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本研究目的在于构建携带GDF-5基因的重组腺病毒表达载体,并用其感染人骨髓来源的间充质干细胞(MSCs)研究GDF-5的表达。从含有人GDF-5基因核心序列的质粒pCMV-SPORT6上通过PCR扩增GDF-5,将其酶切连接到穿梭质粒pAdtrack-CMV上,在BJ5183中和骨架质粒pAdeasy-1同源重组,筛选阳性克隆并酶切鉴定,线性化后磷酸钙法转染入人胚肾293细胞中包装、扩增,得到含有GDF-5基因的重组腺病毒并测定其滴度。用收集的腺病毒感染靶细胞MSCs,在基因水平检测GDF-5的表达情况。结果表明,成功构建了含有GDF-5基因的重组腺病毒载体,病毒滴度为1×109PFU/ml。重组腺病毒能有效感染MSCs并表达目的基因。通过构建该腺病毒并感染人MSCs可得到能持续一定时间表达GDF-5蛋白的MSCs,为这种转基因的MSCs进一步修复组织奠定了基础。
The purpose of this study was to construct a recombinant adenovirus expression vector carrying GDF-5 gene and to investigate the expression of GDF-5 by infecting human bone marrow-derived mesenchymal stem cells (MSCs). GDF-5 was amplified by PCR from the plasmid pCMV-SPORT6 containing the human GDF-5 gene core sequence, ligated to the shuttle plasmid pAdtrack-CMV, homologously recombined with the backbone plasmid pAdeasy-1 in BJ5183, Positive clones were identified by restriction enzyme digestion. After linearization, calcium phosphate was transfected into human embryonic kidney 293 cells for packaging and amplification. The recombinant adenovirus containing GDF-5 gene was obtained and its titer was determined. Target cells MSCs were infected with the collected adenovirus to detect the expression of GDF-5 at the gene level. The results showed that the recombinant adenovirus vector containing GDF-5 gene was successfully constructed and the virus titer was 1 × 109 PFU / ml. Recombinant adenovirus can effectively infect MSCs and express the target gene. By constructing the adenovirus and infecting human MSCs, MSCs expressing GDF-5 protein for a certain period of time can be obtained, which lays the foundation for the further repair of the transgenic MSCs.