CAJ | 학술논문

目的：构建携带人肌浆网钙 ATP酶2 a（SERCA2 a）基因重组腺病毒载体，获得重组腺病毒 rAd-SER-CA2a-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-SERCA2 a，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2998 bp的目的条带，rAd-SERCA2a-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。
목적：구건휴대인기장망개 ATP매2 a（SERCA2 a）기인중조선병독재체，획득중조선병독 rAd-SER-CA2a-EGFP，위진일보재세포화동물수평연구 SERCA2a기인적공능전정기출。방법근거세균내동원중조적방법，사용AdEasy선병독포장계통장인SERCA2a기인극륭도선병독천사재체질립pshuttle-CMV중，선병독천사질립 pshuttle-CMV-SERCA2a선성화후여선병독재체 pAdEasy발생동원중조，통과지질체공전염 HEK293세포，산생중조선병독rAd-SERCA2 a，DNA측서감정정학후진행확증、순화화적도측정。결과통과매절감정화DNA측서감정，증실중조선병독재체 pAdEasy-SERCA2a-EGFP구건성공，PCR감정확증출2998 bp적목적조대，rAd-SERCA2a-EGFP적도고체1×1012μg/mL。결론성공구건화포장휴대 hSERCA2a 기인적 rAd-SERCA2a-EGFP，위 SERCA2a기인치료심력쇠갈동물실험급림상시험연구전정기출。
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 1×1012μg/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.