Immune Protective Efficiency Of Recombinant Actinobacillus Pleuropneumonia LOLA Against Chellenge Infection In Mice

Porcine Contagious Pleuropneumonia(PCP)is a respiratory disease caused by an Actinobacillus pleuropneumoniae(APP),a gram-negative bacterium.Transmission between pigs through contact level,It is one of the five major diseases in pig raising industry in the world.In clinic,the immunoprophylaxis is carried out by inactivated vaccine but it has the disadvantages of narrow antibacterial spectrum and no protection against lung diseases.So it is necessary to find new candidate vaccines.Our previous study found that LOLA of app is an outer membrane protein which may induce effective immune protection.According to the APP Lola gene sequence reported in GenBank,Design primer pairs for the protein.The Lola gene was amplified by PCR using the genome of APP serotype 1 as template,and the sequence was analyzed by bioinformatics.Discovery of the gene(GenBank login number is Bankit MH255942)Composed of 633 bp,it encodes a 210 amino acid protein with a theoretical molecular weight of 2 3458 Kd,pI of 9.70.The protein is an outer membrane lipoprotein and tertiary structure is a reticulated tubular structure consisting of a closed ?-bucket and a ?-helical cap structure composed of a hydrophobic cavity.The structure of the outer membrane is stable and the permeability of the membrane is related to the permeability of the membrane.Homology analysis showed that the coding protein of the gene had high homology among different APP serotypes,which suggested that it could produce good cross-immunological protective effect.the first 26 amino acids of the N-terminal of the protein are signal peptide regions.According to the nucleotide sequence of APPLola gene,Design primer pairs for the protein.The APP genome was used as template PCR to amplify APP Lola and subcloned into pET32a(+).The recombinant strain was transformed into BL21(DE3)and induced by IPTG to express APP rLOLA protein.SDS-PAGE analysis showed that the specific bands were found in 23 kD and West-blot detection showed that the rLOLA expressed by recombinant bacteria could be recognized by the corresponding positive serum.Experimental mice were randomly divided into 5 groups,10 rats in each group.group A.were normal saline control group,group B,Freund's incomplete adjuvant control group,group C,10?g rLOLA protein dosage group,group D,30?g rLOLA protein dosage group,group E,50?g rLOLA protein group,Freund's incomplete adjuvant(50 ? g rLOLA),Freund's incomplete adjuvant(30 ? g rLOLA)and Freund's incomplete adjuvant(10 ? g rLOLA)were immunized subcutaneously in 0,2,and 4 weeks.Three times in total.The level of blood antibody was detected by ELISA before immunization and before infection.Two weeks after the third immunization,each mouse was injected intraperitoneally with Actinobacillus pleuropneumoniae LD50)0.2 ml.The results showed that Freund's incomplete adjuvant +50 mu g rLOLA,Freund's incomplete adjuvant + 30 g rLOLA,Freund's incomplete adjuvant + 10 rLOLA g,30 ug rLOLA could respectively induce mice to produce 40%,20%,10% and 20% survival rates,and the adjuvant group and saline control group all died.The clinical status of the surviving mice gradually returned to normal and began to feed.The results showed that subcutaneous injection of rLOLA could induce immune response and higher antibody titer in mice.The study provides basic information for further screening the molecular vaccine of porcine contagious pleuropneumonia.