编码绿色荧光蛋白和βARKct的重组腺病毒的制备

Preparation of GFP-βARKct-contained Recombinant Adenovirus

【目的】制备编码绿色荧光蛋白(GFP)和G茁γ活性抑制剂茁ARKct的重组腺病毒载体。【方法】构建腺病毒穿梭质粒pAdTrack-CMV-茁ARKct,经PmeI线性化后转化入含腺病毒骨架质粒pAdEasy-I的BJ5183菌,筛选获得重组腺病毒质粒pAdEasy-gfp-茁ARKct。用LipoFectamine2000将经PacI线性化的pAdEasy-gfp-茁ARKct转染入人胚肾293细胞,包装并扩增出重组腺病毒颗粒rAd-gfp-茁ARKct。用氯化铯高速梯度离心、纯化rAd-gfp-茁ARKct。用rAd-GFP-茁ARKct干预去甲肾上腺素诱导的乳鼠心肌细胞肥大过程,观察对茁肌球蛋白重链(茁MHC)基因表达的影响。【结果】将pAdTrack-CMV-茁ARKct转入含pAdEasy-I的BJ5183菌后获得20%的阳性重组体克隆。重组腺病毒质粒在293细胞中包装并扩增出大量的腺病毒,经PCR鉴定含有茁ARKct编码基因。透析纯化得到重组腺病毒滴度为6.0×106pfu/mL。rAd-GFP-茁ARKct表达的茁ARKct可抑制乳鼠心肌细胞茁MHC的高表达。【结论】成功构建重组腺病毒载体,并在293细胞中包装、制备出可表达茁ARKct的重组腺病毒rAd-gfp-茁ARKct。

[Objective]To prepare gfp-βARKct-contained recombinant adenovirus, rAd-gfp-βARKct (beta-adrenergic receptor kinase COOH terminus).[Methods] βARKct gene was digested from pUC57-βARKct and inserted into shuttle plasmid to construct transfer plasmid pAdTrack-CMV-βARKct. pAdTrack-CMV-βARKct was linearized with PmeI and transformed into BJ5183 bacteria containing adenovirus genomic plasmid pAdEasy-I to construct recombinant adenovirus plasmid pAdEasy-gfp-βARKct. The recombinant adenovirus plasmids were identified by PCR amplification and restriction analysis, and the correct one was digested with PacI and transferred into 293 cells mediated by LipoFectamine 2000 to package recombinant adenovirus particles. The βARKct gene was detected by PCR, and the recombinant adenovirus were purified by CsCl banding. Infected by rAd-gfp-βARKct, the β myosin heavy chain (βMHC) gene expression was studied in neonatal hypertrophic cardiac myocytes induced by noradrenaline(NE). [Results] There were about 20% positive recombinant bacterial clones obtained after the transfer plasmid pAdTrack-CMV-βARKct was transformed into BJ5183 containing pAdEasy-I. Recombinant adenovirus particles were produced and further amplified after the transfection of pAdEasy-gfp-βARKct into 293 cells. PCR test showed that the recombinant adenovirus containing βARKct gene. The titer of the purified recombinant adenovirus rAd-gfp-βARKct was 6.0×106 plaque-forming unit (pfu/mL). The βARKct expressed by rAd-gfp-βARKct could reduce the βMHC expression in cardiac myocytes of neonatal Sprague-Dawley. [Conclusion]The recombinant adenovirus plasmid pAdEasy-gfp-βARKct was constructed, and the high titer of recombinant adenovirus rAd-gfp-βARKct, expressing βARKct, were prepared in 293 cells.