Ⅱ型人多巴胺受体逆转录病毒载体的构建及表达

RECONSTRUCTION AND EXPRESSION OF RETROVIRUS VECTOR WITH HUMAN D_2 RECEPTOR GENE

目的构建人全长D2R基因真核表达载体,研究D2R在帕金森病(PD)中的作用和机制。方法将D2RcDNA片段克隆于pLNCX2逆转录病毒载体,转染成纤维包装细胞系PT67,G418筛选单克隆后进行传代培养,通过原位杂交、免疫组织化学和Westernblot检测其表达效果。病毒上清再感染骨髓基质细胞(MSCs),免疫荧光检测D2R基因表达率。结果成功构建了逆转录病毒表达载体pLNCX2_D2R,原位杂交证实D2R基因在PT_67表达的阳性率为80%以上,免疫组织化学检测可见其蛋白分布在胞浆和胞膜,细胞裂解后Westernblot蛋白电泳可检测到约48·84kD的蛋白。经转染D2R基因的PT67细胞能分泌病毒并感染MSCs。结论逆转录病毒介导的D2R基因经PT67细胞包装后,能够产生病毒颗粒,收集含病毒颗粒的上清可进一步感染MSCs,这种经逆转录病毒介导的DR基因可用来作针对帕金森病进行基因治疗的研究。

Objective To construct the eukaryotic expression vector of human full-length D2 receptor gene for further study on role in PD. Methods cDNA fragment of D2 receptor was subcloned into retrovirus vector pLNCX2 which was used to transfected into the PT67 cell line. Cell clones were obtained through G418 selection with passage. D2R gene expression was detected with in situ hybridization, immunocytochemistry and Western blot assay. The packaging cell medium containing virus particles was collected and infected bone marrow stromal cells（MSCs） and the efficiency was measured with immunofluorescence. Results The retrovirus vector pLNCX2-D2R was constructed successfully. More than 80% PT67 cells expressed the target gene. Porteins were detected in plasm and cell membrane was lysed and proteins were harvested for Western blot assays. A fragment of 48.84kD length was manifested with Western blot. Virus particles were secreted from PT-67 cells and were used to further infect MSCs. Conclusion The cloned human D2 receptor gene was expressed in PT67 cells and MSCs was efficiently infected by the recombinant retrovirns, pLNCX2-D2 R could be used to delivery vehicle for gene therapy of PD.