HSV-1 VP22及microdystrophin基因融合表达重组质粒的构建及其生物学特性的初步研究

目的 构建人单纯疱疹病毒1型(HSV-1)病毒蛋白22(VP22)及microdystrophin基因融合表达重组质粒,体外研究其表达及HSV-1 VP22介导的蛋白转导特性,为进一步利用此重组质粒治疗Duchenne型肌营养不良症(DMD)奠定基础.方法 从质粒pSINrep5-VP22中PCR扩增HSV-1 VP22全长基因.然后克隆至真核表达载体pAVX1中,构建重组质粒pAVP22:再用Not I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质粒pAVP22中,获得重组质粒pAVP22-MICDYS:然后将重组质粒pAVP2-MICDYS转染至小鼠成纤维细胞3T3细胞.通过RT-PCR、Western blot及间接免疫荧光检测microdystrophin基因的转录及蛋白表达;最后收集转染后3T3细胞上清,感染人间充质干细胞(MSCs),检测重组蛋白在人MSCs中的表达情况来分析HSV-1 VP22是否可以介导VP22-microdyslrophin融合蛋白的转导.结果 成功构建了含有HSV-1 VP22和microdystrophin基因的重组质粒.并在体外得到了很好表达:同时也证实HSV-1 VP22提高了microdystrophin基因在3T3细胞中的表达效率,并可介导VP22-mizrodystrophin蛋白从3T3细胞到人MSCS间的转导.结论 该重组质粒的构建及体外成功表达和蛋白转导特性的确定为下一步用该重组质粒进行DMD疾病治疗研究奠定了基础.

Construction of a recombinant plasmid expressing microdystrophin gene fused with HSV-1 VP22 and its biological characterization

Objective To construct a recombinant plasmid expressing human microdystrophin gene fused with VP22 ofhmnan herpes simplex virus 1(HSV-1),and investigate the expression of microdystrophin in vitro and the characteristics of VP22-mediated protein transduction.Methods Full length HSV-1 VP22 gene was amplified by PCR from the plasmid pSINrep5-VP22 and cloned into the eukatyotic expression vector pAVXl to conslruct recombinant plasmid pAVP22.Microdystrophin cDNA was obtained from the recombinant plasmid pBSK-MICRO digested with Not Ⅰ,and the product Was inserted into plasmid pAVP22 to construct the plasmid pAVP22-MICDYS. 3T3 cells were transfected with pAVP22-MICDYS,and the expression of microdystrophin was detected by RT-PCR,Western blotting and immtmocytochemislry.The supematant of 3T3 cells transfected with pAVP22-MICDYS were collected to infect human mesenchymal cells(MSCs),and the expression of the fusion protein VP22-microdystrophin in the cells was detected using immunohistochemistry to assess VP22-mediated protein transduction. Results The recombinant plasmid expressing microdystrophin-VP22 fusion gene capable of in vitro expression of the fusion protein was constructed successfully.VP22 was shown to enhance the expression efficiency of microdystrophin in 3T3 cells and transduce VP22-microdystrophin fusion protein from 3T3 cells to human MSCs. Conclusion The recombinant plasmid expressing microdystrophin-VP22 fusion gene with protein transduction capacity provides an important basis for further study of the fusion protein in treatment of Duchenne muscular dystrophy.