Identification And Preliminary Functional Analysis Of Chemokine And Chemokine Receptors From Grouper

Chemokine and chemokine receptors, which build the bridge of immune system from innate immunity to adaptive immunity and active multiple signaling pathways to regulate the proliferation, survival, apoptosis of cells and so on through their interaction, have great roles in the immune system. Many studies had proved that both innate immune system and adaptive immune system have taken part in battle against infection, and previous studies have been revealed that chemokine system may involve in the immune respond of Cryptocaryon irritans' s infection though the mechanism was not clear. In order to reveal whether chemokine and receptors may participate in the immune respond of C. irritans' s infection, we cloned 2 chemokines and 8 chemokine receptors in this paper based on the transcriptome. Based on the sequences have gotten, we carried out many studies to explore the function of these genes, including fluorescence quantitative PCR detecting the transcription level after infected with C. irritans, dual luciferase report gene test and so on. We also conducted recombinant plasmids of chemokine and receptors and have prepared the polyclonal antibody of XCR1-3. The main result were as follows:We cloned some chemokines and receptors for the first time and named them as CCL25, CXCL12-2, CCR9-1, CCR9-2, CXCR3, CXCR4-2, CXCR7, XCR1-1, XCR1-2 and XCR1-3 based on the result of blast and evolutionary tree constructed with all chemokines and receptors of human and mice. The genes cloned all have closer homology with corresponding homologous sequences of fishes.According to the results of fluorescence quantitative PCR, in healthy tissues of grouper, CCL25, CXCR4-2, CCR9-1, CCR9-2, XCR1-1, XCR1-2 and XCR1-3 showed higher expression level in immune tissues than in non-immune tissues. CXCR4-2 exhibited the highest expression level in head kidney and CCR9-1, CCR9-2 and CCL25 was in thymus. After infected with C. irritans, CCL25, CXCL12-2, CXCR4-2, XCR1-1, XCR1-2 and XCR1-3 showed enhanced expression in the entry of pathogen(skin and gills). And the significantly increased expression is CCL25, CXCL12-2, XCR1-1, XCR1-2 and XCR1-3. All the ten genes showed increased expression in immune tissues(head kidney and spleen) except CCR9-2 and CXCR7.We obtained fusion protein p ET32a-CCL25 and p ET32a-CXCL12-2, laid the foundation for the next study. We also prepared polyclonal antibody of XCR1-3 and have succesefully to mark XCR1-3+ cells.We constructed the recombinant eukaryotic expression plasmid pc DNA3.1-HACCR9-1, CCR9-2, CXCR4-2 and CXCR7 and the four plasmid all expressed in both HEK293 T and CLC cell lines. All the four plasmids transfected in HEK293 T and CLC cells showed inhibited the activity of NF-?B, except pc DNA3.1-HA-CXCR7 exhibited enhanced the activity of NF-?B in CLC. And the inhibition was more significant in CLC cells than in HEK293 T cells.