凋亡素基因重组腺病毒构建.docVIP

凋亡素基因重组腺病毒构建 　【摘要】 目的 构建携凋亡素基因的腺病毒载体，为进一步研究凋亡素基因功能及其作用机制建立基础。方法 Pmel酶切线性化已经构建好的含凋亡素基因的穿梭质粒pAdTrackCMVVP3，然后电转化到BJ5183AD1细胞中进行同源重组。PacⅠ酶切出含凋亡素基因的腺病毒DNA，然后转染293细胞进行包装获得重组腺病毒，运用RTPCR鉴定重组腺病毒。用氯化铯梯度离心纯化病毒。用物理和生物方法确定病毒的滴度。结果 PacⅠ酶切证实同源重组成功。绿色荧光观察证实病毒包装成功并且具有感染力，RTPCR证实了重组腺病毒具有凋亡素蛋白的编码。重组腺病毒的浓度为84.52×109 VP/ml，滴度为6.561×109 PFU/ml。结论 成功构建含凋亡素基因的重组腺病毒，它的滴度足以满足体内外实验。 【关键词】 凋亡素基因 腺病毒载体 基因治疗 【Abstract】 Objective To construct a recombinant adenovirus carrying Apoptin gene so as to provide basis for further studying Apoptin gene functions and ascertaining corresponding mechanism. Methods The shuttle vector pAdTrackCMVVP3 containing Apoptin gene was linearized with Pmel and subsequently electrotransformed into BJ5183AD1 cells for homologous recombination. The DNA containing Apoptin gene of the recombinant adenovirus was obtained by digesting with PacI and then transfected into 293 cells, where the recombinant adenovirus containing Apoptin gene was identified by RTPCR and purified by cesium choride density centrifugation. Finally, the titre of the recombinant adenovirus was determined by biological and physical assays. Results Digestion with PacI proved successful homologous recombination. Green fluorescence showed successful recombinant adenovirus with infectiveness. RTPCR confirmed that the recombinant adenovirus had coding of Apoptin gene proteins. The titre of the recombinant adenovirus was 84.52×109 VP/ml and 6.561×109 PFU/ml. Conclusion The recombinant adenovirus vector containing Apoptin gene is successfully constructed, with its titre sufficient for in vivo and in vitro experiment. 【Key words】 Apoptin gene; Adenovirus vector; Gene therapy 凋亡素，又称Apoptin(apoptosis induction)［1］，是来源于鸡贫血病毒(chicken anemia virus,CAV)的小分子蛋白质。它能引发一些肿瘤细胞凋亡和细胞转化，而对正常细胞无此作用。在肿瘤的治疗上具有巨大潜力［1］。由于凋亡素是外来物质，在发挥作用前必须进入细胞，所以我们选择腺病毒作为载体来运载它，以期能探索它的作用机制，为肿瘤的治疗提供新的途径。 1 材料与方法 1.1 含凋亡素基因的穿梭质粒PAdtrackCMVVP3的获得［2］我们已经成功构建了含凋亡素基因的穿梭质粒，并且通过了酶切和测序鉴定，为下一步实验的进行奠定了基础［2］。 1.2 含凋亡素基因的腺病毒基因组(PAdVP3)质粒的