Cloning, Expression And Bioactivity Of Wild Anatidae Interleukin-2

The wild anatide is rare birds, a variety of anatide are protected as an protected animal under the wild animal protection law as category II in china. Wild anatide is a major component of the wetland ecosystem and biological indicator for monitoring wetland environmental change, has important ecological value. The meat of anatide is delicious and rich in nutrition, anatide is one of the main varieties of taming and breeding, therefore the anatide with high economic value. Recent studies showed that the anatide may be the natural host of important viral infectious diseases such as Avian flu is highly valued by the veterinary experts, so it also has important scientific research value. Interleukin-2is one of the important immune cytokines, can promote the survival and proliferation of immune cells, and enhance the level of immune response, regulate the immune. IL-2is applied to enhance the immune response level and establish the immune detection index, prevent or treat the bacterial and viral infections on clinical. In order to understand the molecular differences and the level of genetic variation of different anatide IL-2gene, try to apply cytokine immune treatment on wild anatide and improve the autoimmune level to external environment. This study were to clone the cDNA sequence of anatidae IL-2, to express ruddy shelduck IL-2mature protein in vitro, and to detect the bioactivity of the recombinant protein in vitro.Based on the published nucleotide sequence of muscovy duck IL-2gene in Genbank, a pair of specific cloning primers was designed and synthesized. DNA sequence was amplified by RT-PCR depending on the template of RNA isolated from ConA-stimulated peripheral blood lymphocytes of mallard、spot-billed duck、ruddy shelduck、swan goose、bar-headed goose and white-fronted goose, and cloned to pMD18-T vector and sequenced. The result showed that the ORF of duck is423bp which encoding a protein of140amino acids and the ORF of anser is426bp which encoding a protein of141amino acids. Homology analysis found that anatidae IL-2sequence was nearest to muscovy duck and turkey. By contrast IL-2nucleotide sequence sequence between anatidae and mammalian and rodents are all less than49.3%. Phylogenetic tree analysis indicated the anatidae IL-2has the most closed relationship genetic distance with muscovy duck IL-2among different birds.The ruddy shelduck IL-2mature peptide was ligated to expression vector pET32a(+), recombinant plasmid pET32a-IL-2was constructed, and then transformed to E.coli BL21. The recombinant ruddy shelduck IL-2(rRSIL-2) mature protein was expressed in E.coli induced with IPTG. The bacteria containing rRSIL-2was collected and examined by SDS-PAGE. Result showed that the molecular weight of the protein was32KDa. Soluble analysis of the fusion protein found that the majority of IL-2protein in the supernatant. We successfully purified the recombinant protein using Ni-NTA spin kit. Western Blot showed that rRSIL-2protein was recognized by anti-His Tag mouse monoclonal antibody.MTT colorimetric assay indicated that the purified rRSIL-2protein had the ability to stimulate the proliferation lymphocytes of chicken、duck and goose.The results demonstrated that rRSIL-2stimulated the proliferation of the lymphocytes although the effect on chicken and goose lymphocytes was relatively weak at the same concentration.