Constructions Of Recombinant-plasmid Library And Research On Pseudomonas Aeruginosa Vaccine Of Antigen Screening

ObjectivesAimed for overcoming the deficiency of the present antigen screening strategies,the objectives of this study are to construct genome-wide antigen screening library of Pseudomonas aeruginosa?PA?and to screen their novel candidate antigens,the process of which lay the foundation for the prevention and control of PA infection.Methods1.Construction of genome-wide library: The whole genome of PA XN-1 was extracted and digested into random fragments by Sau3 A I,which were then ligated into pMal-c5 x vector digested with BamH I by ligation mix,thus constructing random recombinant plasmids.The ligation product was transformed into X-blue competent E.coli for selection of random recombinants.Random recombinants were tested by PCR and plasmid digested so as to evaluate features of the genome fragments inserted.Furthermore,random recombinats were induced by adding IPTG and lysed by lysozyme.Lysis supernatants which contained MBP?maltose binding protein?fusion protein were collected and measured by SDS-PAGE.Thus a random library of recombinants from the whole genome of PA XN-1 wasconstructed.2.Screening of candidate protein antigens and evaluation of protection effect: Anti MBP polyclonal antibodies were coated on ELISA plate,and MBP fushion proteins expressed by recombinat plasmids were captured in order to further screen PA vaccine candidate antigens from the random library.After detection of unknown antigens reactivity of in the convalescent serum from PA patients infected,the DNA sequences were determined,and Sequence blast was performed to obtain amino acid information of the strongly reactive antigens.Candidate antigens were producted by E.coli through gengetic engineering methods,with which Balb/c mice were immunized.Enzyme linked immunosorbent assay?ELISA?was employed to measure the antibody tilter of antigen specific IgG.Lethal dose of PA XN-1 was challenged to mice by tracheal incubation,whose survival rate was calculated.The immunoreactivity,immunogenicity and immunoprotective effects of candidate antigens were evaluated comprehensively,and novel PA vaccine candidate antigens were screened out.Results1.After genome of PA XN-1 extracted,it was digested into random fragments by Sau3 A I,whose DNA bands were distributed between 100 and 1000bp.These DNA fragments were ligated to p Mal-c5 x vector to obtain a great number of random recombinant plasmids.Three recombinantplasmids were randomly selected,two of which were inserted with DNA fragments.The size of them was detected by PCR,and it was found that the inserted DNA fragments were distributed between 100 and 1000 bp with good randomness.IPTG was used to induce the expression of recombinant plasmids,and the protein expression of them was detected by SDS-PAGE,which suggested that 4 of the 6 recombinants selected at random express MBP fushion protein successfully.Thes results indicated that PA genome-wide library was successfully constructed,which can be used to screen highly reactive antigens for the next step.2.After 13-turn experiments,2392 recombinant plasmids were screened out and 115 reactive recombinants were obtained,among which were 13 strong reactive antigens?antigens that are more than 10% reaction to PcrV?.These 13 antigens were: PpiA?PtsP?OprP?CAZ10₃4235?HmuU₂?PcaK?CarAd?RecG?YjiR₅?LigD?KinB?RtcA?PscF.ConclusionThe whole genome-wide library of Pseudomonas Aeruginosa?clinical isolated XN-1?was constructed successfully for the first time in this study.Based on the library,thirteen strong reactive antigens were successfully screened immunologically,and finally PscF was identified as a potential candidate protein antigen of PA,as a result of the best immunogenicity and the highest protective rate among animal experiments,which would lay a solid foundation for the development of novel protein vaccines ofpseudomonas aeruginosa in the near future.