Porcine reproductive and respiratory syndrome is caused by the PRRS virus (PRRSV),which has six structural proteins (GP2,GP3,GP4,GP5,M and N). GP5 and N protein are important targets for serological detection by enzym...Porcine reproductive and respiratory syndrome is caused by the PRRS virus (PRRSV),which has six structural proteins (GP2,GP3,GP4,GP5,M and N). GP5 and N protein are important targets for serological detection by enzyme-linked immunosorbent assay (ELISA) and other methods. Toward this goal,we developed an indirect ELISA with recombinant GP5 antigens and this method was validated by comparison to the LSI PRRSV-Ab ELISA kit. The results indicated that the optimal concentration of coated recombinant antigen was 0.2 μg/well for a serum dilution of 1:40. The rate of agreement with the LSI PRRSV-Ab kit was 88.7% (266/300). These results support the potential use of recombinant GP5 as an antigen for indirect ELISA to detect PRRSV antibodies in pigs.展开更多
The GP5 gene which was spanned or truncated with hydrophobic signal peptide(tGP5) was amplified with RT-PCR and cloned into the downstream of promoter(CMV) of the pcDNA3.1,an eukaryotic expressing vector.The construct...The GP5 gene which was spanned or truncated with hydrophobic signal peptide(tGP5) was amplified with RT-PCR and cloned into the downstream of promoter(CMV) of the pcDNA3.1,an eukaryotic expressing vector.The constructed plasmids were transfected into HEK-293A cells with anion lipid transfection reagent.Subsequently,the expression of the mRNA of interest gene was analyzed and confirmed by RT-PCR,and the expression of target protein was confirmed by indirect immunofluorescent assay(IFA).Each mouse was immunized with 100 μg of naked plasmid by intradermal injection.All groups were immunized four times with interval of 15 days.The neutralizing antibody against PRRSV were induced,but the antibody of group immunized with tGP5 gene produced slowly and the titer was lower than that in the group immunized with GP5 gene.It showed that the signal peptide of the GP5 protein of PRRSV is associated with the neutralizing antibody level of DNA immunization of pertinent GP5 gene.展开更多
基金Specific public service sectors of agricultureresearch (200803020)
文摘Porcine reproductive and respiratory syndrome is caused by the PRRS virus (PRRSV),which has six structural proteins (GP2,GP3,GP4,GP5,M and N). GP5 and N protein are important targets for serological detection by enzyme-linked immunosorbent assay (ELISA) and other methods. Toward this goal,we developed an indirect ELISA with recombinant GP5 antigens and this method was validated by comparison to the LSI PRRSV-Ab ELISA kit. The results indicated that the optimal concentration of coated recombinant antigen was 0.2 μg/well for a serum dilution of 1:40. The rate of agreement with the LSI PRRSV-Ab kit was 88.7% (266/300). These results support the potential use of recombinant GP5 as an antigen for indirect ELISA to detect PRRSV antibodies in pigs.
文摘The GP5 gene which was spanned or truncated with hydrophobic signal peptide(tGP5) was amplified with RT-PCR and cloned into the downstream of promoter(CMV) of the pcDNA3.1,an eukaryotic expressing vector.The constructed plasmids were transfected into HEK-293A cells with anion lipid transfection reagent.Subsequently,the expression of the mRNA of interest gene was analyzed and confirmed by RT-PCR,and the expression of target protein was confirmed by indirect immunofluorescent assay(IFA).Each mouse was immunized with 100 μg of naked plasmid by intradermal injection.All groups were immunized four times with interval of 15 days.The neutralizing antibody against PRRSV were induced,but the antibody of group immunized with tGP5 gene produced slowly and the titer was lower than that in the group immunized with GP5 gene.It showed that the signal peptide of the GP5 protein of PRRSV is associated with the neutralizing antibody level of DNA immunization of pertinent GP5 gene.
