Cloning,expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>, and purification of soluble recombinant duck interleukin-2 |
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Authors: | Cuihong du Long Han Zhijun Xie |
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Institution: | (1) School of Biotechnology Engineering, Jimei University, Xiamen, 361021, China |
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Abstract: | Interleukin-2 (IL-2) is a vital cytokine secreted by activated T lymphocytes, and plays an important role in the regulation
of cellular and immunity of animals. In this study, a gene encoding duck IL-2 was cloned and the soluble recombinant duck
IL-2 (rDuIL-2) was expressed in Escherichia coli via fusion with glutathione S-transferase (GST). The results indicated that the GST-rDuIL-2 fusion protein expressed in E. coli Origami (DE3) was confirmed to be of about 40 kDa molecular mass by SDS-PAGE and western blotting. In order to produce soluble
rDuIL-2 in a low-cost, nontoxic and high-level expression process, lactose was used as a substitute for Isopropyl-β-D-thiogalactopyranoside
(IPTG) to induce the above recombinant strain Origami (pGEX-DuIL-2). By optimizing the expression conditions, the production
of soluble GST-rDuIL-2 fusion protein was about 29% of total cellular soluble proteins, which was similar with IPTG used as
inducer. The soluble GST-rDuIL-2 fusion protein was purified by one-step affinity chromatography, and GST was removed by thrombin.
Then rDuIL-2 was purified by a second affinity chromatography. As a result, the 95% pure rduIL-2 was obtained, and the yield
of rDuIL-2 was about 10.6 mg/l bacterial culture. The bioactivity of rduIL-2 was determined by lymphocyte proliferation assay
in vitro. Our study provided a feasible and convenient approach to produce soluble and biologically active rDuIL-2, which
would be used as an immunoadjuvants for enhancing vaccine efficacy. |
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