生物工程学报
生物工程學報
생물공정학보
CHINESE JOURNAL OF BIOTECHNOLOGY
2000年
3期
316-319
,共4页
张毅%屈贤铭%杨胜利
張毅%屈賢銘%楊勝利
장의%굴현명%양성리
融合蛋白%基因表达%细胞因子
融閤蛋白%基因錶達%細胞因子
융합단백%기인표체%세포인자
Fusion protein%gene expression%haemopoietic growth factors
利用PCR扩增得到粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素-3(IL-3)完整基因片段,将其分别克隆至pGEM-T,构建成GM-CSF/IL-3融合蛋白基因,DNA序列与设计预期一致。将得到的融合蛋白基因克隆至T7RNA聚合酶表达载体pT7zz,得到表达质粒pFu,经转化至表达宿主E.coli BL21(DE3),在IPTG诱导下获得融合蛋白目的产物的直接表达。经SDS-PAGE电泳鉴定扫描分析,目的基因产物表达量占菌体总蛋白量的30%以上,目的基因表达产物以包涵体的形式表达。Western-blot鉴定表明,该表达产物可以与GM-CSF抗体及IL-3抗体特异性结合。目的基因表达产物经过包涵体变性、透析复性及柱层析纯化,用GM-CSF、IL-3依赖细胞株TF-l检测,具有明显的生物学活性。
利用PCR擴增得到粒細胞-巨噬細胞集落刺激因子(GM-CSF)、白細胞介素-3(IL-3)完整基因片段,將其分彆剋隆至pGEM-T,構建成GM-CSF/IL-3融閤蛋白基因,DNA序列與設計預期一緻。將得到的融閤蛋白基因剋隆至T7RNA聚閤酶錶達載體pT7zz,得到錶達質粒pFu,經轉化至錶達宿主E.coli BL21(DE3),在IPTG誘導下穫得融閤蛋白目的產物的直接錶達。經SDS-PAGE電泳鑒定掃描分析,目的基因產物錶達量佔菌體總蛋白量的30%以上,目的基因錶達產物以包涵體的形式錶達。Western-blot鑒定錶明,該錶達產物可以與GM-CSF抗體及IL-3抗體特異性結閤。目的基因錶達產物經過包涵體變性、透析複性及柱層析純化,用GM-CSF、IL-3依賴細胞株TF-l檢測,具有明顯的生物學活性。
이용PCR확증득도립세포-거서세포집락자격인자(GM-CSF)、백세포개소-3(IL-3)완정기인편단,장기분별극륭지pGEM-T,구건성GM-CSF/IL-3융합단백기인,DNA서렬여설계예기일치。장득도적융합단백기인극륭지T7RNA취합매표체재체pT7zz,득도표체질립pFu,경전화지표체숙주E.coli BL21(DE3),재IPTG유도하획득융합단백목적산물적직접표체。경SDS-PAGE전영감정소묘분석,목적기인산물표체량점균체총단백량적30%이상,목적기인표체산물이포함체적형식표체。Western-blot감정표명,해표체산물가이여GM-CSF항체급IL-3항체특이성결합。목적기인표체산물경과포함체변성、투석복성급주층석순화,용GM-CSF、IL-3의뢰세포주TF-l검측,구유명현적생물학활성。
A human granulocyte-macrophage colony stimulating factor (GM-CSF)/interleukin-3(IL-3) fusion gene with a short linker between the GM-CSF and IL-3 gene has been successfully constructed and expressed in E. coli under the control of T7 promoter. The recombinant fusion protein was expressed as inclusion bodies after the IPTG induction. The yield of the GM-CSF/IL-3 fusion protein was over 30 % of the total cellular proteins. Western-blotting results showed that the fusion protein could specifically combined with GM-CSF antibody and IL-3 antibody. The biological activity was detected by the GM-CSF and IL-3 dependent cell line TF-1. After solubilizing with 8mol/L urea and renaturing with dialysis against Tris. HCl solution,the refolded fusion protein showed obvious activities to maintain the growth of TF-1 cell.