Cloning、Prokaryotic Expression and Preparation of Polyclonal Antibody of Chicken IL-2 Gene

Abstract: Interleukin-2 is a lymphokine synthesized and secreted primarily by T lymphocyte or T helper lymphocytes that have been activated by stimulation with certin antigens or mitogens.It is one of the important T cell growth factors.Because of its key role in immune response,it is always a center in the study of cytokines.Compared with human and mammalian,the study of chicken IL-2 is very slowly. Acquirement of recombinant protein or polyclonal and monoclonal antibody of chicken IL-2 by eukaryotic and prokaryotic express system is of important effection in study of biology activity of chicken IL-2 and the regulation of immune response.In this study,we designed a pair of primers based on the sequence of the upstream and downstream of chicken IL-2 gene.About 600 bp chicken IL-2 cDNA fragment was cloned from ConA-stimulated chicken splenocytes by reverse transcription-polymerase chain reaction (RT-PCR) and was subcloned into PUC18 vector.Recombinant clone was demonstrated by restriction enzyme digestion and DNA sequencing.Next,we construct recombinant plasmid pPROEX橦T-IL-2.The cDNA of chicken IL-2 gene was subcloned into BamH I/Hind III sites of vector.The recombinant plasmid pPROEX橦T-IL-2 was transformed into E. coli DH5a and the bacteria was induced with IPTG.It was demonstrated by SDS-PAGE and Western blot that a 18kDa protein which was equal to chicken IL-2 protein in molecular weight was expressed in E.coli DH5a.The expression level was up to 30% of the total bacterial proteins.The purified protein was used to prepare the antibody against chicken IL-2 protein. The result showed that the antibody could react with standard chicken IL-2 protein.This study paved the way for further study of biological function and application of chicken IL-2…
Key words: Chicken IL-2 gene; RT-PCR; Molecular cloning; DNA sequencing; Chicken IL-2 fusion protein; Prokaryotic expression; Antibody preparation

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