Construction and identification of an adeno-associated viral vector serotype 2/9 containing human thyrotropin receptor A subunit / 西安交通大学学报(医学版)

ABSTRACT

Objective: To construct an adeno-associated viral vector serotype2/9 containing human thyrotropin receptor (rAAV2-9-hTSHR289-IRES-ZsGreen) so as to provide a better means for establishing an ideal animal model and the gene prevention against Graves' disease. Methods: AAV skeleton plasmid pAAV-IRES-ZsGreen and PDC315 plasmid containing hTSHR289 were digested by EcoR+BamH. The digestion products were connected. And pAAV-hTSHR289-IRES-ZsGreen vector was generated after transformation of JM109 by ligation products. The recombinant plasmid was identified by gel electrophoresis and sequencing. Three plasmids (pAAV-hTSHR289-IRES-ZsGreen, pHelper, and pAAV-2/9) were transfected into 293AAV cell line by calcium phosphate method and a large amount of rAAV2/9-hTSHR289-IRES-ZsGreen was obtained. After purification, the packaging efficiency of recombinant adeno-associated virus was observed by using fluorescence microscope, and its titer was determined by rQ-PCR. HEK293 cells were transfected with rAAV2/9-hTSHR289-IRES-ZsGreen; then the expression level of hTSHR289 was analyzed by ELISA at different time points. Results: The success of hTSHR289 gene inserted into the AAV skeleton vector was confirmed by double digestion and sequencing. After 72 h of transfection of 293AAV cells, the packaging efficiency of the virus was 92%-94% under fluorescence microscope. The titer of the recombinant adeno-associated virus was 1×1013vg/mL. ELISA assay showed that the expression level of hTSHR289 protein mediated by rAAV2/9 was significantly higher at 96 h than that at 72 h and 48 h. Conclusion: The rAAV2/9-hTSHR289-IRES-ZsGreen was successfully constructed. Furthermore, it could be transfected and expressed in cells effectively.