| Summary: | 碩士 === 國立中興大學 === 獸醫學系 === 91 === Chicken IL-8 (chIL-8) is a proinflammatory cytokine. It is important to defend virus and bacterial infection. We obtained chIL-8 from macrophages stimulated with bacterial lipopolysaccharide (LPS). The chIL-8 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and was inserted into expression vector pET32a and expressed in Escherichia coli. The molecular weights of the recombinant protein (r-chIL-8) protein was 31 kDa. The r-chIL-8 was purified by affinity chromatography. The results showed that the r-chIL-8 protein can be purified by the elution with 150 mM imidazole . BALB/c mice were immunized with the purified r- chIL-8 and were used for hybridomas generation. The antibodies to r-chIL-8 were screened by enzyme-linked immunosorbent assay (ELISA) and further identified by indirect immunofluorescence staining assay. Four hybridoma cell lines secreting antibody against r-chIL-8 were obtained. Their isotypes were determined to be IgG1 subtype. All monoclonal antibodies (mAb) recognize the conformational form epitope. Four mAbs were used to dected the IL-8 expression in the LPS-stimulated macrophages derived from duck﹑goose﹑pigeon﹑and turkey by using indirect immunofluorescence staining. The results showed that IL-8 protein could be detected from all avian species tested, suggesting that the epitopes recognized by these mAbs existed among these avian species. In addition to chicken, RT-PCR was also used to amplify the IL-8 gene of the other four avain species. The amplified products were tested with four restriction enzymes. The results indicated that the restriction sites of IL-8 gene among these avian species did not show to differ and the restriction sites for one of 4 enzymes, Ava Ⅱ, did not exist. The amplified products were further sequenced and their sequences of nucleotide and amino acid were compared. The sequences of nucleotide and amino acid were analyzed by DNA STAR® software .The results showed that the homologies of the nucleotides about and the amino acids were about 84.6% to 98.7% and 82.7 to 98.1% respectively.
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