携载人基质金属蛋白酶组织抑制剂-2基因重组腺病毒载体的构建

The construction of adenovirus vector carrying human tissue inhibitor of metalloproteinase-2 gene

目的 构建携载人基质金属蛋白酶组织抑制剂 2 (hTIMP 2 )基因的重组腺病毒载体 ,为基因治疗提供实验基础。方法 利用基因重组技术将腺病毒骨架质粒 pAdEasy 1以及线性化的重组穿梭质粒 pTrack CMV hTIMP 2共转化BJ5 183受体菌并在其中发生同源重组 ,利用重组前后抗性的改变筛选出重组子 ,重组腺病毒质粒AdhTIMP 2经过 2 93细胞的包装 ,扩增和纯化后 ,测定病毒滴度。一步法提取细胞总RNA ,RT PCR检测hTIMP 2的mRNA。收集细胞上清 ,进行Western杂交检测TIMP 2蛋白。结果 得到了携带hTIMP 2基因的重组腺病毒 ,纯化后滴度为 4× 10 11pfu/ml,应用RT PCR和Westernblot方法 ,在转染 2 93细胞后 2 4h即可检测到hTIMP 2的表达。结论 成功地构建了携带hTIMP 2的重组腺病毒载体 ,为下一步的基因治疗提供了基础。

Objective To construct an adenoviral vector carrying human tissue inhibitor of metalloproteinase-2 (TIMP-2) gene for gene therapy. Methods A recombinant adenovirus (AdhTIMP-2) containing human TIMP-2 cDNA fragment was generated by homologous recombination in BJ5183 bacteria. Recombinant plasmids were screened by alteration of antibiotic. The adenovirus vector was then packaged and amplified in 293 cells. The expression of TIMP-2 was detected by the techniques of Western blot and RT-PCR. Results The recombinant adenoviral vector carrying human TIMP-2 was constructed. The titer was 4×10 11pfu/ml after purification. The expression of TIMP-2 gene in 293 cells was detected by RT-PCR. After the 293 cells were transfected with AdhTIMP-2 24 hours, TIMP-2 protein could be detected in the medium by Western blot. Conclusions The recombinant adenoviral vector carrying human TIMP-2 is successfully constructed and paved the way for further application in vascular disease gene therapy.