摘要
构建含有人microdystrophin基因的重组腺病毒,来感染dystrophin基因敲除小鼠mdx的骨髓间充质干细胞(MSC)进行基因修饰,为同种异体基因修饰的干细胞移植治疗DMD疾病奠定基础。用NotⅠ酶切含microdystrophin基因的pBSK-MICRO质粒,获得microdystrophin基因。片段回收后定向插入腺病毒穿梭质粒pShuttle-CMV,获得重组质粒pShuttle-CMV-MICRO。PmeⅠ线性化重组质粒pShuttle-CMV-MICRO,去磷酸化后回收后与腺病毒骨架质粒pAdeasy-1共电转化BJ5183感受态细胞。同源重组后用选择性培养基筛选阳性克隆,提取质粒,用脂质体介导转染293细胞,通过观察293细胞病变及PCR扩增目的基因等方法鉴定重组的腺病毒。然后将病毒上清转染DMD模型鼠mdx小鼠的骨髓间充质干细胞,通过RT-PCR以及间接免疫荧光检测microdystrophin的转录及蛋白表达。成功构建了含有microdystrophin基因的重组腺病毒,病毒滴度为5·58×1012vp/mL。间接免疫荧光检测可见microdystrophin蛋白在mdx小鼠MSCs中高效表达。该重组腺病毒载体的构建及成功转染到mdxMSCs内表达为下一步用microdystrophin基因修饰的mdxMSCs进行同种异体移植治疗DMD疾病奠定了基础。
Construction of recombinant adenovirus, which contain human mierodystrophin, and then transfeetion into mesenehymal cells （MSCs） of mdx mice were done, and genetically-corrected isogenie MSCs were acquired; the MSCs transplantation into the mdx mice was then done to treat the Duehenne muscular dystrophy（DMD）. Mierodystrophin cDNA was obtained from recombinant plasmid pBSK-MICRO digested with restrictive endonuelease Not Ⅰ; the production was inserted directionally into pShuttle-CMV. The plasmid of pShuttle-CMV-MICRO was digested by Pme Ⅰ, the fragment containing microdystrophin was reclaimed and transfected into E. coli BJ5183 with plasmid pAdeasy-1. After screening by selected media, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by observing the CPE of cells and by the PCR method. Finally, MSCs of mdx mice were infected with the culture media containing recombinant adenovirus, and the expression of microdystrophin was detected by RT-PCR and immunocytochemistry. Recombinant adenovirus including microdystrophin was constructed successfully and the titer of recombinant adenovirus was about 5.58×10^12 vp/mL. The recombinant adenovirus could infect MSC of mdx mice and microdystrophin could be expressed in the MSC of mdx mice. Recombinant adenovirus including microdystrophin was constructed successfully, and the microdystrophin was expressed in the MSC of mdx mice. This lays the foundation for the further study of microdystrophin as a target gene to correct the dystrophin - defected MSC for stem cell transplantation to cure DMD.
出处
《生物工程学报》
CAS
CSCD
北大核心
2007年第1期27-32,共6页
Chinese Journal of Biotechnology
基金
国家自然科学基金(No.30170337)
教育部博士点基金(No.20030558058)资助~~
