及两步氯化铯超速离心方法获得纯化的rAd.SERCA2a,紫外分光光度计比色法测定病毒DNA质粒数。结果:rAd.SERCA2a成功在HEK293细胞表达呈现绿色荧光,纯化的rAd-SERCA2a-GFP DNA质粒数为1.3±0.58×1012 pfu/mL,OD260/OD280比值为1.57±0.49(n=50)。结论:建立了稳定可靠的借助HEK293细胞培养扩增rAd.SERCA2a的实验方法,纯化后的高效价的SERCA2a基因的重组腺病毒可直接用于心力衰竭的实验研究,对重组腺病毒携带其他基因的扩增与提纯方法也具有一定的参考价值。
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.
出处
《生物技术》
CAS
CSCD
北大核心
2011年第2期53-56,共4页
Biotechnology
基金
新疆维吾尔自治区自然科学基金项目("肌浆网钙ATP酶基因转导治疗急性心肌梗塞的实验研究"
2009211B18)资助
关键词
HEK293细胞
腺病毒
SERCA2A
扩增
纯化
HEK-293 cells
adenovirus
sarcoplasmicreticulum Ca2＋ ATPase
amplification
purification
