Immunogenicity of DNA vaccine expressing GP5 of porcine reproductive and respiratory syndrome virus fused with VP22 of bovine herpesvirus 1 / 生物工程学报

To enhance the immuogenicity of DNA vaccines expressing the GP5 protein of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the tegument protein VP22 (encoded by VP22 gene) of Bovine Herpesvirus 1 (BHV-1), which has been demonstrated to exhibit the unusual protein transduction property, was fused to N-terminus of GP5 of DNA vaccine construct pCI-ORF5M, resulting in pCI-VP22-ORF5M expressing VP22-GP5 fusion protein. The expression of VP22-GP5 fusion protein was confirmed by both indirect immunofluorescence assay (IFA) and Western blot. To investigate its immunogenicity, BALB/c mice were immunized with the fusion expression plasmid pCI-VP22-ORF5M and non-fusion expression plasmid pCI-ORF5M, respectively. The GP5-specific ELISA antibodies, neutralizing antibodies and lymphocyte proliferative responses were evaluated at various time points after primary immunization. The results showed that GP5-specific ELISA antibodies, neutralizing antibodies, and lymphocyte proliferative responses induced by DNA vaccine pCI-VP22-ORF5M were higher significantly than those of DNA vaccine pCI-ORF5M, indicating that fusion expression with BHV-1 VP22 significantly enhances the immuogenicity of DNA vaccine expressing the PRRSV GP5 protein, and that this strategy may also be useful to develop more efficient DNA vaccines against other pathogens.

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus / 生物工程学报

Pseudorabies virus (PRV), an alpha-herpesvirus, has been used as a vector for live-viral animal vaccines. The recombinant PRV TK- / gE- / GP5+, which expressing GP5 of PRRSV, is developed based on the PRV genetic-depleting vaccine-virus strain, TK- / gE- /LacZ+. However, this strain stimulated poorly the vaccinated animals to produce neutralizing antibodies against PRRSV. In order to develop a booster specific immunized response of the PRV recombinant, the ORF5 gene of PRRSV TK- / gE- / LacZ+ was substituted by a modified ORF5 gene, ORF5m. The resultant recombinant PRV, TK- /gE- / GP5m+, was verified by PCR, Southern blotting and Western blotting. TK- / gE- / GP5m+ and TK- / gE- / GP5+ expressed GP5 proteins were inoculated into balb/c mice to evaluate their immunogenicity. The results demonstrated that the amount of neutralization antibodies and cell-immunity responses induced by TK- / gE- /GP5m+ against PRRSV were higher than that of TK- / gE- / GP5+. This study indicated that the new recombinant PRV expressing the modified GP5m protein is a candidate for the development of bivalent genetic engineering vaccines against PRRSV and PRV.

Expression of GP5-M fusion protein of porcine reproductive and respiratory syndrone virus (PRRSV) and establishment of ELISA diagnose based on the recombinant fusion protein / 生物工程学报

The cDNA fragment encoding the truncated GP5 and the full-length M protein of Porcine Reproductive and Respiratory Syndrone Virus (PRRSV) were orderly fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pKG-56. After transformed into E. coli BL21 (DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-GP5-M fusion protein was expressed in high level. Western-blot was performed to confirm that the expressed fusion protein could specifically react with antiserum against PRRSV. The fusion protein was further purified and used as an antigen to establish a novel PRRSV ELISA diagnose assay (P56-ELISA). Comparison between P56-ELISA and the abroad kit IDEXX-ELISA showed the two methods had 94.1 percent agreement by detecting 205 serum samples, indicating that the indirect P56-ELISA was specific and sensitive. The correlation between virus neutralization antibody of the infected pigs (not convalescent pigs) and antibody response to the fusion protein GP5-M was further studied. The regression function analysis suggested that there was no significant correlation between ELISA antibody response (OD630 nm) to the fusion protein GP5-M in clinical serum and their specific neutralizing titers.