| Forest musk deer (Moschus berezovskii) is a precious animal, and has been listed as a category I species under the Wild Animal Protection Law of China. The musk, which is the natural product of male musk deer, is one of the most expensive traditional Chinese medicines and prevalently used in Southeast Asia countries. The market requirements for musk have resulted in severe problems, such as over-hunting for wild musk deer, which has leaded to steeply declining of the wild musk-deer populations with the concomitant habitats lose. Captive breeding for forest musk deer has been started since 1958 in China, but the population sizes and the treatments for severe diseases have not been improved dramatically until now. Many of the musk deer in zoos and on farms still died of infectious diseases. Researches showed that the purulent infectious diseases killed the largest number of farmed musk deer, with the percentage of 50-70% in died individuals.Interferon gamma is an important mediator of the immune system and produced primarily by activated T lymphocytes and natural killer (NK) cells. IFN-γcan promote the activation of T and B lymphocytes and the expression of the Fc receptor of IgG, boost up the MHC antigen expression of the antigen submitting cells, enhance the interaction of antigen submitting cells and T lymphocytes, potentate the generating of T lymphocytes assistant antibody and helper cytotoxic T lymphocytes, and induce the humoral immune response of macrophage. Therefore, IFN-γcan reinforce the immune ability of the economy observably. In human and other species, IFN-γhas been used for clinic therapy of tumor and diseases caused by virus infection, as well as many other diseases. In order to apply this cytokine to the immune therapy of forest musk deer, we conduct a research on cloning and characterization of IFN-γgene from forest musk deer, and its expression in E.coli and yeast.Two cloning primers were designed from conserved regions of the Bos taurus IFN-γcDNA (Genbank accession No. M29867), then the cDNA encoding M. berezovskii IFN-γ(MB_IFN-γ) was isolated from concanavalin-A stimulated peripheral blood mononuclear cells (PBMC) using RT-PCR. PCR products were purified and ligated into pMD18-T vector and transformed into JM109 competent cells for proliferation. Following PCR verification, sequencing, and sequence analysis, it turned up that the cDNA we cloned from M. berezovskii was the IFN-γcoding sequence. The predicted IFN-γprotein consists of 166 amino acids (23 aa signal peptide and 143 aa mature protein). MB_IFN-γshares a high degree of identity with that of Cervus elaphus (L07502), Bos taurus (M29867), and Capra hircus (U34232).The DNA fragment encoding mature MB_IFN-γwas subcloned into pGEX-4T-l expression vector and transformed into E.coli BL21, and highly expressed with IPTG induction. The expressed protein fused to GST were found to have a molecular weight of about 43 kDa by SDS-PAGE, which is in accordance with predicted molecular weight and account for 26.9% of the total bacterial proteins. The NaOH method was employed to solubilize the inclusion bodies, and the proteins were further purified using a GST purification column.The DNA fragment encoding mature MB_IFN-γwas subcloned to pPIC9K expression vector and transformed into E.coli JM109. Verified by colony PCR, a positive clone was selected for isolating plasmids. The linearized recombinant plasmid was then transformed into the Pichia pastoris strain GS115 by electrotransfromation. Many recombinants were recognized through G418 resistance selection, yeast colony PCR verification. The protein was expressed in the supernatant after induction by methanol for 4 d, and then analyzed by SDS-PAGE. The protein was detected with a molecular weight of 17.3 kDa. As a result, the amount of MBIFN-γwas low in the Pichia pastoris strain GS115, although a higher production has been found in the expression system E.coli BL21.These results provided a basis for further studies on the biological activity of the MB_IFN-γand its clinic therapy for infectious diseases. We expected this protein could be developed into medicine which would be useful in the treatment of forest musk deer diseases, and be benefit to the conservation of this precious and endangered animal.
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