Abstract：Abstract Porcine contagious pleuropneumonia (PCP) is a serious respiratory disease with great harm to pigs (Sus scrofa), caused by Actinobacillus pleuropneumoniae (APP) infection. It is one of the major epidemic diseases in the world pig industry. Existing vaccines can not give full protection against App infection, so it is necessary to screening new vaccines. In this study, immune protection of recombinant NLPI (rNLPI)vaccination against App challenged infection was detected. Based on APP nlpi sequences in GenBank, the primers were designed. APP nlpi sequence was obtained by PCR from APP genome. Sequence analysis and function predication was made by bioinformatics software. The results revealed that the length of this gene (GenBank No.: MH027649) was 900 bp and its predicat protein was a 299 amino acid resides of an puptative outer membrane lipoprotein. Homology analysis showed that it shared high sequence similarity between different serotypes of APP. The APP nlpi was inserted into pET32a (+). Recombinant protein rNLPI was produced in Escherichia coli BL21 after induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and then determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The 34 kD-protein was detected and showed good immunogenicity. The protective efficacy of rNLPI experiment was tested in 6-week-old specific-pathogen-free BALB/c mice (Mus musculus). The serum immunoglobulin G (IgG) level was assayed by enzyme-linked immune sorbent assay (ELISA). The results showed that 50 μg rNLPI+Frund's incompleted adjuvant, 30 μg rNLPI+ Frund's incompleted adjuvant and 10 μg rNLPI+Frund's incompleted adjuvant and 30 μg rNLPI respectively induced 41%, 22%, 10% and 20% survival rate. No mice survived the challenge in adjuvant group and control group. The rest living mice of experiment groups slowly returned to normal health status. From above, rNLPI induced a partial immune protection of mice against App challenged infection. The study accumulated data for exploring new vaccines of porcine contagious pleuropneumonia.
