细菌内同源重组构建人p21^(WAF1)基因重组腺病毒

Construction of Recombinant Adenovirus Vector Carrying Human p21~ WAF1 Gene by Homologous Recombination in Bacteria

目的构建人p21WAF1基因重组腺病毒载体,并在293细胞中包装制备重组腺病毒。方法将人p21WAF1基因亚克隆连接到腺病毒转移质粒pshuttle-cmv,得到阳性重组转移质粒psh-p21。先将腺病毒骨架质粒pAdEasy-1转化BJ5183菌,筛选得到AdBJ5183菌,再将PmeⅠ线性化的psh-p21转化AdBJ5183菌,经细菌内同源重组法构建重组腺病毒质粒pAdp21。经PacⅠ和BstXⅠ酶切及PCR鉴定,将正确的重组腺病毒质粒pAdp21转染293细胞包装病毒,用RT-PCR和PCR鉴定重组腺病毒及其稳定性。结果所构建的重组腺病毒质粒pAdp21,经PCR鉴定,可扩增出约500bp的片段,与预期大小相符。转染293细胞可包装成p21WAF1基因重组腺病毒,经电镜观察,具有典型的腺病毒特征。结论已成功地构建了重组腺病毒质粒pAdp21,并制备出重组腺病毒rAdp21。

Objective To construct recombinant adenovirus vector carrying human p21^WAF1 gene by homologous recombination in bacteria. Methods p21^WAF1 gene was subcloned into pshuttle-cmv transfer vector, and positive recombinant psh-p21 was linearized with Pine Ⅰ ,then transformed to competent AdBJ5183 cells transformed with adenovirus skeletal vector pAdEasy-1 in advance. The recombinant adenovirus vector pAdp21 was identified by digestion with Pac Ⅰ and Bstx Ⅰ as well as amplification by PCR,then linearized with Pac Ⅰ and transfected to 293 cells to package recombinant adenovirus particles. The recombinant adenovirus was identified by RTPCR, and its stability was analyzed by PCR. Results A 500 bp fragment was amplified by PCR from the constructed recombinant adenovirus vector pAdp21 ,which was consistent with that expected. The rAdp21 showed typical character of adenovirus under electron microscope. Conclusion Recombinant adenovirus vector pAdp21 was successfully constructed, and recombinant adenovirns rAdp21 was prepared.