中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2008年
2期
114-118
,共5页
王勇军%颜江华%王阶平%王臻%黄志平%陈彩霞
王勇軍%顏江華%王階平%王臻%黃誌平%陳綵霞
왕용군%안강화%왕계평%왕진%황지평%진채하
通用载体%单域抗体%核心链酶亲和素%肺癌
通用載體%單域抗體%覈心鏈酶親和素%肺癌
통용재체%단역항체%핵심련매친화소%폐암
Universal carrier%Single-domaJo antibody%Core-streptavidin%Lung cancer
目的 制备一种新型的肺癌靶向通用载体hu3D3与核心链霉亲和素(core-streptavidin,cSA)的融合蛋白hu3D3/cSA,并鉴定其活性.方法 PCR扩增cSA基因,克隆至质粒hu3D3/pET-22b(+)而获得表达载体hu3D3/cSA/pET-22b(+),并在E. coli BL21(DE3)中表达,镍亲和层析柱纯化hu3D3/cSA融合蛋白,采用TEA缓冲液进行复性.SDS-PAGE和Western blot鉴定融合蛋白四聚体的形成.FITC标记hu3D3/cSA,通过荧光显微镜分析hu3D3组分的抗原反应性和特异性,流式细胞术比较hu3D3/cSA和hu3D3与靶点的结合能力;ELISA和Western blot鉴定cSA组分结合生物素(biotin)的能力.结果 获得序列正确的hu3D3/cSA/pET-22b(+)重组子,融合基因在E.coli BL21(DE3)中主要以包涵体的形式表达.纯化复性后的融合蛋白能够形成四聚体复合物,保留了cSA结合生物素的活性,hu3D3组分能与肺癌细胞表面抗原结合,且结合能力与单体hu3D3相比有明显增强.结论 成功制备了具有结合靶抗原和生物素活性的hu3D3/cSA融合蛋白,同时改善了hu3D3组分结合其靶点的亲合力(avidity).因此,hu3D3/cSA可以作为一个通用的肺癌靶向载体,与多种生物素化的抗肿瘤药物或试剂联用,有望为多药物联合靶向治疗肺癌提供一种可行的方法.
目的 製備一種新型的肺癌靶嚮通用載體hu3D3與覈心鏈黴親和素(core-streptavidin,cSA)的融閤蛋白hu3D3/cSA,併鑒定其活性.方法 PCR擴增cSA基因,剋隆至質粒hu3D3/pET-22b(+)而穫得錶達載體hu3D3/cSA/pET-22b(+),併在E. coli BL21(DE3)中錶達,鎳親和層析柱純化hu3D3/cSA融閤蛋白,採用TEA緩遲液進行複性.SDS-PAGE和Western blot鑒定融閤蛋白四聚體的形成.FITC標記hu3D3/cSA,通過熒光顯微鏡分析hu3D3組分的抗原反應性和特異性,流式細胞術比較hu3D3/cSA和hu3D3與靶點的結閤能力;ELISA和Western blot鑒定cSA組分結閤生物素(biotin)的能力.結果 穫得序列正確的hu3D3/cSA/pET-22b(+)重組子,融閤基因在E.coli BL21(DE3)中主要以包涵體的形式錶達.純化複性後的融閤蛋白能夠形成四聚體複閤物,保留瞭cSA結閤生物素的活性,hu3D3組分能與肺癌細胞錶麵抗原結閤,且結閤能力與單體hu3D3相比有明顯增彊.結論 成功製備瞭具有結閤靶抗原和生物素活性的hu3D3/cSA融閤蛋白,同時改善瞭hu3D3組分結閤其靶點的親閤力(avidity).因此,hu3D3/cSA可以作為一箇通用的肺癌靶嚮載體,與多種生物素化的抗腫瘤藥物或試劑聯用,有望為多藥物聯閤靶嚮治療肺癌提供一種可行的方法.
목적 제비일충신형적폐암파향통용재체hu3D3여핵심련매친화소(core-streptavidin,cSA)적융합단백hu3D3/cSA,병감정기활성.방법 PCR확증cSA기인,극륭지질립hu3D3/pET-22b(+)이획득표체재체hu3D3/cSA/pET-22b(+),병재E. coli BL21(DE3)중표체,얼친화층석주순화hu3D3/cSA융합단백,채용TEA완충액진행복성.SDS-PAGE화Western blot감정융합단백사취체적형성.FITC표기hu3D3/cSA,통과형광현미경분석hu3D3조분적항원반응성화특이성,류식세포술비교hu3D3/cSA화hu3D3여파점적결합능력;ELISA화Western blot감정cSA조분결합생물소(biotin)적능력.결과 획득서렬정학적hu3D3/cSA/pET-22b(+)중조자,융합기인재E.coli BL21(DE3)중주요이포함체적형식표체.순화복성후적융합단백능구형성사취체복합물,보류료cSA결합생물소적활성,hu3D3조분능여폐암세포표면항원결합,차결합능력여단체hu3D3상비유명현증강.결론 성공제비료구유결합파항원화생물소활성적hu3D3/cSA융합단백,동시개선료hu3D3조분결합기파점적친합력(avidity).인차,hu3D3/cSA가이작위일개통용적폐암파향재체,여다충생물소화적항종류약물혹시제련용,유망위다약물연합파향치료폐암제공일충가행적방법.
Objective To prepare a novel fusion protein of hu3D3 and core-streptavidin(hu3D3/cSA) as a universal carrier targeting to lung cancer,and analyze its activities. Methods cSA gene was acquired by PCR,and inserted into plasmid hu3D3/pET-22b(+)to construct a recombinant plasmid hu3D3/cSA/pET-22b(+)．The fusion gene was expressed in E. Coli BL21(DE3).The fusion protein was purified through nickel-affinity chromatography column and renatured using TEA buffer. The tetrameric hu3D3/cSA complex was analyzed by SDS-PAGE and Western blot.The hu3D3/cSA protein was labeled with FITC,then its antigen-binding activity was analyzed using fluorescence microscopy,and the functional affinity of hu3D3/cSA and hu3D3 were analyzed and compared by flow cytometry. The biotin-binding activity of hu3D3/cSA was tested bv EUSA and Western blot. Results The recombinant plasmid hu3D3/cSA/pET-22b(+)with correct sequence was obtained. The fusion protein was found after expression in E. Coli BL21 (DE3)mainly in the form of inclusion body. After being purified and refolded,tetrameric complex was formed. The purified tetrameric hu3D3/cSA complex retained both antigen-binding activity of hu3D3 moiety and biotin-binding activity of cSA moiety:furthermore,the avidity of the hu3D3/cSA to its target antigen increased by about 14 times as compared with that of monomeric hu3D3. Conclusion The fusion protein hu3D3/cSA with both antigen- and biotin-binding activity is SHCCessfully prepared,and the avidity of hu3D3 moiety to 3D3 antigen is enhanced. Consequently,hu3D3/cSA could be a universal carrier targeting to lung cancer, and could be an alternative,convenient method to realize target therapy to lung cancer by the combination of multiple biotinylated anti-tumor drugs or agents.
