CAJ | 학술논문

目的　在甲醇营养型毕氏酵母蛋白质表达系统中高效表达重组人白介素-13（rhIL-13），便于进一步研究开发。方法　根据酵母对密码子的偏爱性设计并人工合成了人IL-13编码基因片段，经T4DNA连接酶、PCR技术连接形成IL-13 cDNA全长序列；将其插入pPICZαA质粒中，构建成pPICZαA-IL-13表达载体；该载体经SacI线形化后以电转化法导入GS115酵母菌株中，经YPD平板Zeocine抗性筛选获得IL-13表达阳性菌株；用Western Blotting 和ELISA检测发酵上清中IL-13的抗原性和表达量，B9-11细胞株分析其生物学活性。结果　15% SDS-PAGE显示重组人IL-13分子量约13kd，Western-blotting证实为人 IL-13，ELISA测定在摇瓶培养上清表达量达15μg/ml，其生物学活性同标准品一样具有剂量依赖性的刺激B9-11细胞株增殖的作用。结论　成功获得IL-13人工基因和稳定分泌蛋白的基因工程菌株，该重组蛋白的生物学活性达到标准品的要求。
목적　재갑순영양형필씨효모단백질표체계통중고효표체중조인백개소-13（rhIL-13），편우진일보연구개발。방법　근거효모대밀마자적편애성설계병인공합성료인IL-13편마기인편단，경T4DNA련접매、PCR기술련접형성IL-13 cDNA전장서렬；장기삽입pPICZαA질립중，구건성pPICZαA-IL-13표체재체；해재체경SacI선형화후이전전화법도입GS115효모균주중，경YPD평판Zeocine항성사선획득IL-13표체양성균주；용Western Blotting 화ELISA검측발효상청중IL-13적항원성화표체량，B9-11세포주분석기생물학활성。결과　15% SDS-PAGE현시중조인IL-13분자량약13kd，Western-blotting증실위인 IL-13，ELISA측정재요병배양상청표체량체15μg/ml，기생물학활성동표준품일양구유제량의뢰성적자격B9-11세포주증식적작용。결론　성공획득IL-13인공기인화은정분비단백적기인공정균주，해중조단백적생물학활성체도표준품적요구。
Objective　To express recombinant human interleukin-13(rhIL-13) in methylotropic yeast Pichia Pastoris. Methods　An artificial gene with the optinal codon usage of Pichia Pastoris for IL-13 was designed and synthesized by T4DNA ligase and PCR. The expression vector pPICZαA-IL-13 was constracted and introduced into Pichia Pastoris GS115 by electroporation after linearized with SacI.Recombinants were selected by plating cells on YPD/Zeocine plates. The recombinant protein secreted by the yeast was identified by 15% SDS-PAGE, ELISA and Westren Blotting .The bioactivity was analyzed by B9-11 cell line. Results　Based on the result of the 15% SDS-PAGE and Westren Blotting assay，an about 13kd recommbinant protein expressed in the yeast supernatant was identified to be recombinant human IL-13.The secreted yeild of rhIL-13 in flask reached 15μg/ml.The biology activity of the IL-13 in yeast supernatant was the same as that of E. coli standard determined by stimulating the proliferation of B9-11. Conclusion　The gene of recombinant human IL-13 can be obtained and its protein successfully expressed in the Pichia Pastoris. The biological activity of the IL-13 in yeast supertant is the same as that of E.coli standard.