| Cytokines play important roles on innate immunity and primarily involved in inflammation and immune responses. Researches showed that the transcription and expression levels of certain cytokines in the body would be significantly changed responding to external stimuli or infection. Although cytokines are well described in several mammalians but very few study has been done in poultry. Therefore, in this study, we tried to clone the duck Mitogen-activated protein kinase 1 (MAPK1) and Oligonucleotides domain 1 (NOD1) gene, and established the real-time PCR methods to for the detection. In addition, recombinant duck MAPK1 protein was expressed in a prokaryotic expression system, and used for preparation of monoclonal and polyclonal antibodies. This study provides technical supports for study of poultry cytokines involved in the regulation of signaling pathways of innate immunity.1. Cloning, expression and purification of duck MAPK1 and NOD1 The specific primers were designed according to the chicken MAPK1 and NODI gene sequences published in GenBank. Total RNA was extracted from Cherry Valley duck spleen tissue by RT-PCR and cDNA ends RACE technology. The duck MAPK1 and NOD1 gene were successfully cloned. Sequence analysis indicated that duck MAPK1 cDNA was 1,557 bp long which cloned by RT-PCR and RACE method, with an open reading frame of 1,107 bp and encodes 368 amino acids. The duck NODI cDNA was 2,756 bp long which amplified by RT-PCR. Homology analysis demonstrated that duck MAPK1 showed a 85.4%,84.5% and 97.3% nucleotide homology and a 96.4% 97.2% and 98.6% amino acid homology with human, mouse and chicken MAPK1 respectively. Duck NOD1 showed a 59.2%,57.4%, and 88.8% nucleotide homology and a 62.9% 61.1% and 91.9% amino acid homology with human, mouse and chicken NOD1 respectively. The duck MAPK1 was cloned into the prokaryotic expression vector pET-28a and the prokaryotic expression vector pET-MAPKl was constructed and expressed in E. coli Rosetta by IPTG induction at low temperature. The expressed proteins were purified by affinity chromatography. SDS-PAGE analysis showed that, the MAPK1 was expressed as a soluble protein in E. coli Rosetta and the molecular weight of recombinant protein was 42 kDa, which could be used for preparation of monoclonal antibodies and polyclonal antibodies.2. Production of antibodies to duck MAPK1 1) Production of monoclonal antibodies (MAbs):The objective of this study is preparation of MAbs against duck MAPK1. Purified recombinant MAPK1 protein was used as an antigen for immunization and BALB/c mice were immunized for 3 times. After the third immunization, the spleen cells were collected from immunized ducks with highest antibody titer, and fused with myeloma SP/20 cells. The positive monoclonal hybridoma cells secreting specific MAbs were screened by indirect ELISA and cloned for three times using limited dilution method. As a result, two monoclonal antibodies were successfully prepared. The titers of both MAbs in the ascites were all measured over 1:12,800 by indirect ELISA. Western blot further showed that both MAbs reacted with the duck rMAPKl very well. identified the two monoclonal antibodies The subclass of both MAbs was identified as IgGl type using monoclonal subtype kit.2) Production of polyclonal antibody:Purified recombinant MAPK1 protein was used as an antigen for immunization and New Zealand white rabbits were immunized four times subcutaneously at two weeks intervels, and the serum was collected after 4th immunization. The antibody titer was measured as 1:25,600 by ELISA. Western blot analysis showed that the rabbit antisera against duck MAPK1 have a good specificity.3. Establishment and preliminary application of a quantitative real-time PCR method for detecting the transcription of duck MAPK1 and NODI gene In order to establish a real-time PCR method to detect the transcription of duck MAPKl and NOD1 genes, specific primers of duck MAPK1, NODI and apoptosis-related basic protein (Arbp) were designed according to the respective gene sequences. Total RNA was extracted from the duck spleen and reverse transcriped into cDNA as a template, the positive standard plasmid was constructed and the real-time PCR method to detect duck MAPK1 and NOD1 transcription level was established. The results showed that the minimum detectable amounts for duck MAPK1 and NOD1 mRNA were 103 and 102 copies respectively, with a good linear relationship between 103 to 1012copies, R2 were greater than 0.996 and with a good specificity and repeatability. Following Riemerella anatipestifer infection by virulent strain Yb2, MAPK1 and NOD1 mRNA level increased significantly in the duck spleens, suggesting that increased duck MAPK1 and NOD1expression can be used as indicators of bacterial infection. Our results provide groundwork to warrant further studies of duck MAPK1 gene in bacterial pathogenesis. |
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