rAAV 2-GPⅡb/GPⅢa载体构建和体外表达、功能实验

为了研究rAAV载体介导的血小板无力症基因治疗的可行性,构建了由CMV启动子调控的rAAV2-Ⅱb、rAAV2-Ⅲa病毒载体,并采用多种方法验证了rAAV2-Ⅱb和rAAV2-Ⅲa病毒载体在真核细胞中的表达能力,其中包括用RT-RCR方法检测BHK-21细胞感染24小时(MOI=1×105 v.g/cell)后目的基因在mRNA水平的表达,用流式细胞术的方法检测不同MOI和目的基因表达之间的量效关系,采用Western blot的方法检测BHK-21细胞感染48小时后(MOI=1×105v.g/cell)目的基因在蛋白水平的表达,用免疫荧光法检测BHK-21细胞感染48小时后(MOI=105 v.g/cell)目的基因在细胞表面的表达,以及应用PAC-Ⅰ结合试验验证所表达的GPⅡb/Ⅲa蛋白功能.结果表明,在MOI=1×105v.g/cell条件下,在BHK-21细胞感染24小时后即可在mRNA水平上检测到目的基因的表达,在48小时可在蛋白水平检测到目的基因的表达;在一定剂量范围内目的基因的表达量随着MOI的增加而增加,但MOI过高反而使表达量下降,目的基因表达产物活化之后能与PAC-I结合,并具有正常的生物学活性.结论rAAV2载体能有效地介导GPⅡb、GPⅢa基因在真核细胞内表达,所表达的产物都具有正常的生物学活性,此结果为rAAV2载体在血小板无力症基因治疗中的应用打下了基础.

Construction of rAAV2-GP Ⅱ b/Ⅲ a Vector and Test of Its Expression and Function In Vitro

This study was aimed to explore the possibility of rAAV2 vector-mediating gene therapy for Glanzmann' s thrombasthenia. The rAAV2-GPIIb/IIIa vector was constructed. The GPIIb/IIIa gene expression in mammal cell were examined by different methods, such as: detection of mRNA expression in BHK-21 cells after 24 hours of infection (MOI = 1 x 10(5) v.g/cell) was performed by RT-PCR; the relation between MOI and quantity of GPII6/IIIa gene expression was detected by FACS after 48 hours of infection; GPIIb/IIIa protein expression in BHK-21 cells after 48 hours of infection (MOI = 10(5) v x g/cell) was assayed by Western blot, GPIIb/IIIa protein expression on cell surface was detected by immunofluorescence, and the biological function of expressing product was determined by PAC-1 conjunct experiments. The results showed that GPIIb/IIIa gene expression in mRNA level could be detected in BHK-21 cells after 24 hours of infection at MOI = 1 x 10(5) v x g/cell and the GPIIb/IIIa gene expression in protein level could be detected in BHK-21 cells after 48 hours of infection at MOI = 1 x 10(5) v x g/cell. In certain range, quantity of GPIIb/IIIa gene expression increased with MOI, but overdose of MOI decreased quantity of GPIIb/IIIa gene expression. Activated product of GPIIb/IIIa gene expression could combined with PAC-I, and possesed normal biological function. In conclusion, rAAV2 vactor can effectively mediate GPIIb and GPIIIa gene expressing in mammal cells, and the products of these genes exhibit biological function. This result may provide a basis for application of rAAV2 vector in Glanzmann's thrombasthenia gene therapy in furture.