Cloning And Expression Of Duck Cytokines And Develoment Of Elisa

Cytokines are proteins secreted by cells that play an important role in the activation and regulation of other cells and tissues during inflammation and immune responses. Although well described in several mammalian species, the role of cytokines and other related proteins is poorly understood in avian species. Progress in isolating avian homologues of mammalian cytokines has been slowed by the generally low level of sequence similarity. Recent advances in avian genetics and immunology have begun to allow the exploration of cytokines in health and disease. Being one of important varieties in poultry breeding, little was known about the cytokines of Cherry Valley Duck. At present, the data showed that the cytokines were undergoing obviously changes with disease development. Therefore, the cDNA of duck interferon-gama (IFN-y), interleukin-2(IL-2) and tumor necrosis factor-alpaha (TNF-a) were cloned in this report, then recombinant proteins were produced in prokaryotic expression system; on this basis, monoclonal antibodies and polyclonal antibodies specific for the three cytokines were generated, and Ag-capture ELISA was established. This study facilitates basic immunobiological studies of the role of cytokines in avian immune system.1Cloning, expression and purification of duck IFN-γ、IL-2and TNF-αThe specific primers were designed according to the sequence of Anas platyrhynchos IFN-y gene, Cairina moschata IL-2gene and Gallus gallus TNF-a gene reported in GenBank. Then extracting total RNA from Cherry Valley Duck spleen lymphocytes, IFN-y, IL-2and TNF-a gene were amplified by RT-PCR (Reverse Transcription-PCR),3’RACE and5’RACE approach. Sequence analysis indicated that the length of cloned DuIFN-y, DuIL-2(excluded the signal peptide sequence) and DuTNF-a gene ORF sequence were respectively about495bp,363bp,447bp and encoded164,120and148amino acids, respectively; the full-length cDNA sequence of duck TNF-a was1049bp. Sequence analysis showed that99.6%homologies in nucleotide sequence and98.8%in amino acid sequence respectively between Cherry Valley Duck and Anas platyrhynchos IFN-y gene,98%homologies in nucleotide sequence and94%in amino acid sequence respectively between Cherry Valley Duck and Cairina moschata IL-2gene,99.6%homologies in nucleotide sequence and100%in amino acid sequence respectively between Cherry Valley Duck and Gallus gallus TNF-α gene. After cloning and sequencing, recombinant expression plasmid pCold-DuIFNy, pCold-DuIL2and pCold-DuTNFa were constructed and expressed in E.coli BL21(DE3) by IPTG induction at low temperature (15℃). The expressed proteins were purified by affinity chromatography. SDS-PAGE analysis showed that the three proteins were expressed as soluble forms in E.coli strain BL21(DE3), and the molecular weights of recombinant proteins were70kDa,65kDa and68kDa, respectively. It is concluded that the recombinant proteins have been successfully expressed and purified in monoclonal antibodies and polyclonal antibodies production.2Production of antibodies to duck IFN-y、IL-2and TNF-a1) Production of monoclonal antibodies:BALB/c mice were immunized biweekly with recombinant proteins emulsified in adjuvant by subcutaneous injection and boosted intraperitoneal injection with recombinant proteins without adjuvant3days prior to fusion. Mice producing high serum Ab titers against cytokines were selected by indirect ELISA, and their spleen lymphocytes were fused with nonsecreting mouse myeloma SP2/0cells. Hybridomas were selected in medium supplemented with hypoxanthine, aminopterin, and thymidine, and supernatants screened by indirect ELISA. The hybridomas secreting antibodies were cloned by limiting dilution. Three hybridomas secreting mAbs against duck IFN-y, duck IL-2or duck TNF-a were obtained. Indirect ELISA showed that the titer of ascites fluids was between1:25,600and1:51,200. Western blot analysis confirmed that the mAbs had good immuno-reactivity.2) Production of rabbit antiserum against recombinant proteins. Antiserum against recombinant proteins were generated by tri-weekly immunization of New Zealand white rabbits with recombinant proteins emulsified in adjuvant by subcutaneous injection. Immunized rabbits were bled14days after last injection and the serum was collected. The titer of the antiserum was1:12,800～1:51,200detected with indirect ELISA. In conclusion, this study has successfully obtained monoclonal antibodies and polyclonal antibodies against cytokines, which can lay a foundation for measuring duck cytokines. 3Development of an antigen capture ELISA for duck IFN-yUsing the monoclonal antibody and polyclonal antibody described above, the sensitive Ag-capture ELISA for duck IFN-y was developed. The optimal coating concentration for monoclonal antibody was0.8μg/ml, the optimal concentration for the polyclonal antibody was0.8μg/ml, the optimal coating time was4℃overnight, the optimal blocking solution was1%BSA, the optimal developing time was10min, and the minimum limit of detecting was3.9ng/mL. This study has provided a groundwork for developing Ag-capture ELISA kit for detecting duck IFN-y.