RESUMO
Objective: To investigate the expression and expression efficiency of recombinant adeno-associated viral containing human thyrotropin receptor A subunit (rAAV2/9-hTSHR289-IRES-ZS Green) in mouse embryonic fibroblasts (NIH3T3) or in mice. Methods: NIH3T3 was infected by rAAV2/9-hTSHR289-IRES-ZsGreen and its TSHR289 protein expression was detected by Western blotting. Female BALB/c mice were intramuscularly injected with different doses of rAAV2/9-hTSHR289-IRES-ZsGreen; the expressions of the target protein in different organs were determined by immunofluorescence while the serum TSHR antibody (TRAb) titer was determined by radioimmunoassay. Results: NIH3T3 cells transfected with rAAV2/9-hTSHR289-IRES-ZsGreen expressed hTSHR289 protein both at 48 h and 72 h. The expression of target protein at 48 h or 72 h was significantly higher than that at 24 h (P<0.05). There were different levels of TSHR289 expression in leg skeletal muscle, heart, liver and spleen under fluorescence microscope. The results of radioimmunoassay showed that the higher dose injection produced a higher titer of TSHR antibody, but only the TRAb level in high dose and control injection exhibited a statistical difference (P<0.05) at week 4, week 8 and week 12. Furthermore, strong antibody response was observed in the mice injected with high dose and medium dose of rAAV2/9-hTSHR289-IRES-ZsGreen at week 4 and gradually weakened between 4 and 8 weeks. In addition, the antibody lasted for 12 weeks. Results: rAAV2/9-hTSHR289-IRES-ZsGreen can highly express hTSHR289 protein in vivo and in vitro, and the hTSHR289 protein displays strong immunogenicity. It indicates that rAAV2/9-hTSHR289-IRES-ZsGreen might become a more effective means of preparing Graves' disease model.
RESUMO
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.