Development Of Monoclonal Antibodies Against ChPLA2-IB Protein And Identification Of Antigenic Epitopes Recognized By MAbs

Secreted phospholipases A2(sPLA2)catalyze the hydrolysis of the glycerophospholipids at the sn-2 position,leading to release fatty acids and lysophospholipids.PLA2 is extensively spread in mammalian tissues,plants,bacteriaand insect venom.Amino acids sequence and physiological functions divide PLA2 into three major groups:secretory PLA2(sPLA2),cytosolic PLA2(cPLA2)and calcium-independent PLA2(iPLA2).sPLA2 play a role in inflammatory;cPLA2 were involved in signal transduction,while iPLA2 were involved in cell death and neurodegenerative processes.sPLA2-IB is one of the best-known secret PLA2.sPLA2-IB has been isolated from the pancreas and characterized in various mammal species.In contrast,few studies have been performed on the chicken PLA2-IB(ChPLA2-IB).In this study,ChPLA2-IB encoding gene was digested with Nde I/EcoR I and inseted into the prokaryotic expression vector pET-11b(+)to construct the plasmid pET-11b-ChPLA2-IB.The recombinant plasmid was introduced into E.coli BL21(DE3).The ChPLA2-IB protein was expressed in inclusion bodies at the expected molecular weight(18kDa)analyzed by SDS-PAGE and Western blot.Similarly,the eukaryotic expression plasmid pLVX-AcGFP1-N1-ChPLA2-IB was obtained using Xho I/BamH I.The plasmid was transfected into 293T cell to provide amaterial for screening of monoclonal antibodies(MAbs)in indirect immunoinfluscent assay(IFA).6-8 weeks old BALB/c mice were injected with 75 μg of purified ChPLA2-IB in Freund’s adjuvant.The mouse serum antibody specific to ChPLA2-IB was determined by ELISA and reached at a liter of 1:51200.Based on hydridoma technique,four hybridoma cell lines secrecting MAbs against ChPLA2-IB were selected by ELISA,Western blot and IFA,and designated as 1D1,2E1,3G9 and 4C3.The MAb immune globin(Ig)subclass identification kit was used for Ig subclass identification.The results indicated the MAbs 1D1 and 2E1 belonged to IgG1 while the MAbs 3G9 and 4C3 were IgG2b.The light chain subtypes of 2E1,3G9 and 4C3 belonged to κ chain and 1D1 belonged to λ chain.The titers of the four hydridoma supernatants reached 1:6400 determined by ELISA.Besides,the ascite titer of 1D1 and 4C3 was 1:204800 while that of 2E1 and 3G9 was 1:102400.A series of overlapping peptides were constructed.Truncated ChPLA2-IB DNA fragment were cloned into pET-11b(+)at Nde I/EcoR I restriction enzyme sites and expressed in E.coli BL21(DE3).Epitope recognized by MAbs was analyzed by Western blot.The results showed that the epitope of MAbs 2E1,3G9 and 4C3 was loacated on 101 DEEITC106.However,the epitope of MAb 1D1 was situated on 139RLDKKK144.Provided that sPLA2 are involved in inflammatory processes,the expression of sPLA2 at both transcription and translation level were evaluated in chickenchallenged with avian pathogenic Escherichia coli(APEC)strain E058.The results suggested that the expression of ChPLA2-IB at transcription level was upregulated in heart,kidney and jejunum of birds post APCE infection.By contrast,we noticed a down regulation of ChPLA2-IB in lungs and spleen of chicken post APEC infection at transcription level.Western blot analysis showed that Ch PLA2-IB protein only expressed in the pancreas of chicken,line with the ChPLA2-IB transcription level determined by quantitative real time PCR(qPCR).