|Other Abstract||The valuable explore was performed in the reptile immunology, which was the least studied. By using the suppressive subtractive hybridization technique, a cDNA library was first constructed from the main internal organs of Aeromonas hydrophila infected Chinese soft-shelled turtles. 42 genes were identified from more than 200 clones, and 16 of them were immune-relevant genes and firstly identified in reptile. Then, IL-8, SAA, CD9, Toxin-like protein, ATF4 and CL genes were further observed to be up-regulated in the infected tissues of turtles by virtual Northern hybridization and RT-PCR assays.
By using RACE-PCR, the full-length cDNA and turtle IL-8, SAA, Toxin-like protein, CD9 and Fg were isolated, and the molecular and phylogenetic characteristics were analyzed. The genome DNA and promoter sequences of the first three genes were amplified by genome-working. The turtle IL-8 gene spans 4924 kb, contains three introns and four exons. The full-length cDNA was 1188 bp contained an open reading frame (ORF) of 312 bp, which coding a protein of 104 amino acids (aa). The CXC domain, four cysteines and the ELR motif (involved in IL-8 founction) were well conserved in turtle IL-8. The genome sequence of turtle SAA was 3153 bp, contained two introns and three extrons. The 554 bp cDNA sequence is obtained with an ORF of 381 bp encoding 127aa. The N-terminus of turtle SAA was composed of hydrophobic residues; the C-terminus amino acids were well conserved. The full-length genome DNA of Toxin-like protein was 1995 bp and occupied two introns and three extrons. The full-length cDNA was 580 bp and contained an ORF of 267 bp coding for a protein of 89 aa. There were 8 conserve cycteines and the consensus sequence motif -CCXXXCN- at the C-terminal end, which was a marker sequence of the “three-finger” protein family. The full-length cDNA of turtle CD9 was 1146 bp and contained a 672 bp ORF coding for a protein of 224 amino acids (aa). Turtle CD9 contained four TM domains, large and small extracellular loops (LEL and SEL). The conserved CCG motif and four cycteines were found within the LEL, and a potential N-glycosylation site was found in the SEL of turtle CD9. The full-length cDNA of turtle Fg was 1605 bp and contained a ORF of 1320 bp, which coding a protein of 440 aa. It contained the FReD, C terminal globular domain, Ca2+ binding site and a polymerization pocket. Phylogenetic analysis showed that, except Toxin-like protein, the rest four proteins of soft-shelled turtle were clustered with their birds’ homologues.
RT-PCR analysis identified the transcripts of IL-8, CD9 and Toxin-like protein in the tissues of control turtles. IL-8 was constitutively expressed; CD9 was mainly expressed in liver and spleen, and Toxin-like protein was mainly expressed in liver and kidney. RT-PCR also showed that, except SAA, the rest four APP genes (Fg, C3, CL and ALB) were all constitutively expressed in liver.
After infected with A. hydrophila, RQ-PCR analysis showed that there were dynamic changes of IL-8, CD9 and Toxin-like protein mRNA expression in related tissues of soft-shelled turtles. The most induction of turtle IL-8 gene was found in liver, spleen and kidney 4 d after infection. CD9 gene was significantly up-regulated in liver, spleen and blood during 2-4 d after infection. So was the Toxin-like protein gene during 1-2 d after infection. Except the ALB gene was observably suppressed, the rest four APP genes were induced at different levels in liver after infection, and SAA was induced and reached to about 2000-fold at 2 d. These results suggested that the dynamic changes of these genes expression were closely related to the anti-bacteria immune responses of reptiles.
The recombinant turtle IL-8 protein was expressed in E. coli and purified. Chemotaxis experiments showed that the turtle rIL-8 induced a migration of turtle neutrophils. There attraction of lymphocytes was not obvious. This was the fist identification of the chemotaxis effects of reptile rIL-8 to neutrophils.|