抗KDRscFv复制缺陷型腺病毒载体的构建及鉴定

Construction of replication-defective recombinatant adenovirus vector containing anti-KDRscFv gene

目的构建抗KDRscFv复制缺陷型腺病毒载体,为抗KDRscFv抗肿瘤血管生成的体内应用打下实验基础。方法采用重叠延伸PCR方法构建IL-2信号肽及抗KDRscFv融合基因,克隆于腺病毒穿梭质粒pAdTrack-CMV,转染含pAdEasy-1的BJ5183细菌,经细菌内同源重组产生携带抗KDRscFv基因(含信号肽)的重组腺病毒载体pAd-抗KDRscFv,经脂质体法转染293细胞,包装产生携带抗KDRscFv基因的重组腺病毒Ad-抗KDRscFv。结果构建了表达抗KDRscFv基因的复制缺陷型腺病毒,且该重组腺病毒能在肝癌细胞株HepG2中高效表达。结论成功构建表达抗KDRscFv基因的复制缺陷型腺病毒载体,为进一步研究抗KDR-scFv在肿瘤治疗中的作用研究提供实验基础。

To construct the replication-defective recombinatant adenovirus containing anti-KDRscFv gene the homogenous recombination method. Methods Anti KDRscFv containing IL-2 signal peptide was cloned by gene splicing overlap extension （SOE） method and then was linked with T vector. After sequenced, the shuttle plas-mid pAdTrack-CMV KDRscFv was constructed and linearized with Pme I, then transformed into ultracompletent BJ5183 bacterial cell containing pAdeasy-1. The positive clone of homologous recombination was selected. The cDNA of identi-fied recombinant plasmid was digested with Pac I and transferred to HEK293 cells to package adenovirus particles. PCR technique was used to detect target gene. Results Replication-defective recombinatant adenovirus for KDRscFv gene was constructed and the adenovirus could infect HepG2 cells efficiently. Conclusion The replication-defective recom-binatant adenovirus containing anti-KDRscFv gene is successfully constructed by the method of homogenous recombination in bacteria, which provides the possibility for gene therapy of tumor.