Cloning And Expression Of Muscovy Duck Interleukin-2 Cdna

Interleukin-2 (IL-2) is the most vigorous T cell growth factor, belonging to Th1 cytokine, which is secreted by T leukomonocyte and lymphocyte series. IL-2 is the glycoprotein, having biological extensive activity, elevating immunological function efficiently, inducing T cell multiplication and differentiation, inducing the expression of Interleukin-2 receptor(IL-2R); IL-2 could enhance B cell differentiation, hyperplasic and secretary antibody, at the same time, IL-2 can activate B cell to express highly congeniality IL-2R; IL-2 can induce and strengthen the kill activity of NK cell and histomonocyte, enhancing others cytokine to secrete and stimulate the T cell to grow in vitro. Enhancing leukomonocyte and endotheliocyte surface adhesion factor expression,facilitating the adherence and movement of leukomonocyte and other activation cell; Increasing the MHCⅠandⅡmolecule quantity, which belong to antigen presenting cell surface, enhancing antigen presentation. IL-2 is a native immunological adjuvant, playing a important part in immunologic system.Based on the published nucleotide sequence of Anas platyrhynchos interleukin-2, a pair of primers was designed and synthesized by myself. with the technology of RT-PCR, a cDNA fragment of 423bp was amplified from the spleen lymphocytes total RNA template ,which actived with ConA of 30-day-old poult Muscovy duck, and cloned to pMD-18T vector by the strategy of T-A. They are identified to be interleukin-2 cDNA with the methods of PCR, restriction enzyme digestion and sequencing. They are identical to the nucleotide sequences reported in gene bank. It covers all Muscovy duck interleukin-2 ORF. Encoding the protein composed from 140 amino acids. Comparing with the sequences of Anas platyrhynchos reported in gene bank, there are mutation nucleotides of seven, but the predicted second structure of encoding amino acid is similar. The predicted second structure of encoding amino acid indicated, they haveα-helices being similarity to the ones of mammals. IL-2sequence analysis indicated that Muscovy duck interlink-2 have higher homology with other Avian, which accordingly certificated Muscovy duck interlink-2 have fairly conservatism.By the technology of DNA recombination, the IL-2cDNA was cloned to expression plasmid pGEX6P-1 vector, and transformed to BL21 (DE3) competent cell. The recombinant protein was expressed by the inducing of IPTG. The results of SDS-PAGE indicated that the production is 40KD(fusion protein), and the production of recombinant IL-2 reached its peak about 3 hours after inducing at 35℃,0.1mM IPTG. The results manifested that pGEX-IL2 can express GST-IL2 fusion protein, and the molecular weight of Muscovy duck interleukin-2 is 14KD, which is identical to the expectancy of Muscovy duck interleukin-2 maturation protein. Initial abstraction by supersonic indicated that most of recombinant expression protein exists in sentiment. The success expression of Muscovy duck gene that provide the potential application prospect for immunopotentiating agent and immunologic adjuvant.