Screen Of Heterologous Protective Antigen Peptide Of Actinobacillus Pleuropneumoniae And The Expression Of Apxiv In Vitro

Actinobacillus pleuropneumoniae is the causative agent of acute and chronicpleuroneumonia that is responsible for substantial morbidity and mortality in the pigindustry. New improved vaccines that can protect against all serotypes and preventcolonization are required. In a previous study we showed that whole cells ofPropionibacterium acnes protected pigs from A. pleuropneumoniae serotype1and5and, there are common antigens between P.acnes and A.pleuropneumoniae, therefore,the basis for a promising heterologous vaccine. The aim of this study was to analysisthose protein antigens of P. acnes responsible for protection against A.pleuropneumoniae infection. Six P. acnes protein antigens that were recognized duringour priory study by sera raised against A. pleuropneumoniae were identified by2-DEand immunoblotting. Recombinant versions of all P. acnes proteins gave partialprotection (10-80%) against A. pleuropneumoniae serotype1and/or5infection in amouse challenge model. The best protection (80%serotype1;60%serotype5) wasobtained using recombinant P. acnes single-stranded DNA-binding protein. In part,protection against A. pleuropneumoniae infection may be mediated by small peptidesequences present in P. acnes single-stranded DNA-binding protein that arecross-reactive with those present in the A. pleuropneumoniae-specific RTX toxinApxIV and the zinc-binding protein ZnuA. The results suggest that P. acnes may be auseful vaccine to protect against different serotypes ofA. pleuropneumoniae.Nanoluc is a newly reported luciferase which catalysis furimazine substrate andglow luminescence100times stronger than commonly used firefly luciferase. It couldbe used as a reporter in mammalian cell. In this study, we are trying to integrate theNluc into of Actinobacillus pleuropneumoniae serotype15(HS143) genome under thecontrol of sodC promoter by construct a homologous recombination reporter plasmid and a mutant strain to evaluate if this new luciferase could expression and detect in A.pleuropneumoniae. Mutant strain APPS15::SodCNK cells PBS dilution andfurimazine substrate solution mixture could glow luminescence and detect by96wellsplate reader. Detect limitation is500CFU per well (1:50diluted substrate) or1:5,000diluted substrate (5*107CFU per well). Luminescence signal count will slowlyclimbed to the top in120minutes after sample loaded with NanoGlo buffer dilutedsubstrate. Another METK buffer diluted substrate reaction reach to signal top in60minutes and the signal variation is bigger than NanoGlo buffer. Single colonies placedon agar plate sprayed with substrate solution could be detect with IVIS imagingsystem, available for detect reporter gene expression. Our results indicate thatNanoluc luciferase could express in A. pleuropneumoniae and the luminescence ofthat could be detected by plate reader (suspension cells) or IVIS imaging system(colony) after catalysis furimazine substrate.ApxIV is one of the four A. pleuropneumoniae RTX toxin, generally consideredApxIV is unique only in the in vivo expression of A. pleuropneumoniae toxins. Theexistence of ApxIV has played an important role on A. pleuropneumoniae virulenceeffecting. Recent studies reported by gene chip and two-dimensional electrophoresisand mass spectrometry in the detection of a large number of genes or proteins isdetected in vitro transcription and protein ApxIV exist, but not specifically for theexpression of ApxIV further understanding. To confirm this conclusion, we used A.pleuropneumoniae serotype1,8and15were cultured in vitro, using reversetranscription PCR, quantitative PCR, SDS-PAGE, Western Blot and MALDI massspectrometry and other methods, from transcription, protein level and theantigen-antibody reaction proved that the ApxIV expression under in vitro cultureconditions. A. pleuropneumoniae in vitro expression under different conditions isquite different, with the culture medium and the calcium ion concentration. Under theconditions of PPLO broth contains L-cysteine hydrochloride, L-cysteinehydrochloride, glucose, Tween-80,5mM calcium, it can be detected the highestApxIV protein bands. Different concentrations of calcium ions on ApxIV protein leveldetection have an important influence, but weaker effect on the transcriptional level. Therefore, we speculate that calcium may be an important regulatory factor ApxIVposttranslational modification.In this study, cloning and expression of the six common antigen proteins betweenA. pleuropneumoniae and P. acnes and screen out two small peptides fragment of aprotein is a potential vaccine candidate, from the preliminary interpretation of theprotein levels of heterologous P, acnes immune protection Actinobacilluspleuropneumoniae infection mechanism. For the first time verified for the newluciferase Nanoluc expression and work effectively in A. pleuropneumoniae, prove itcan be a regulation studies tool and provides a new and convenient research method.From multiple angles proved A. pleuropneumoniae important virulence factor ApxIVexpression in vitro, initially discussed its expression regulation mechanism for thefurther study of this virulence factor and some help on A. pleuropneumoniaepathogenicity study and vaccine development.