小鼠白细胞介素23基因在毕赤酵母中的诱导表达及初步活性研究

Expression of mice interlecukin-23 in pichia and preliminary studies on its biology function

目的:克隆小鼠白细胞介素23(mice interlecukin-23,mIL-23)基因,构建高效稳定的毕赤酵母表达菌株,并对得到的蛋白进行生物活性的初步测定。方法:采用聚合酶链式反应(PCR)技术从pcDNA3-mIL-23上分别扩增获得IL-23的两亚基p19和p40,并通过重叠PCR(over-lap PCR)技术获得含有连接子的p1940,构建pPICZαA-IL-23重组质粒;采用甲醇诱导毕赤酵母表达重组蛋白,MTT方法检测诱导表达蛋白的促淋巴细胞增殖情况。结果:SDS-PAGE分析显示在诱导24小时和36小时后的上清及24～96小时的沉淀中均可以检测到约70 kD的诱导条带。Western blot印迹法证实了重组蛋白为特异性蛋白。上清和沉淀中表达的重组蛋白IL-23能促进外周血单个核细胞的增殖,OD570 nm分别达到(0.235±0.029)和(0.216±0.035),而未刺激的对照组只有(0.135±0.008)和(0.164±0.017)。结论:成功构建出mIL-23的酵母表达载体,诱导产生的蛋白可以明显地促进外周血单个核细胞的增殖。

Objective：To clone the mice interleukin-23（mIL-23） and express it in Pichia efficiently. Methods： Two subunits of IL-23 were amplified by PCR from pcDNA3/mIL-23, and ligated together with adaptor by over-PCR. The IL-23 eDNA confirmed by sequencing was inserted into expressing vector pPICZαA and expressed in Pichia X-33 strain. IL-23 protein expression was induced by methanol and ammonia. The recombinant IL-23 was identified by immunoblot and its biological activity was analyzed. Results： DNA sequencing confirmed that cloned eDNA was identical to the published sequence of mIL-23. The recombinant plasmid pPICZαA/mIL-23 was electroprated into X-33. An expected 72 kD protein of mIL-23 was founded both in the induced pellets and supermatants by SDS-PAGE and coomassie blue staining. The 72 kD protein could be recognized in Western blot. While we could detect bands at 72 kD in cell pellets induced more than 24 h by Westem blot. Detected by Westem blot using anti-His antibody, we could see positive band at 72 kD from supematants induced 24 and 36 h. While we could detect band at 72 kD in cell pellets induced between 24 h and 96 h. Meanwhile, we could see obviously proliferation of mice peripheral blood mononuclear cells（PBMC） treated with the induced pellets and supermanants. Conclusion：We have successfully expressed mIL-23 protein in Pichia and the expressed product has its bioactivity in promoting the proliferation on PBMC.