| The disease caused by Haemonchus contortus,a blood-feeding nematode of small ruminants,is of major economic importance worldwide.Four main drug classes are available for the treatment of this parasite,and drug resistance worms are spreading widly.It is highly desirable to discover new drug targets and vaccine candidates.During the lifecycle,the second-stage larva(L2)molts to third-stage larva(L3)and become infective.Once the L3 is ingested by the host,the worm undergoes an exsheathment process that marks the transition from the free-living stage to the parasitic stage.In this study,the differential protein expressions between L2 and L3,L3 and exsheathed L3(xL3)were explored.1.Identification of proteins differential expressed between free-living and activated third-stage larvae of Haemonchus contortusProteins extracted from free living L3 and xL3 were analyzed by two-dimensional differential gel electrophoresis(2D-DIGE).More than 2200 protein spots were recognized,and 124 of them was found to be differential expressed(Average Ratio of xL3/L3>1.5 or xL3/L3<-1.5,p<0.05).Among which,83 spots were up-regulated and 41 spots were down-regulated in xL3 when compared with L3.These 124 differential expressed spots were analyzed by Matrix-assisted Laser Desorption Ionization Time of Flight Mass Spectrometry(MALDI-TOF-MS)or MALDI-TOF-MS/MS and a total of 40 proteins were identified.To predict the functions of these identified proteins,they were assigned for gene ontology(GO)annotation.Results showed these proteins may involve in biological processes of reproduction,cellular component organization or biogenesis,multi-cellular organismal process,single-organism process,metabolic process,signaling,biological regulation,response to stimulus,cellular process,biological adhesion,growth,locomotion,localization,developmental process and multi-organism process.Kyoto Encyclopedia of Genes and Genomes(KEGG)annotations were also performed,the results were helpful for exploring the process of metabolism and signal transduction pathways.2.Identification of proteins differential expressed between the second-stage and the third-stage larvae of Haemonchus contortusProteins extracted from L2 and L3 were analyzed by two-dimensional differential gel electrophoresis(2D-DIGE).More than 2200 protein spots were recognized,and 110 of them was found to be differential expressed(Average Ratio of L2/L3>1.5 or L2/L3<-1.5,p<0.05).Among which,45 spots were up-regulated and 65 spots were down-regulated in L3 when compared with L2.These 110 differential expressed spots were analyzed by Matrix-assisted Laser Desorption Ionization Time of Flight Mass Spectrometry(MALDI-TOF-MS)or MALDI-TOF-MS/MS and a total of 44 proteins were identified.This study indicated that some key alterations taking place during the transition from L2 to L3,and some proteins involved in this process might be candidate antigens for vaccine development.3.Differential transcriptions of CPN60,Proteasome,Transthyretin and Peroxidase between free-living and activated third-stage larvae of Haemonchus contortusAccording to the sequences of CPN60,Proteasome,Transthyretin,Peroxidase and housekeeping gene β-tubulin of H.contortus,five pairs of genes primers were designed.We quantified the expression of L3 and xL3 by Real Time PCR.Results showed that the expression of Proteasome,Transt.hyretin and Peroxidase in xL3 were higher than L3 in gene level.While,the expression of CPN60 in xL3 were lower than L3 in gene level.4.cDNA Cloning and prokaryotic expression of CPN60,Proteasome,Transthyretin,Peroxidase(1293bp)of Haemonchus contortusFour gene fragments of 1710bp,696bp,411bp,1293bp were produced by RT-PCR,using four pair of primers based on the four sequences of H.contortus.The four fragments were cloned into pMD19-T vector.Then the four fragments were transferred into the vector pET-32a(+)of Escherichia coli BL21.SDS-PAGE analysis showed that the four recombinant proteins weght of 81 KDa,43.5 KDa,33 KDa and 65 KDa,respectively.Results of Western blot showed that all of the four recombinant proteins can be recognized by the serum from the goat infected with H.contortus. |
