Analysis Of Antigens From Cryptocaryon Irritans Based On Transcriptomics And Proteomics Technique

Cryptocaryon irritans is a parasitic ciliate causing severe cryptocaryoniasis or “wihte spots disease” of culturled marine fish with heavy economic loss for commercial aquaculture in southeast coast of China. Immunoprophylaxis is considered as one of safe and effective therapeutic methods for treating cryptocaryoniasis. So the main work of vaccine development is to determine potential protective antigens from C.irritans. The rapid advance of genomics, transcriptomics and proteomics facilitates us to screen large number of potential immunogenic antigens from C.irritans.Based on the genome of Tetrahymena thermophila, the transcriptome of three developmental stages of C.irritans, theront, trophont and tomont, was subject to Solexa mRNA sequencing. The data of three development stages was pooled and produced 49104 unigene transcipts(UTs). BLASTx analysis showed that 42.85% of UTs had available annotation in Uniprot database and 48.5% of UTs had significant similarity to the sequence from T.thermophila and Paramecium tetraurelia. Based on gene ontology annotation, 12565 UTs were assigned to 25 functional groups of KOGs. Four signal pathways, including metabolic pathways, ribosome, oxidative phosphorylation and microbial metabolism in diverse environments, were selected in KEGGs because these pathways were involved with the lagrest number of UTs and might be relative with occurance of cryptocaryoniasis. A total of 27 UTs were classified as encoding proteins involved in cryptocaryoniasis including 40 S ribosomal protein, 60 S ribosomal protein, ribosomal protein, ubiquitin conjugating enzyme, agglutination/immobilization antigen precursor, protein kinase domain protein, dnaK protein, TNFR/NGFR cysteine-rich region family protein.The proteins isolated from theronts, trophonts and tomonts were performed by 2-DE coupled with MALDI-TOF MS based on pairwise comparation. Comparative analysis showed 86 protein spots with differential abundance and 44 of them were indentified as 12 proteins such as tubulin, actin, enolase, V-type ATPase catalytic subunit α, heat shock protein 70, protein kinase containing protein, Mcm2-3-5 family proteins and 26 S proteasome subunit P45 family protein. These idenified proteins might be considered as potential protective antigens and utilized as therapeutic targets for C.irritans.The proteins extracted from theronts, trophonts and tomonts were also carried out by 2-DE coupled with immunoblotting with corresponding rabbit anti-sera. A total of 84 protein spots were immunoreactive with rabbit anti-sera and 60 of them were identified as 9 proteins including actin, tubulin, heat shock protein 70, V-type ATPase catalytic subunit α, mitochondrial heat shock protein 70, dnaK and 2 kinds of uncharacterized proteins. Meanwhile, the theront proteins was immunoreactive with grouper anti-sera prepared by natural infection and intraperitoneal injection with live theronts respectively. 28 common proteins spots were immunoreactive with these two kinds of grouper anti-sera and 27 of them were identified as 11 proteins including β-tubulin, enolase, polypyrimidine tract-binding protein, protein kinase containing protein, TNFR/NGFR cysteine-rich region family protein, V-type ATPase catalytic subunit α, NADH-ubiquinone oxidoreductase 75 kDa subunit, glutamine synthetase catalytic domain containing protein, malate dehydrogenase and 2 kinds of uncharacterized proteins.These above potential antigens discovered by transcriptomics and proteomics were amplified by PCR based on corresponding templates from transcriptome database. Finnaly, the ORFs of actin, enolase, dnaK and 26 S proteasome subunit P45 family protein were successfully obtained with 3, 3, 6, 3 common conserved antigenic determinants respectively, which might be used as multivalent vaccines against C.irritans.