Isolation And Identification Of Mycoplasma Gallisepticum Adhesin Interacting Protenins

Mycoplasma gallisepticum（MG） is a pathogenic bacteria of chicken Chronic respiratory disease（CRD）.It is widespread to exist in the world and causes heavier economic loss in poultry industry.Many researches show that MG combines with chicken respiratory mucosa superficial acceptor through superficial protein mycoplasma gallisepticum AdheA-In（pMGA） to infect host,so one of the important channels to prevent and cure CRD is to block the combination of MG and acceptor.In order to isolate and identify the interacting protein of pMGA1.2,we apply virus overlay protein blot assay（VOPBA）,GST-pull down technology and immunoblot assay to accomplish.Through mass spectrum sequencing of pMGA1.2 interacting protein,we clone and express it,the protein interaction is ApoA-I by VOPBA and indirect Dot-ELISA to verify.ApoA-I carried out the bioinformatics analysis,cloning and expression of the domain containing part of the gene fragments.these results establish groundwork for further studying the mechanism of action of pMGA1.2 and receptor protein,and following as the results.1.Isolation and identification of pMGA1.2 interacting proteinWe apply virus overlay protein blot assay（VOPBA）,GST-pull down technology and immunoblot assay to isolate and identify receptor protein,identify amino acid composition through protein mass spectrum technology,and carry out bioinformatics analysis of it.The best reaction condition of VOPBA:transfer protein with damp-dry blotting,followed by sealing NC membrane in 5%skim milk（prepared by PBS, containing 0.02%sodium azide）;add 1 ml purified pMGA1.2,37℃for 1h.Add 1:100 dilution of chicken-anti-MG polyclonal antibody,37℃for 2h;add 1:500 dilution rabbit-anti-chicken Ig,room temperature for 1h;incubate in color reagent following washing NC membrane,and then observe one evident band with molecular weight about 30.6kD.It is evident by GST-pull down test that two obvious protein bands with molecular weight 92ku and 30.6kD respectively,coincident with VOPBA results.Through mass spectrum of 30.6kD protein,we infer the protein may be apolipoprotein A-I（apo-A-I）. 2.Clone,expression and identification of ApoA-I geneFirstly,design two pairs of primer according to polyclonal restriction enzyme cutting site of ApoA-I gene sequence and vector,attain ApoA-I gene from chicken trachealis total RNA by RT-PCR,and then clone it to pMD18-T.After enzyme cutting analysis and sequencing,we further subclone to prokaryotic expression vector pGEX-KG and eukaryotic expression vector pcDNA3.1.Rseults show that the study clones chicken ApoA-I gene with molecular weight 795bp successfully.Its sequence similarity is 99% after sequencing and alignmenting,and it has only two ribonucleotide changes which lead to one amine acid change.Constructed successful prokaryotic expression recombinant plasmid pGEX-KG-ApoA-I is transfered to E coli BL21（DE3） to induce expression through isopropylthio-b-D-galactoside（IPTG）.The expression level is highest with the induced temperature 34℃for 4 h,IPTG concentration 0.50～1.00mmol/L and pH 7.0～7.8.After GST purification by affinity column,purity is 97%.VOPBA results indicate no chromogenic band appears by pGEX-KG blank vector and obvious chromogenic band appears by expression protein.It is evident specific binding of acquired GST-ApoA-I fusion protein and purified pMGA1.2 protein.Constructed eukaryotic plasmid pcDNA3.1-ApoA-I is transfected to chick embryo fibroblast with lipidosome.Collect cells,extract RNA and identify transfected cell of apoA-I by PCR after cultivation for 48h and trypsinization.Extracted cell expressed protein are broken by ultrasonic waves and fixed to nitrocellulose filter after treatment. Employing purified pMGA1.2 as target protein,we detect the reaction of expressed protein in cells and pMGA1.2 by chicken MG positive serum and rat-anti-chicken IgG. Deep blue spot appears by indirect Dot-ELISA of cell extracted protein,and control groups which are untransfected cell protein group and corresponding buffer group have no spots,this explains that the combination of expressed ApoA-I protein in cells and pMGA1.2 is specific.3.Clone,expression of ApoA-I fragment gene（ApoA-I₂）Design two pairs of primer according to functional structural domain of apoA-I and polyclonal restriction enzyme cutting site analysed by bioinformatics,attain ApoA-I₂ gene fragment from chicken tracheal total RNA by RT-PCR,clone ApoA-I₂ fragment to pMD 18-T,and further subclone it to prokaryotic expression vector pGEX-KG following restriction enzyme cutting and sequencing.Results show that we clone ApoA-I gene fragment successfully.Sequence similarity of ApoA-I₂ with molecular weight 177bp is 100%through sequencing.Above-mentioned constructed successful prokaryotic expression recombinant plasmid pGEX-KG- ApoA-I₂ is transfered to E coli BL21（DE3）, and attain fusion protein GST- ApoA-I₂ induced by IPTG.The expression level of soluble fusion protein is highest with the induced temperature 28℃for 7 h,concentration of IPTG 0.50～0.75mmol/L and pH 7.0～7.8.After GST purification by affinity column, purity is 96%.VOPBA results indicate no chromogenic band appears by pGEX-KG blank vector and obvious chromogenic band by soluble expression protein.It illustrates specific binding of acquired GST-ApoA-I₂ fusion protein and purified pMGA1.2 protein,and further indicates the protein fragment contains functional structural domain conjuncted with pMGA1.2.