| Interleukin (IL) plays an important role in the immune responses, inflammatory and hematopoietic systems, and it is also of great value in controling disease. In recent years, the research of poultry interleukin developped rapidly, and it involved many breeds of birds, but few interleukins of each species were studied, and the research was not systematic and comprehensive. Lushi chicken, known as its unique green eggs, is one of excellent chicken breeds in Henan Province. Currently interleukins of Lushi chicken is not reported in China and other countries. Cloning of Lushi chicken interleukin genes and studying their biological activity systematically was very important not only for chicken immune system and immune mechanism, but also for immunology and biological evolution comparison with human and another mammalian. IL-21 has a wide range of biological functions. It can stimulate T cells, B cells, NK cell to proliferation, and enhance the cytotoxic activity of NK cells. It has an important role in host defense and anti-tumor function. Therefore, IL-21 has great potential applications for it as a new immunomodulatory agent and adjuvant.In this study, IL-1β, IL-2, IL-3, IL-4, IL-9, IL-12B, IL-15, IL-17, IL-18, IL-21 were cloned from total RNA of Lushi chicken spleen lymphocytes stimulated by ConA via RT- PCR techique. Then by using a variety of softwares and online servers, the signal peptide, protein molecular weight, isoelectric point, hydrophilicity and tertiary structure prediction of each IL were analyzed. The study provided a good foundation for researching protein function. Meanwhile, among the Lushi chicken interleukin genes and other avians and mammals, homology analysis were proceeded and the phylogenetic trees were constructed. The results showed that the similarity of nucleotide and amino acid sequences of interleukins were very high, close to 100%, between Lushi chicken and other chickens. This indicated that avian interleukin genes were conserved highly. The nucleotide sequences of IL-1β, IL-15, IL-12B changed but the amino acid did not change. In contrast with mammals, the corresponding nucleotide similarity was about 50%, while there are more different in the amino acid sequence similarity: the similarity of IL-2, IL-3, IL-4 and IL-18 were from 15% to 25%, while the similarity of IL-9, IL-12B and IL-17 were from 40% to 50%.Then, the prokaryotic expression vector pET-28a-chIL-21 was constructed, and efficient expression of chIL-21 was achieved in E.coli Rosetta(DE3). The expression amount of recombinant protein was about 33.9% of the total protein. Recombinant chIL-21 was purified and refolded by Ni-NTA affinity chromatography. MTT test was used to detect the effect of recombinant protein on chicken peripheral blood lymphocyte proliferation, and ELISA test was used to examine the effect of recombinant chIL-21 on stimulating chicken peripheral blood lymphocyte to secret IL-2 and IFN-γ. The results showed that recombinant chIL-21 was able to significantly stimulate the proliferation of the lymphocyte, and promote the secretion of IL-2 and IFN-γin peripheral blood lymphocytes.In conlusion, the data of this study show the differences between the Lushi chicken interleukin and other animals interleukin in the molecular structure. The prokaryotic expression of chicken IL-21 was reported at first. The recombinant chicken IL-21 was expressed in E.coli Rosetta(DE3) efficiently, and its biological activity was determined. The study provide a solid foundation for further study of the structure and biological function of chIL-21 and application of avian cytokines. |
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