关键词: 副猪格拉瑟菌, 寡肽渗透酶, 过表达, 基因工程, 弱毒疫苗

Abstract:

The study aims to construct Glaesserella parasuis (G. parasuis) mutants overexpressing oligopeptide permease gene through improvement of its expression capacity, and to evaluate its immunogenicity and efficacity in mice. G. parasuis CF7066 strain being used as the parent strain, the overexpressing mutans were constructed via natural transformation and Flp-FRT markless mutation system. The OppA expression capacity of the overexpression strains was detected by WB, and then strains were induced by arabinose to excise Kan^R cassette and were cultured at high temperature to eliminated Flp expression plasmid. The mutant of ΔnanH :: oppA^R was selected for conductind immunization and challenge experiments in mice. Comprehensive evaluations were conducted on serum antibody levels, spleen lymphocyte proliferation and the murine survival rate. Three overexpressing mutans of ΔnanH :: oppA^R, ΔnanH :: oppA andΔnanH : : P_qseB-oppA were constructed. Because ΔnanH :: oppA^R had the highest capacity for expression of OppA, it wase used for murine immunization experiment. The rOppA-ELISA showed that the mice immunized with ΔnanH :: oppA^R strain produced the higher level of OppA antibody than CF7066 and ΔnanH, but lower than the commercial inactivated vaccine group. After being stimulated with rOppA, the number of lymphocyte proliferation were significantly larger than before immunization among the mice immunized with ΔnanH :: oppA^R and with CF7066(P < 0.05), but not in the mice immunized with ΔnanH, the inactivated vaccine and PBS control. The challenge experiment showed that the survival rate were 100% in the mice immunized with ΔnanH :: oppA^R and CF7066, and it was 75% in ΔnanH group. Three overexpressing mutants of G. parasuis were constructed, in which ΔnanH :: oppA^R has the highest expression capacity of OppA. Immunization and challenge experiments showed that ΔnanH :: oppA^R elicited murine antibody production and splenic lymphocyte proliferation specific for OppA protein, and simultaneously provided substantial protection against the wild strain CF7066. Thus, ΔnanH :: oppA^R possessed potential as an attenuated vaccine strain for G. parasuis.

Key words: Glaesserella parasuis, oligopeptide permease, overexpression, genetic engineering, attenuated vaccines