Cloning, Expression Of Chicken Interleukin 18 Mature Protein Gene And Preparation Of Its Antibody

Interleukin-18 (IL-18) is a novel discovered cytokine, which is the potent inducer of IFN-Y production. It has been demonstrated that interleukin-18 exist in cells of human, mammal and some kinds of birds. Interleukin-18 plays an important role in host defense against various microorganisms. In addition, interleukin-18 is expected to be a target for tumor immunotherapy. Cytokines including interleukin-18, as natural mediators of the immune response, offer exciting alternatives to conventional therapeutics in control of disease in intensive livestock and poultry industries. The discovery of chicken interleukin-18 gene is very meaningful for both the study on comparative immunology and the therapeutics for poultry diseases.In this study, specific primers for chicken interleukin-18 (ChIL-18) cDNA have been designed and synthesized according to the previously reported nucleic acid sequence of chicken interleukin-18 cDNA. Using total RNA from ConA-stimulated chicken spleen cells, chicken IL-18 cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis indicated that there is one nucleotide different from the published ChIL-18 cDNA sequence in 482 (T-C).To highly express chicken interleukin-18 in E.coli and purify the fusion protein, cDNA fragment encoding the mature ChIL-18 was subcloned into the prokaryotic expression vector pPROEX橦Tb. The recombinant plasmid was transformed into E.coli, and then induced by IPTG at 37 C. The recombinant ChIL-18 (rChIL-18) was expressed efficiently in forms of inclusion body with the yield accounting for 30% total bacteria protein. SDS-PAGE and Western blot analysis showed that the recombinant fusion protein had a molecular weight approximately 23ku. The inclusion body was solubilized by 6M Guanidine hydrochloric acid and purified by ProBond?Resin. The antiserum against rChIL-18 was obtained by injecting the guinea pig with fusion ChIL-18 protein and its purified form.As a result, we successfully cloned the ChIL-18 mature protein gene, expressed and purified the rChIL-18 fusion protein from E.coli and obtained the polyclonal antibody against rChIL-18, laid a solid foundation and prepared experimental material for the future studies on the bioactivity of ChIL-18.Candidate: Pan Weiqi Major: Basic Veterinary Advisor: Vice Prof. Li Guangxing...