rAAV 2-GP Ⅱ b - GP Ⅲ a vector and in vitro expression functional experiments.docVIP

 PAGE \* MERGEFORMAT 35 rAAV 2-GP Ⅱ b / GP Ⅲ a vector and in vitro expression functional experiments Author: Wang Kai, Peng Jianqiang, Chen Fang-ping, Wu Xiaobing Abstract To study the rAAV vector-mediated gene therapy for Glanzmann the feasibility of building regulation and control by the CMV promoter of the rAAV2-Ⅱ b, rAAV2-Ⅲ a viral vector, and verified using a variety of ways rAAV2-Ⅱ b and rAAV2 - Ⅲ a viral vector in eukaryotic cells, the expression of capabilities, including detection by RT-RCR method of BHK-21 cells infected 24 hours (MOI = 1 × 105 vg / cell) after the target gene expression at the mRNA level, using flow cytometry technique used to detect gene expression of different MOI and purpose of the dose-effect relationship between the use of Western blot method to detect infection of BHK-21 cells after 48 hours (MOI = 1 × 105 vg / cell) gene expression at the protein level, with immunofluorescence assay BHK-21 cells 48 hours after infection (MOI = 105 vg / cell) gene expression at the cell surface, and the application of PAC-I binding assay validate the expression of GP Ⅱ b / Ⅲ a protein function. The results showed that MOI = 1 × 105 vg / cell conditions, in BHK-21 cells 24 hours after infection can be detected at the mRNA level of target gene expression, a 48-hour protein levels detected in the expression of target genes ; in a certain dose range of target gene expression increases with the MOI, the MOI is too high the contrary, the expression decreased after the activation of target gene expression product with the PAC-I binding, and has a normal biological activity. Conclusion: rAAV2 vectors can effectively mediate GP Ⅱ b, GP Ⅲ a gene expression in eukaryotic cells, the expression of the product has a normal biological activity, this result is rAAV2 vectors in gene therapy of Glanzmann laid the foundation for the application. Keywords: adeno-associated virus vector Construction of rAAV2-GP Ⅱ b / Ⅲ a Vector and Test