摘要
目的构建携带人肌浆网钙ATP酶2a(SERCA2a)基因重组腺病毒载体,获得重组腺病毒rAd-SERCA2a-EGFP,为进一步在细胞和动物水平研究SERCA2a基因的功能奠定基础。方法根据细菌内同源重组的方法,使用AdEasy腺病毒包装系统将人SERCA2a基因克隆到腺病毒穿梭载体质粒pshuttle-CMV中,腺病毒穿梭质粒pshuttle-CMV-SERCA2a线性化后与腺病毒载体pAdEasy发生同源重组,通过脂质体共转染HEK293细胞,产生重组腺病毒rAd-SERCA2a,DNA测序鉴定正确后进行扩增、纯化和滴度测定。结果通过酶切鉴定和DNA测序鉴定,证实重组腺病毒载体pAdEasy-SERCA2a-EGFP构建成功,PCR鉴定扩增出2 998bp的目的条带,rAdSERCA2a-EGFP滴度高达1×1012μg/mL。结论成功构建和包装携带hSERCA2a基因的rAd-SERCA2a-EGFP,为SERCA2a基因治疗心力衰竭动物实验及临床试验研究奠定基础。
Objective To construct a recombinant adenovirus vector carrying the sarcoplasmic reticulum Ca2＋ ATPase （hSERCA2a ）gene,and use rAd-SERCA2a-EGFP for further research SERCA2a gene func-tion in cell and animal levels.Methods According to the method of homologous recombination in bacteria, using AdEasy adenovirus packaging systems,we cloned the hSERCA2a gene into adenovirus shuttle vector.Adenovirus shuttle plasmid pshuttle-CMV-SERCA2a was linearized and it occurred homologous re-combination with adenoviral vectors pAdEasy.Liposomes were co-transfected into HEK293 cells so as to produce the recombinant adenovirus.Amplification,purification and titer determination were carried out after correcting identification by DNA sequencing.Results The results of restriction endonuclease digestion and DNA sequencing showed that recombinant adenovirus vector pAdEasy-SERCA2a-EGFP was constructed successfully;the positive amplification bands could be seen in PCR analysis,and the titer of rAd-SERCA2a-EGFP was determined to be up to 1×10^12μg/mL.Conclusion The building and packaging of rAd-SERCA2a-EGFP carrying the hSERCA2a gene was successful.SERCA2a gene therapy for heart failure animal experiments and clinical trials research lay the foundation.
出处
《新疆医科大学学报》
CAS
2014年第10期1305-1307,1311,共4页
Journal of Xinjiang Medical University
基金
新疆心血管病研究实验室开放课题(XJDX0903-2013-02)
新疆医学动物模型研究重点实验室开放课题(XJDX1103-2013-06)
关键词
肌浆网钙ATP酶
腺病毒
构建
sarcoplasmic reticulum Ca^2＋ ATPase
adenovirus
building
