Xiao Shaobo

Development of a latex agglutination test using the major epitope domain of glycoprotein E of pseudorabies virus expressed in E. coli to differentiate between immune responses in pigs naturally infected or vaccinated with pseudorabies virus.

A 0.8 kb DNA fragment encoding the major epitope domain of glycoprotein E (gE) of pseudorabies virus (PRV) was inserted downstream of the T7 promoter of an expression vector, pET-28b, to yield the recombinant plasmid pETgE804. After induction by isopropy1-β-D-thiogalactopyranoside (IPTG), a high level expression of fusion protein was obtained. SDS-PAGE and western immunoblotting analysis showed that the fusion protein was 38 kDa and could bind with antisera against PRV. The protein existed mainly in the form of the inclusion body. After being denatured and renatured, the protein was used to prepare the latex antigen. The concentration of antigen, temperature and time for sensitization were optimized. The latex agglutination test (LAT) was able to differentiate sera of PRV-infected pigs from those of gE-deletion vaccine-immunized pigs. The diagnostic specificity and sensitivity of the developed gE latex agglutination test (gE-LAT) were also evaluated by using sets of sera. The diagnostic specificity and diagnostic sensitivity of the gE-LAT were 96.77% and 95.76%, respectively. For comparison between gE-LAT and a commercial blocking enzyme-linked immunosorbent assays (ELISA), 260 serum samples were tested. The coincidence frequency of both assays was 96.94% (252/260). No significant difference was found between the two methods (p>0.05). For comparison between the abilities of gE-LAT and gE-ELISA to detect sera with low titres of gE-specific antibody, 66 sera from 22 pigs were tested. The data indicate that the gE-LAT is of similar sensitivity to gE-ELISA. These results indicate that gE-LAT using recombinant gE might be very useful as a routine screening method for the differential diagnosis of PRV infection.

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus (PRV), an alpha-herpesvirus, has been developed as a live viral vector for animal vaccines. However, the PRV recombinant virus TK−/gE−/GP5+ expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV), based on the PRV genetically depleted vaccine strain TK−/gE−/LacZ+, scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV. To develop a booster-specific immune response of such PRV recombinants, the ORF5m gene (the modified ORF5 gene having better immune responses) was substituted for the ORF5 gene and introduced into PRV TK−/gE−/LacZ+, resulting in a PRV recombinant named TK−/gE−/GP5m+, which expressed the modified GP5m protein. The recombinant virus was confirmed using PCR, Southern blotting and Western blotting. TK−/gE−/GP5m+ and TK−/gE−/GP5+ expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses. The results indicated that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16) and cell immune responses induced by TK−/gE−/GP5m+ against PRRSV were higher than that induced by TK−/gE−/GP5+. Thus, the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV.