Biochemical Characteristics Of Fasciola Hepatica CD59 Like Proteins

The tegumental proteins of trematodes react with host’s immune cells and antibodies directly and these proteins were taken to be potential vaccine candidates and targets of new drugs. In this thesis, we studied on the Fasciola hepatica (Fh) CD59-like protein which was identified from Fh tegumental protein named FhCD59-like-1 protein. Sequence analysis shown there are 11 different CD59-like proteins and two most important are FhCD59-like-1 and FhCD59-like-5, we performed the FhCD59-like-1 and FhCD59-like-5 transcriptomic cDNA cloning, sequencing and analysis, vitro recombinant eukaryotic expression and detecting the differential expression levels in new excysted juvenile (NEJ) and adult stage by Real time PCR. In addition, other six trematodes CD59-like sequences from different databases were found and analysed.A cDNA encoding a FhCD59-like-1 sequence was cloned and sequenced and shown to comprise a 366bp open reading frame, encoding a 122 amino acid residue putative with 10 cysteines and a conserved CD59/Ly-6 family motif "CCXXXXCN". Analysis of 34 cDNA sequences from two different adult flukes revealed a polymorphic family of FhCD59-like-1 proteins with 21 different cDNA sequences identified showing 22 polymorphic positions. The results of FhCD59-like-1 protein genomic DNA amplification, cloning and sequencing shown that there were two different Genomic DNAs transcripted FhCD59-like-1 protein RNA, one is 554bp without any introns and the other is 3437bp included three exons and two introns. Comparison of the polymorphic sites of transcriptomic cDNA and genomic DNA did not strongly support the RNA editing occurred in FhCD59-like-1 protein.In order to harvest different kinds of FhCD59-like proteins, a pattern searching was used to blast within three Fh RNA-seq databases and 11 different FhCD59-like proteins were found after analysis. Construction and analysis of phylogenetic tree shown FhCD59-like-1 and FhCD59-like-5 proteins related with Schistosoma mansoni two tegument surface-associated CD59-like proteins (CD59a and CD59b); Databases searching and analysis of CD59-like sequences revealed 5,6,8,9,8 and 3 different CD59-like sequences in Fasciola gigantica, Fasciolae magna, Schistosoma mansoni, Schistosoma japonicum, Clonorchis sinensis and Opishitorchis viverrini. All 50 CD59-like sequences from 7 different parasites were constructed an unroot phylogenetic tree by MrBayes based on the MAFFT alignment and all the sequences were divided into seven clades.Recombinant expression of FhCD59-like-1 and FhCD59-like-5 used pPICZa A vector and Pichia pastoris(P.pastoris). Both FhCD59-like-1 and FhCD59-like-5 like proteins were expressed and these proteins can reacted with anti-6×His monoclonal antibody which are bigger sizes than expected proteins and these expressed proteins can not be purified with Ni-NTA column. The FhCD59-like-1 expressed proteins can not do mass spectrometry sequencing cause they hard to reduction and trypsin digestion; Applying real time PCR to detect the differential expression of FhCD59-like-1 and FhCD59-like-5 in newly excystment juvenile (NEJ) and adult of Fh. Results shown that FhCD59-like-1 protein e expressed much lower level in NEJ stag than adult with ratio 0.0058:1, while FhCD59-5 was expressed higher lever in NEJ than adult with ration 15.42:1.In this study, at least 11 different CD59-like proteins were found in Fh, FhCD59-like-1 shown very high polymorphism. Recombinant expression and detected the differential expression of two most important proteins FhCD59-like-1 and FhCD59-like-5 in NEJ and adult. Databases searching and analysis confirmed there are different CD59-like proteins in F. gigantica, F. megna, S. mansoni, S. japonicum, C. sinensis and O. viverrini. The 50 different CD59-like proteins from 7 trematodes could be divided into 7 clades by phylogenetic analysis that indicated these CD59-like proteins should contained different functions. Our study firstly looking at these FhCD59-like proteins, set a foundation for the future functional research and application of these FhCD59-like proteins.