中国医学科学院学报
中國醫學科學院學報
중국의학과학원학보
ACTA ACADEMIAE MEDICINAE SINICAE
2001年
2期
127-131
,共5页
朱剑昆%许志祥%黄伟达%闵太善%谢炜%徐颖%张学光
硃劍昆%許誌祥%黃偉達%閔太善%謝煒%徐穎%張學光
주검곤%허지상%황위체%민태선%사위%서영%장학광
重组人白细胞介素-11(rhIL-11)毕氏酵母表达分离纯化
重組人白細胞介素-11(rhIL-11)畢氏酵母錶達分離純化
중조인백세포개소-11(rhIL-11)필씨효모표체분리순화
目的在甲醇营养型毕氏酵母蛋白质表达系统中高效表达人白细胞介素-11(rhIL-11),便于进一步开发。方法以人工设计合成的rhIL-11基因,构建表达载体pPICZαA-IL-11,经线性化后电转化导入毕氏酵母菌株KM71,甲醇诱导表达,用ELISA和SDS-PAGE检测发酵上清中IL-11的抗原性和表达量,用IL-11依赖的B9-11细胞株分析其生物学活性,采用疏水层析、离子交换和凝胶过滤纯化发酵上清中的IL-11。结果序列分析表明,克隆载体中IL-11人工基因序列与设计相符;基因工程菌株KM71-2424在摇瓶培养上清中IL-11的表达量超过60mg/L,生物学活性测定显示其比活性为5.5×107U/mg,而标准品的生物学活性为2.2×107 U/mg。经过三步层析纯化得到电泳纯的rhIL-11蛋白质。结论成功获得IL-11人工基因和稳定分泌重组蛋白的基因工程菌株KM71-2424,该重组蛋白的生物学活性显著高于大肠杆菌表达的标准品,并获得较高纯度的重组蛋白。
目的在甲醇營養型畢氏酵母蛋白質錶達繫統中高效錶達人白細胞介素-11(rhIL-11),便于進一步開髮。方法以人工設計閤成的rhIL-11基因,構建錶達載體pPICZαA-IL-11,經線性化後電轉化導入畢氏酵母菌株KM71,甲醇誘導錶達,用ELISA和SDS-PAGE檢測髮酵上清中IL-11的抗原性和錶達量,用IL-11依賴的B9-11細胞株分析其生物學活性,採用疏水層析、離子交換和凝膠過濾純化髮酵上清中的IL-11。結果序列分析錶明,剋隆載體中IL-11人工基因序列與設計相符;基因工程菌株KM71-2424在搖瓶培養上清中IL-11的錶達量超過60mg/L,生物學活性測定顯示其比活性為5.5×107U/mg,而標準品的生物學活性為2.2×107 U/mg。經過三步層析純化得到電泳純的rhIL-11蛋白質。結論成功穫得IL-11人工基因和穩定分泌重組蛋白的基因工程菌株KM71-2424,該重組蛋白的生物學活性顯著高于大腸桿菌錶達的標準品,併穫得較高純度的重組蛋白。
목적재갑순영양형필씨효모단백질표체계통중고효표체인백세포개소-11(rhIL-11),편우진일보개발。방법이인공설계합성적rhIL-11기인,구건표체재체pPICZαA-IL-11,경선성화후전전화도입필씨효모균주KM71,갑순유도표체,용ELISA화SDS-PAGE검측발효상청중IL-11적항원성화표체량,용IL-11의뢰적B9-11세포주분석기생물학활성,채용소수층석、리자교환화응효과려순화발효상청중적IL-11。결과서렬분석표명,극륭재체중IL-11인공기인서렬여설계상부;기인공정균주KM71-2424재요병배양상청중IL-11적표체량초과60mg/L,생물학활성측정현시기비활성위5.5×107U/mg,이표준품적생물학활성위2.2×107 U/mg。경과삼보층석순화득도전영순적rhIL-11단백질。결론성공획득IL-11인공기인화은정분비중조단백적기인공정균주KM71-2424,해중조단백적생물학활성현저고우대장간균표체적표준품,병획득교고순도적중조단백。
Objective To express recombinant human interleukin-11 (rhIL-11 ) in methylotropic yeast Pichia pas toris. Methods By designing and synthesizing an artificial gene for IL-11, the expression vector pPICZα-A-IL-11 was constructed and introduced into Pichiapastoris by linearized electroporation. The rhIL-11 pro tein was identified by ELISAand SDS-PAGE analysis. The bioactivity was analyzed by B9-11 cell line. A combination of liquid chromatography was developed to purify the rhIL-11 from ferment supernatant. Results The nucleotide sequence analysis indicated that the sequence of cloned artificial IL-11 gene accorded with that of designed; the secreted yield of rhIL-11 by yeast Pichia pastoris KM71-2424 in flask
reached 60 mg/L. The biological activity of IL-11 in yeast supernatant and E. coli standard determined by B9-11 was 5.5×107 U/mg and 2.2×107 U/mg respectively. The rhIL-11 was purified to electrophoretic purity by a combination of liquid chromatography. Conclusion The human IL-11 artificial gene was obtained and successfully expressed in the Pichia pastoris(KM71-2424). The biological activity of IL-1l in yeast supematant was significantly higher than that of E. coli standard. The rhIL-11 was purified to electrophoretic purity.