Comparative Proteomics Between Muscle Larvae And Newborn Larvae,Female And Male Adult Of Trichinella Spiralis

Trichinella spiralis is a zoonotic parasitic nematode.Humans and more than 150 kinds of animal could be infected by consumption of raw or insufficiently cooked meat.For this parasite,infective muscle larvae(ML),newborn larvae(NBL)and adult worms(Ad)were singificant stages in the life cycle.Female and male adults palyed important roles in generatiion of newborns.To identify and characterize the proteins that differential expressed between ML,NBL,female Ad and male Ad,label-free quantitation tequcniqe(a kind of method used in comparative proteomics)combined with bioinformatic analysis was perfomed in this paper.The results might provide a fundation for further studies of the biological functions on invasion,survival,and reproduction of T.spiralis.1.Comparative proteomics analysis of T.spiralis ML and NBL using label-free quantitationIn total,1936 proteins were identified from ML and NBL.By using T-test(both side)statical analysis,1180 proteins were found to be differential expressed between T.spiralis ML and NBL.Among which,395 proteins were unique(291 proteins in ML and 104 proteins in NBL)expressed.The other 785 proteins were significant differences(ratio>2.0 or ratio<0.5,and P value<0.05,in which,196 proteins have a differential ratio>10,22 proteins have a differential ratio>100).All of 1003 differentially expressed proteins were analysed by GO and KEGG annotation.Results showed that these proteins may involve in molecular function,biological process and cellular localization.574 proteins were mapped to 444 signal/metabolism pathways.2.Comparative proteomics analysis of T.spiralis female and male Ad using label-free quantitation1877 proteins were identified from female and male Ad totally.By using T-test(both side)statical analysis,279 proteins were found to be differential expressed between T.spiralis female and male Ad.Among which,92 proteins were unique(55 proteins in female Ad and 37 proteins in male Ad)expressed.The other 187 proteins were significant differences(ratio>2.0 or ratio<0.5,and P value<0.05,in which,5 proteins have a differential ratio>10).All of 139 differentially expressed proteins were analysed by GO and KEGG annotation.Results showed that these proteins may involve in molecular function,biological process and cellular localization.68 proteins were mapped to 101 signal/metabolism pathways.3.Clone,expression and biological characteristics of T.spiralis FBPTo study the biological characteristics of TsFBP,cDNA from T.spiralis was cloned and expressed.To amplify the target gene,a pair of gene specific primers was designed based on the TsFBP ORF,and a RT-PCR was performed.The PCR product was cloned into pMD19-T vector.Sequence analyzing showed that the ORF of TsFBP sized 1026bp and encoding 341 amino acids.Results of BLAST proved that the gene sequence was highly homology with that available on the GenBank.TsFBP gene was subcloned into pET-32a vector and transferred into E.coli BL21(DE3).To induce the expression,IPTG was added to the bacterial cultures.Results of SDS-PAGE showed that recombinant protein TsFBP was expressed in the insoluble body,sized 55.4kDa as a fusion protein.The result of western blot showed that recombinant TsFBP can be recognized by serum from T.spiralis infected mice.Enzyme catalytic experiment indicated that the optimum pH was 7.0 and the optimum temperature was 37? for the recombinant protein.