Construction Of A CDNA Library Of The Trionyx Sinensis Stimulated With Aeromonas Hydrophila And Analysis And Expression Study Of CCL20

In the first place, the same size soft-shelled turtle were hatched at the different temperatures but the same humidity, and the effects of different temperature on embryonic development, hatchling traits and hatchling moving of Trionyx sinensis were researched. To understand anti-infectious response to bacteria in the Chinese soft shell turtle (Trionyx sinensis), a full Length cDNA library was constructed for it by SMART technique experimentally infected with Aeromonas hydrophila. Firstly, to synthesize the double-strand cDNA using SMARTTM PCR cDNA systhesis kit. Then, the ds cDNA was separated into two parts based on the size distribution of amplified ds cDNA by agarose gel size fractionation. The part that shorter than 500 bp discards and the other one which longer than 500bp was ligated to the pGEM-T vector. The ligation mixture was transformed into E.coli JM109 by electroporation. The cDNA library contained 4.3×105 independent clones with DNA inserts of 0.5 kb-2 kb. The recombination rate was 92%. We sequenced 164 cDNA clones about 1 kb, most of the genes are found for the first time in reptiles, and classified in function.We identified one cDNA encoding a macrophage inflammatory protein-3α (CCL20) from the cDNA library (designated as CCL20). The CCL20 cDNA had an open reading frame(ORF) of 303bp encoding a polypeptide of 100 amino acides of the polypeptide chain, the molecular weight of CCL20 was 11429.3, theoretical isoelectric point was 8.71. The homology analysis showed that the Chinese soft shell turtle(Trionyx sinensis) and Western painted turtle(Chrysemys picta bellii) and Red jungle fowl (Gallus gallus) Mississippi crocodile(Alligator mississippiensis) and Zebra finch (Taeniopygia guttata) had closest relationship, the similarity was between 71%-84%, and had lower relationship with mammal. It was predicted that the protein was unstable and hydrophilic amino acid, so the protein was hydrophilic ptotein. The signal peptide was between 1-28 sites of amino acid, and cleavage site was between 28-29 amino acid. Subcellular localization was in cytoplasm and nucleus. At the same time, CCL20 had no transmembrane structure, structure function domain SMALL-CYTOKINES-CC which was between 34-77 amino acid was also called chemokine, and at the conserved domain of CCL20, Chemokine superfamily existed.The secondary structure of CCL20 had more a-helix, random coil and the extension of the chain. They consisted the majority of the teriary structure.The PCR results showed that:In the normal group, CCL20 expressed in the liver, kidney, heart, spleen, the spleen, the highest expression level, the heart, the lowest expression, the relative expression level was spleen (12.9 times)>liver (1.5 times)>kidney (1.0 times)>heart (0.75 times). In the experimental group, the CCL20 expressed more when the Chinese soft shell turtles were infected with Aeromonas hydrophila 12h,24h,36h later, and significantly higher than the control group, liver was the highest expression tissue and with time continued, the CCL20 expression level showed a slow decline.