Study On The Combined Immune Effect Of Apx Toxin And Outer Membrane Protein In Actinobacillus Pleuropneumonia

Porcine contagious pleuropneumonia(PCP)is a highly contagious respiratory disease caused by Actinobacillus pleuropneumonia(APP),which has caused significant economic losses worldwide.Therefore,this research project aims to screen out well-protected subunit vaccine antigens and their combinations through genetic engineering techniques and mouse immune challenge experiments,which are divided into the following three parts:1.To enhance APP toxin-like protein Apx In this study,the genetic engineering method was used to achieve the fusion expression of Apx I-C and Apx III-C with Escherichia coli heat-labile toxin type B subunit(LTB)in the form of inclusion bodies.Recombinant protein and ISA201 adjuvant were formulated into vaccinated mice,and peritoneal challenge was performed with LD₅₀ of 3 times the serotype 1APP-LY strain.The experimental results show that:LTB can improve the immune protection effect of Apx I-C protein,the protection rate increased from 83.3%to 100%;after the fusion expression of LTB and Apx III-C,the immune protection effect has not changed,and the protection rate is still 66.7%.2.Select the 6 protective antigens of the Apx toxin proteins LTB-Apx I-C,LTB-Apx III-C and the Omp outer membrane proteins LTB-APJL?0382,LTB-Irp,LTB-Rcp A and LTB-Omp D15 stored in the laboratory As research objects,Apx toxin group vaccine,Omp outer membrane protein group vaccine and Apx+Omp combination group vaccine were prepared separately,and mice were subjected to immune challenge protection experiments.According to the challenge protection results and antibody detection results,the best combination of antigens to be screened is LTB-Apx I-C,LTB-Apx III-C and LTB-Omp D15.3.Using bioinformatics methods to screen the Lam B sequence of APP's maltosporin gene,a prokaryotic expression vector was constructed for this gene and successfully expressed in the form of inclusion bodies.After the recombinant protein was formulated into a subunit vaccine,the mouse immune challenge test and a serum antibody detection test were carried out.The results showed that maltosporin has good immunogenicity and can stimulate mice to produce higher levels of antibodies.The protection rate for mice is 0%,but it significantly slows the morbidity of mice and prolongs the death time of mice.