成骨细胞特异性转录因子Cbfa1重组腺病毒质粒的构建与鉴定

摘要

Objective: To construct the recombinant adenovirus expressing the osteoblast specific transcription fac-tor Cbfa1(core binding factor alpha 1) for gene therapy of osteonecrosis of the femeral head, fracture and bone de-fect. Methods: The Cbfa1 gene fragment was amplified by PCR according to the cDNA of Cbfa1, then the frag-ment was cloned to pShuttle-CMV. The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in E.coli BJ5183. The candidate clone was further analyzed by restriction endonuclease digestion, PCR, and se-quence scan. Then the recombined adenovirus was transfected into HEK293 cells for packaging and amplifying. Ad-Easy1/Cbfa1 was purified by CsCl banding. Infection titer and rate were monitored by green fluorescent protein expression. The protein expression of Cbfa1 was detected by Western-blotting after AdEasy1/Cbfa1 infected mesen-chymal stem cell(MSC). Results: Sequence scan, restriction endonuclease, and PCR confirmed that Cbfa1 was cloned to the adenovirus vector successfully. The protein expression of Cbfa1 was detected after AdEasy1/Cbfa1 in-fected MSC by Western-blotting. Conclusion: The recombined adenovirus AdEasy1/Cbfa1 was constructed success-fully, which could enhance the ability of MSC differentiation to osteoblast, and will be benefit for treatment of os-teonecrosis of femoral head, fracture and bone defect in the future.%目的：构建成骨细胞特异性转录因子--核心结合因子α1（Cbfa1）重组腺病毒载体，为后期应用Cbfa1基因治疗股骨头坏死、骨折和骨缺损等疾病奠定基础。方法：以目的基因Cbfa1全长cDNA为模板进行PCR扩增，将扩增产物克隆到pShuttle-CMV载体的相应酶切位点，获得目的基因载体pShuttle-CMV-Cbfa1，在大肠杆菌BJ5183中和pAdEasy-1同源重组，筛选阳性克隆，经酶切、PCR及测序鉴定，线性化后用脂质体法转染HEK293细胞进行包装、扩增，用报告基因GFP对病毒滴度和感染效率进行监测，酚氯仿抽提纯化病毒，AdEasy1/Cbfa1感染间充质干细胞（MSC）后检测Cbfa1的表达。结果：测序、酶切及PCR证实Cbfa1基因重组腺病毒载体构建成功，AdEasy1/Cbfa1感染MSC后Cbfa1的表达显著升高。结论：构建了含Cbfa1基因的重组腺病毒载体，该基因能促进MSC向成骨细胞分化，预示其可作为治疗基因应用于股骨头坏死、骨折和骨缺损的治疗。