构建重组人SERCA2a基因9型腺相关病毒载体和病毒包装

Building of recombinant human SERCA2a gene-mediated 9 adenovirus-associated virus vector and virus packaging

目的探讨构建重组人SERCA2a基因9型腺相关病毒载体和病毒包装的方法,并包装出rAAV9-hSERCA2a-EGFP。方法根据细菌内同源重组的方法使用腺相关病毒包装系统,将hSERCA2a基因克隆到腺相关病毒载体中,与腺相关病毒包装质粒pAAV pHelper发生重组,通过脂质体共转染人胚肾293(HEK293)细胞,产生重组腺相关病毒,通过酶切鉴定和DNA测序鉴定正确后进行扩增、纯化和滴度测定(采用荧光滴度检测法)。结果酶切鉴定和DNA测序证明重组腺相关病毒载体rAAV9-hSERCA2a-EGFP构建成功,PCR鉴定可见到阳性扩增条带,rAAV9-hSERCA2a-EGFP滴度高达1×1012 vg(virus genomes,vg)/mL。结论本方法成功构建和包装滴度为1×1012 vg/mL携带hSERCA2a基因的rAAV9-hSERCA2a-EGFP。

Objective To explore the way of constructing a recombinant adeno-associated virus vector carry ing the hSERCA2a gene, and to package rAAV9-hSERCA2a-EGFP successfully. Methods According to the method of homologous recombination in bacteria, we used the adeno-associated virus packaging sys terns, we cloned the hSERCA2a gene into the adeno-associated virus vector in recombination with adeno-associated virus packaging plasmid pAAV pHelper. Liposomes were co-transfected into HEK293 cells so as to produce the recombinant adeno-associated virus. After correct identification by restriction endonucle ase digestion and DNA sequencing, amplification, purification and titer determination were detected using the method of fluorescence titer test. Results The results of restriction endonuclease digestion and DNA sequencing showed that recombinant adeno-associated virus vector rAAV9-hSERCA2a-EGFP was con structed successfully; the positive amplification bands could be seen in PCR analysis, and the titer of rAAV9-hSERCA2a-EGFP was determined to be up to 1× 10^12 vg/mL. Conclusion The aging of rAAVg-hSERCA2a-EGFP carrying the hSERCA2a gene is successful at a titer of 1× 10^12 vg/mL.