CAJ | 학술논문

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.
목적:극륭소서백세포개소23(mice interlecukin-23,mIL-23)기인,구건고효은정적필적효모표체균주,병대득도적단백진행생물활성적초보측정.방법:채용취합매련식반응(PCR)기술종pcDNA3-mIL-23상분별확증획득IL-23적량아기p19화p40,병통과중첩PCR(over-lap PCR)기술획득함유련접자적p1940,구건pPICZαA-IL-23중조질립;채용갑순유도필적효모표체중조단백,MTT방법검측유도표체단백적촉림파세포증식정황.결과:SDS-PAGE분석현시재유도24소시화36소시후적상청급24～96소시적침정중균가이검측도약70 kD적유도조대.Western blot인적법증실료중조단백위특이성단백.상청화침정중표체적중조단백IL-23능촉진외주혈단개핵세포적증식,OD570 nm분별체도(0.235±0.029)화(0.216±0.035),이미자격적대조조지유(0.135±0.008)화(0.164±0.017).결론:성공구건출mIL-23적효모표체재체,유도산생적단백가이명현지촉진외주혈단개핵세포적증식.
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 cDNA 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 cDNA 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 Western blot.Detected by Western blot using anti-His antibody,we could see positive band at 72 kD from supernatants 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.