Analysis On Structure And Immune Function Of The Gene Cyclophilin A From Red Swamp Crayfish,Procambarus Clarkia

Procambarus clarkiihas become an important freshwater aquaculture species in China in recent years,which yield has increased by an average of 24.56%in the past 10 years.But with the continuous expansion of farming,it frequently suffers froma multitude of diseases,leading to catastrophic economic losses.Therefore,it is of great significance to understand the immune responses of the red swamp crayfish against pathogen challenges.Cyclophilins(Cyps)are a family of highly conserved and multi-functional proteins that exist extensively from microorganisms to mammals.They serve as molecular chaperones and possess the activity of peptidyl-prolyl cis-trans isomerase that catalyzes protein folding and conveying.In addition,binding to cyclosporin A(CsA),they have the effect of immunity suppression that mediates immunosuppressive agents in T cells.Discovered in 1984,CypA was the first member of cyclophilins.Recent researches has indicated that CypA may trigger an immune response induced by infections or stresses in some aquatic organisms.In this paper,CypA of P.clarkii(referred to as PcCypA)was identified from cDNA library of hepatopancreas and its structural features and functions were analyzed by bioinformatics methods.The amplified fragment was cloned into vector pET30a(+)and transformed into E.coli BL21(DE3)for prokaryotic expression.Then the recombinant protein was used to immunize New Zealand white rabbits to prepare the polyclonal antibodies.The gene transcriptional and translational distributions in the different tissues were analyzed by qRT-PCR and Western blot and then the immune response of PcCypA genes in the different tissues of P.clarkii following challenge with WSSV infection.It is anticipated that the results will lay a foundation for the research on the functions of immune-relevant genes in crustacean and shed light on immunological mechanisms of invertebrates.The results are summarized as follows:1.The total length consisted of 884 nucleotides(GenBank accession number:JX878886),including a 495bp single open reading frame,a 108bp 5’untranslated region(UTR)and a 281 bp 3’UTR.The ORF encoded a protein of 164 amino acids with a calculated molecular mass of about 17.3kDa.There existed a Cyp-type peptidyl-prolyl cis-trans isomerase signature(YKGSTFHRIIPGFMCQGG).PcCypA peptide had no obvious hydrophobic domains,transmembrane domains or signal peptide sequence.Irregular curls(the main structures),8 β foldings and 2 α helices were present at the predicted secondary structure.It supports the notion that PcCypA is a PPIase belonging to the Cyp family.However,PcCypA conserves Gly70 instead of His70 in mammalian and teleost fish.Whether the Gly70 residue is critical to chemotaxis in crustacean CypA remains to be determined.Phylogenetic analysis showed that the relationships displayed in the CypA phylogenic tree was in general agreement with traditional taxonomy.2.The recombinant prokaryotic expression vector pET30a(+)-PcCypA with the his-tag was constructed and transformed into E.coli BL21(DE3).After being induced by IPTG,the infusion protein was highly expressed,purified by high affinity Ni-NTA resin and identified by SDS-PAGE and Western blot.The result showed that 23kDa His-PcCypA protein in the form of inclusion bodies was the interest protein.The rabbit specific polyclonal antibody against PcCypA was prepared and identified by the Western blot using recombinant CypA protein.The antibody can also have a specific,high-affinity interaction with His-PcCypA fusion protein(23kDa)and the interest protein(17kDa)from hepatopancreas of P.clarikii.It is the first CypA antibody in the crustacean,which can provide a material basis for the following protein expression and immunohistochemistry researches.3.qRT-PCR assays showed that CypA was transcribed into mRNA in all the observed tissues.The highest expression level was detected in the heart,followed by the muscle,the gill,the ovary,the testis and thehepatopancreas.The Western blot analysis results demonstrated that there was a specific immunoreactive band of approximately 17 kDa in the hepatopancreas,the gill,the testis,the heart and the muscle.They were basically of the same size as the predicted CypA amino acid.However,the immunoreactive band of 17kDa was not observed in the ovary,while 35kDa,47kDa,51 kDa and 55kDa took its place.It might suggest a complicate expression pattern in the ovary.4.P.clarkii is susceptive to white spot syndrome virus(WSSV).Histologic observation under a light microscope showed that the heart,the hepatopancreas and the striate muscle of the abdomen were severely affected.PcCypA transcriptional levels were significantly up-regulated in the heart and the hepatopancreas tissues at 0-24 hpi.After 48hpi,CypA transcripts in the gill and the muscle were significantly increased,while transcripts in the hepatopancreas and the testicle decreased.During 96-120hpi,CypA transcripts in the hepatopancreas and the intestine were significantly increased again.The results suggested that CypA might be involved in the immune response of P.clarkii.5.By RT-PCR from the total RNA,the gene sequence variations 60 of individuals from 3 populationswere analyzed.The results illustrated that PcCypA was a highly conserved fragment,but its ORF region had 2 mutations and 3’UTR had at least 1 mutation.8 haploid types revealed a high genetic diversity in the 3 populations,testifying that PcCypA was suitable for SNP analysis of the red swamp crayfish.