Construction Of CDNA Libraries Of Peripheral Blood Leucocyte Of Carp(Cyprinus Carpio L.) And Screening Of The Differential Expression Genes

Peripheral blood leucocyte in fishes are important components of humoralimmunity and cellular immunity just like white blood cells in vertebrates. Whenpresented with antigen or mitogens, a antigenic determinant is identified inresponding leucocytes, resulting in the activation, proliferation and differentiation ofthese cells. Immune molecules such as antibody and cytokine were also generated asresults. During the course of this activating, the leucocytes are induced to express orto up-regulate the expression of many genes named leucocytes activated genes whenpresented with mitogens. The possibility that the activation processes of leucocytesin fish and the genes whose expression is affected during fish leukocyte activation ismostly unknown. Elucidating the molecular events of leucocytes activation isessential for a complete understanding of the mechanisms of immune response offish.To obtain sufficient numbers of highly purified leucocytes seemed to be of majorimportance to study the mechanisms of immune response of fish. In order to cloneand study immune response related genes in carp (Cyprinus carpio L.) leucocytes,the cDNA libraries of normal and mitogens stimulated carp leucocytes wereconstructed with HybriZAP-2.1 cDNA construction kits. The carp leucocytes wereisolated from the peripheral blood of normal carp and cultured in vitro with orwithout stimulation in different time (4h, 12h and 24h) by treatment with LPS,PHAand Con A, respectively. Total RNA of leucocytes was then extracted, pooledtogether and mRNA was isolated with a column of oligo (dT) cellulose. The first andsecond strand of cDNA were synthesized using Stratagene HybriZAP-2.1 XR cDNAsynthesis kit, then ligated to EcoR I adapters and digested with Xho I. After sizefractionated with CHROMA Spin-400 column cDNA were ligated intoHybriZAP-2.1 vector and packaged in vitro to construct cDNA libraries. Themitogens stimulated carp leucocyte cDNA primary library contains 1.67×106recombinants with the average inserts size between 0.4×103～3.0×103 bp and 98.9%of recombinant clones. After amplification, the titer of amplified library was6.16×109pfu/ml. The normal carp leucocyte cDNA primary library contains 5.79×106recombinants with the average inserts size between 0.4×103～3.0×103 bp and 99.4%of recombinant clones. After amplification, the titer of amplified library was4.22×109pfu/ml.DDRT-PCR is an effective and quick method to study gene different expressionin the same cell under different physiological status and different stages anddevelopment. In this study, fluorescence DDRT-PCR was used to compare mRNAfrom leucocytes from peripheral blood of carp with and without mitogens (LPA,ConA and PHA) stimulation in different time such as 4h, 12h and 24h. The resultsindicated 92 different fragments and 87 of them were re-amplified. After cloning andsequencing 60 fragments were identified and sequence analysis revealed 5 cDNAfragments were immune response related genes, which were highly homologous withThymosin beta, proteasome activator complex PA28 β subunit, CXC chemokine,translation elongation factor-1β(EF-1β) and Matrix metalloproteinase (MMPs). Thefull-length thymosin β cDNA was 528 bp with the open reading frame encoding 46amino acid residues and submitted to GenBank with accession number AY457946.The cDNA library of mitogen LPS,PHA and ConA stimulated carp leucocyteswas screened by a DIG labeled DNA probe C15. The positive clone obtained has ainsert sequence of 1097 bp in length with a full ORF encoding 249 amino acids ofproteasome activator PA28 of Carp, including PA28 α and β subunit motifs. Theprotein sequence showed significant homologues (93% identity) with zebrafishproteasome activator PA28, which has 248 amino acids. Multiple sequence alignmentwith other species showed that carp PA28 sequence has the highest homologue withthat of zebrafish, and has the lowest homologue with woodchuck, which is 57%identity. This gene was submitted to GenBank with accession number DQ453126.Semi-quantitate RT-PCR indicated the expression of PA28 mRNA in mitogenstimulated carp leucocytes is higher than in normal carp leucocytes(p>0.05), andthe expression of PA28 mRNA in tissues sush as spleen, heart, liver,headkidney ,brain,musle of normal carp is significantly lower than those tissues ofstimulated by Aeromonas hydrophila(p<0.01). Northern blots showed a specificband in the mRNA of mitogen stimulated carp leucocytes and southern blot indicatedthis gene was not a single copy in the genome of carp.In order to study immune related genes in carp leucocytes, IL-8 were clonedfrom the cDNA library of mitogen stimulated carp leucocytes by a probe A26 gottenfrom the DD-RTPCR. IL-8 cDNA was 753 bp in full length with an ORF of 294 bpencoding 98 amino acids with molecular weight of 10848.9 Da and isoeletric point of8.68. This gene was submitted to GenBank with accession number DQ453125.Semi-quantitate RT-PCR indicated the expression of IL-8 mRNA in mitogen LPS andPHA stimulated carp leucocytes is higher than in normal carp leucocytes at4h(p<0.01). Northern blots showed a specific band only in the mRNA of mitogen(LPS and PHA) stimulated carp leucocytes and southern blot indicated this genewas not a single copy in the genome of carp.