摘要
目的:原核表达抗克伦特罗重组单链抗体,对表达的包涵体蛋白进行提取和纯化,并鉴定重组抗体特性。方法:通过温度诱导大肠杆菌表达抗克伦特罗单链抗体,超声破碎菌体细胞,采用不同洗涤溶液提取包涵体。以不同变性剂溶解包涵体,Ni-NTA亲和柱纯化后,经脉冲稀释法将其复性。超滤及透析浓缩蛋白复性液,经Ni-NTA亲和柱二次纯化,得到纯化重组目的蛋白。通过SDS-PAGE电泳分析重组目的蛋白纯度,采用Western blotting和间接竞争ELISA对所制备的抗克伦特罗重组单链抗体特异性进行鉴定。结果:以大肠杆菌Es-cherichia coli BL21(DE3)为宿主菌诱导表达重组单链抗体,产率最高,占菌体蛋白的31%。以1mol/L尿素洗涤包涵体,6mol/L盐酸胍为变性剂,经Ni-NTA亲和柱纯化,所得复性重组单链抗体蛋白的纯度达96%。Western blotting分析显示,纯化并复性后的单链抗体可与鼠抗His标签蛋白单克隆抗体发生特异性的结合反应。间接竞争ELSIA结果表明,该重组抗体IC50值为3.35ng/mL,与沙丁胺醇等结构类似物无交叉反应。结论:原核表达制备的抗克伦特罗重组单链抗体具有较好的抗原结合活性及特异性,为进一步开展克伦特罗的免疫快速检测研究奠定基础。
Objective： To extract and purify the inclusion bodies of single chain Fv （scFv） antibodies against clenbuterol （CBL） expressed in prokaryotic cells and to identify the characteristics of the target recombinant antibody. Methods： The expression of anti-CBL scFv antibodies in Escherichia coli （E.coli） was induced by temperature. The inclusion bodies were extracted through sonication lysis and washed with different detergents. Different denaturants were used to dissolve the inclusion bodies. The solubilized inclusion bodies were purified by Ni-NTA column chromatography and then refolded by pulsed-dilution method. After concentrated by ultra.filtration and dialysis, the renaturated recombinant target proteins were purified by Ni-NTA column chromatography once again. The final purity of the recombinant scFv antibodies was identified by SDS-PAGE analysis and the specificity was identified by Western blotting analysis and indirect competitive ELISA. Results： The 6xHis-tagged anti-CBL scFv antibodies were expressed with the yield of 31% of the total cell proteins in E.coll BL21 （DE3） strain cells. The detergent containing 1 mol/L urea and the denaturant containing 6 mol/L guanidinium chloride were optimal. The purity of the recombinant scFv antibodies was about 96% by SDS-PAGE analysis. Western blotting analysis showed the obtained anti-CBL scFv antibodies posed HRP-anti-His-tag antibody-recognized activity. Moreover, an indirect competitive ELISA revealed that the IC50 value of the anti-CBL scFv antibody was 3.35 ng/ mL. The cross reactivity studies showed that the anti-CBL scFv antibody poses high specificity to CBL and has little reactivity to the analogues. Taken together, these findings suggest that the prepared recombinant antibody can be used for future immunoassay detection for CBL.
出处
《中国食品学报》
EI
CAS
CSCD
北大核心
2009年第4期45-50,共6页
Journal of Chinese Institute Of Food Science and Technology
基金
国家自然科学基金(30871755
30400354)
国家"863"计划(2007AA10Z437)
广东省自然科学基金(06025825)
关键词
克伦特罗
单链抗体
原核表达
纯化
Clenbuterol Single chain Fv Prokaryotic expression Purification
