人突变p27重组腺病毒的构建及其在结肠癌细胞SW480中的表达

Construction of Recombinant p27mt Adenovirus and Expression in colorectal cancer cell SW480

目的 :为了探讨腺病毒载体用于基因治疗的可行性及突变 p2 7的抗肿瘤特性 ,构建复制缺陷型重组hp2 7mt基因腺病毒。 方法 :hp2 7mt自载体pORF9-hp2 7mt中切出 ,经 2次亚克隆 ,插入到穿梭质粒 pShuttle -CMV中 ,形成转移质粒 pShuttle -CMV -hp2 7mt;然后再用PmeI线性化的 pShuttle -CMV -hp2 7mt转化含腺病毒骨架质粒 pAdeasy - 1的超感受态BJ5 183,使其在细菌内发生同源重组。重组质粒鉴定正确后经PacⅠ酶切 ,转入Ad2 93细胞 ,包装成重组腺病毒Ad -hp2 7mt。采用PCR方法对重组腺病毒进行鉴定 ,用紫外分光光度计进行滴度测定。用重组 p2 7mt的腺病毒Ad - p2 7mt感染大肠癌细胞SW 4 80 ,WesternBlot检测p2 7蛋白的表达。结果 :用含pShuttle -CMV -hp2 7mt转化含 pAdeasy - 1的超感受态BJ5 183后 ,可获得约 30 %的阳性重组质粒。PCR检测表明重组腺病毒DNA中含有目的基因。重组腺病毒滴度为 7.95× 10 15pfu/L。转染大肠癌细胞后 ,p2 7在SW4 80中获得了高表达 ,而未转染细胞和Ad -LacZ转染的细胞中仅有低表达。结论 :腺病毒作为一种基因转移载体 ,可有效介导 p2 7在肿瘤细胞中的表达 ,在肿瘤基因治疗方面具有很大的应用前景。

Objective To explore the protein expression of p27 in colorectal cancer cell SW480 and the potential of adenovirus vector in gene therapy. Methods hp27mt gene was digested from plasmid of pORF9- hp27mt and subcloned into shuttle vector and formed transfer plasmid of pShuttle-CMV-hp27mt; Adenovirus genomic DNA plasmid of pAdeasy-1 was transformed into BJ5183 bacterial cells and ultracompetent BJ5183 containing pAdeasy-1 was prepared. pShuttle-CMV-hp27mt was linearized with PmeI and transformed into ultracompetent BJ5183 bacterial cells containing pAdeasy-1,Posi-tive clone of homologous recombination was selected;The identified recombinant plasmid was digested with PacI and transfered to Ad293 cells to package Adenovirus particles. PCR technique was used to detect target gene. The titer of the recombinant Ad was determined by measuring the absorbance at 260nm.Ad-p27mt infected SW480 cell,Western Blot was used to detect the protein expression of p27. Results there was over 30% postive recombinant bacteria clones after cotransformation of BJ5183 bacterial cells with pShuttle-CMV- hp27mt and pAdeasy-1. The titre of purified recombinant adenovirus was 7.95×10 15 pfu/L.the protein of p27 expressed highly in SW480 cell. Conclusion adenovirus is an efficient vector for mediating transfer and expression of tumour suppressor gene in human tumor cells and prepared recombinant p27 adenovirus could be used for cancer gene therapy.