Immunoprotective Study Of GalT-GalU Soluble Recombinant Protein From Actinobacillus Pleuropneumoniae

Porcine contagious Pleuropneumoniae,caused by Actinobacillus pleuropneumoniae,is a highly infectious disease that has caused severe economic losses worldwide^[1,2].The most effective preventive measure against APP infection is vaccination,and the most widely used vaccine is the whole-cell inactivated vaccine.However,these vaccines can only protect against homologous serotypes,and there are many serotypes of APP.To develop a cross-protective vaccine against APP infection,this experiment prepared the APP GalT-GalU soluble recombinant protein and evaluated its immunogenicity and protective effect against virulent strains of multiple serotypes of APP.1.Research on the expression of soluble recombinant proteins of GalT-GalU in the APP.Firstly,the APP GalT-GalU plasmid was successfully constructed.The GalU gene of 888bp was amplified using APP DNA as a template,and a linearized vector p ET-28a-GalT of 6391bp was obtained by double enzyme digestion.The GalU fragment was connected with the linearized vector p ET-28a-GalT by homologous recombination to obtain the p ET-28a-GalT-GalU plasmid,with a size of 1950bp after fusion.Secondly,the APP GalT-GalU soluble protein was successfully expressed.The p ET-28a-GalT-GalU was transformed into BL21（DE3）competent cells and induced at a low temperature of 18°C for24 hours with IPTG after shaking the culture for 6 hours at 37°C.After breaking the cells using an ultrasonic disruptor,the supernatant was collected by centrifugation,and the protein was purified using a nickel column and ultrafiltration.The successful expression of the APP GalT-GalU soluble protein with a size of 73k Da was confirmed by SDS-PAGE analysis.2.Research on immunoprotection of soluble recombinant protein APP GalT-GalU.Firstly,it was confirmed that the purified APP GalT-GalU recombinant protein prepared in this study has good immunogenicity.After two immunizations with the purified soluble protein of APP GalT-GalU in 5-week-old female BALB/c mice,the results showed that the Ig G antibody titer in the serum of the mice was greatly increased.The GalT-GalU protein could specifically bind to APP2701,APP5b,and APP1516,and the proliferation rate of spleen lymphocytes stimulated by GalT-GalU was 51.5%.The cytokine levels of IL-4,IL-12,and IFN-γin the serum were significantly up-regulated,indicating that the APP GalT-GalU fusion protein has good antigenicity and can significantly activate humoral and cellular immunity in the body.Secondly,it was confirmed that the APP GalT-GalU recombinant protein has good multi-serotype immune cross-protection.In the cross-protection test against multiple serotypes of APP,GalT-GalU provided 62.5%,75%,and 87.5%resistant protection against infection of APP2701（serogroup 1）,APP5b（serogroup 5）,and APP1516（serogroup 7）,respectively.It Confirmed that GalT-GalU could provide effective immune cross-protection against infection of the three predominant serotypes（1,5,and 7）of bacterial strains in China.