Gene Cloning, Polymorphisms And Expression Of Interleukin10in Rabbits

Cytokines are a class of small peptides that are secreted by immune cells and can regulate cell functions, thus they play an important role in cell growth and differentiation processes. Interleukin10(IL-10) is a typical representative cytokine. It is secreted by a variety of cells and function in the regulation of immune and inflammatory responses. Recently, studies are focused on the association between human IL-10gene promoter SNPs and diseases, while studies on rabbit IL-10gene exon SNPs and cellular localization are rarely reported. Therefore, the coding sequence of rabbit IL-10gene was cloned and analysed; prokaryotic expression vector was constructed and further induced in order to prepare polyclonal antibody; eukaryotic expression vector was also constructed and transfected into DF-1cells in order to study the subcellular localization of IL-10gene; PCR-SSCP method was used to detect the five exons polymorphisms of IL-10gene in New Zealand white population and Fujian yellow population. The main research contents and results were as follows:1. Cloning and bioinformatics analysis of IL-10gene. The coding sequence of IL-10gene was amplify from the total RNA of rabbit spleen by RT-PCR method and then cloned into pMD19-T Simple vector. Bacteria PCR, restriction enzyme digestion and sequencing results all showed that pMD19-T-IL-10vector was constructed correctly. Analysis on IL-10gene showed that, the cDNA and CDS are1,227bp and537bp in length, respectively, which encodes178amino acid residues. Also, one functional site exists between the75th and95th amino acids. The molecular mass of IL-10protein is about20.15kDa and the theoretical isoelectric point is8.20. This protein is unstable and fat-soluble, which has signal peptides in the position of the first21amino acids and multiple potential phosphorylation sites, but no transmembrane domains. In addition, the secondary structure of the protein contains only a helix and free curl. Homology and phylogenetic analysis showed that rabbits were close to mammalians, while distant from poultry and fishes. 2. Correlation analysis of IL-10gene polymorphisms in exons and serum IL-10index. PCR-SSCP results showed that, there were no SNPs on exon1,2,4or5. Three genotypes (AA, BB and AB) and1SNP loci (A2150G) were detected on exon3, and this mutation was a synonymous one that did not cause any changes in the encoded amimo acids. The detection results of serum IL-10showed that, New Zealand white rabbits had significantly higher IL-10level than Fujian yellow rabbits, and IL-10levels among genotypes of these two groups were not significant.3. Prokaryotic expression and polyclonal antibody preparation of IL-10gene. IL-10gene was subcloned into the prokaryotic expression vector pET-32a and then transformed into the expression strain BL21(DE3). IL-10gene was successfully expressed through the induction by using IPTG. The result of SDS-PAGE electrophoresis showed that, the fusion protein was37.7kDa in size and mainly existed as inclusion body. Fusion protein was purified and immunized into2guinea pigs so as to prepare polyclonal antibody. As a result, the anii-IL-10polyclonal antibody showed high sensitivity (1:56000) and specificity, which lays the foundation for further studies on rabbit IL-10gene functions.4. Expression and Sub-cellular Localization of Rabbit IL-10and EGFP Fusion Protein. IL-10gene was subcloned into the eukaryotic expression vector pEGFP-Cl. DF-1cells were transfected pEGFP-IL-10through Lipo2000and then observed under inverted microscope to identify recombinant proteins localization. Total RNA and total protein were extracted from the transfected cells48h later. As a result, the fusion proteins mainly expressed in cytoplasm. The expression of IL-10gene can be detected at both mRNA and protein levels, and pEGFP-IL-10protein was about69.6kDa. All these above provides basis for further study on the biological functions of IL-10.