The Disvovery Of Type 1 Phosphodiesterase From Riemerella Anatipestifer And The Cloning And Expression And Its Applicable Study Of PDE1 Gene

A positive clone which was identified by blue/white selection in Expression DNA Library of CH-1 Strain of Riemerella anatipestifer Serotype 1 was screened by in situ Hybridization using anti-RA rabbit sera.After five rounds of immunoscreening,in vivo excision of a positive phage plague,restriction enzyme digestion analysis and sequencing,an insert sequence of 4434bp was obtained.The sequence was the submitted to GenBank with accession number of EF030421.BLASTN,BLASTP and ORF Finder provided by American National Center for Biotechnology information(NCBI) and FASTA provided by the European Molecular Biology Laboratory were applied to analyze one of the three Open Reading Frames which was denominated ORF1689.The result displayed that the complete ORF contains 1689bp and encodes 562 amino acids.The molecular weight is 59.5kD and the theoretical pI is 5.82. Ribosome Binding Site(RBS) was found in the front of the initiator.The gene contains a conserved domain of phosphodiest.And the amino acid sequence deduced from ORF displayed homologies of 49.283%,38.716%,38.693%and38.313%to those from Caulobacter sp.K31,Sphingomonas sp.,Sphingopyxis alaskensis and Novosphingobium aromaticivorans,so the gene is a new of Type 1 phosphodiesterase genes.A pair primer was designed according to nucleotide sequence of RA serotype 1 which was submitted to GenBank with accession number of EF030421.The PDE1 gene was obtained by PCR with the RA serotype 1 isolated from ducks as template and cloned into pMD18-T vector.It was identified by PCR,restriction endonuclease analysis,DNA sequencing and then subcloned into BamHâ… /Hindâ…¢site of prokaryotic expression vector pET-32a(+) and recombinant expression plasmid pET-32a-PDE1 was constructed successfully. The plasmid was transformed into Eschercha coli BL21(DE3) and 80kD PDE1 recombinant protein was expressed with IPTG inducer.The expression product were mainly non-soluble inclusion body.The expression conditions were determined by optimizing induction temperature,induction time and concentration of IPTG.The recombinant protein was purified by Ni-NTA Superflow affinity chromatograghy and used to prepare the polyclone antibody.The results of immunoblotting test showed that the recombinant protein had good immnuogenicity with using anti-RA rabbit sera.In order to study the function of the gene,the polyclone antibody was purified by High-Q affinity chromatograghy and its antibody titer was detected by an indirect ELISA method.An indirect ELISA method detecting RA infection was established based on PDE1 recombinant protein as coating antigen.It is positive when the OD₄₅₀＞0.271,and it is negative when OD₄₅₀≤0.271.The result showed that the method can differentiate RA infection and other infections,such as Duck plague virus infection,Duck hepatitis B virus infection, Duck virus hepatitis infection,Duck backwoods coli infection,Duck salmonella bacillus infection.The method can be applied in diagnosis of and detection of RA.The indirect immunoperoxidase staining technique and the indirect fluorescent antibody technique of RA were constructed by using anti-PDE1 serum.The two methods can detect RA in the paraffin wax tissue splice distinctively.The tissues of ducklings infected with Duck plague virus,Duck avian influenza virus,Duck virus hepatitis,Duck backwoods coli,Duck salmonella bacillus,Duck pasteurella multocidahas not shown positive results.The tissues of artificial infected ducklings showed specific and positive results.RA antigen could be detected in the heart,Iiver,spleen,lung,pancreas kidney,bursa of Fabricius,Harderian gland,thymus, brain,duodenum,rectum and cecum of the infected ducklings.It may be used to diagnosis of RA infection and for detecting the distribution of RA antigen.The distribution of RA antigen detected by the two methods is identical.These results proved that the PDE1 gene was a characteristic gene of RA.And the function of the PDE1 gene played an important role of the RA's metabolism and RA infection.Therefore,the PDE1 gene was a target gene which cures RA infection.The result of western blotting showed that the anti-PDE1 serum specific binding with the recombinant protein.The PDE1 recombinant protein was prepared as vaccine with Freud's complete adjuvant.The duckings were immunized with the vaccine at 7 and 14-old-day respectively.Two weeks after the last immunized,the antibody titer of duck serum was detected agglutination test and the ducklings were challenged with 10⁹pFU enteropathogenic RA Serotype 1 CH-1 strain.The immunoprotection effect of PDE1 recombinant protein vaccine was evaluated according to the mortality,re-isolated rate of RA and grades of pathological changes.The results show that the antibody titer detected by agglutination test is 1:32 in the group of PDE1 recombinant protein vaccine.The results showed that PDE1 recombinant protein has some immunoprotection effect with challenging of virulent strain of RA CH-1.It suggested to be a qualified candidate of subunit vaccine.