抗克伦特罗单链抗体在毕赤酵母GS115中的表达  

Expression of a single chain Fv antibody against clenbuterol in Pichia pastoris

作　　者：陈舒迟[1] 何永盛[1] 徐小艳[1] 陆田珍[1] 王弘[1] 

机构地区：[1]广东省食品质量与安全重点实验室/华南农业大学食品质量与安全研究所,广东广州510642

出　　处：《食品工业科技》2011年第7期217-219,236,共4页Science and Technology of Food Industry

基　　金：国家自然科学基金(30400354);广东省自然科学基金(04300502)

摘　　要：构建表达抗克伦特罗单链抗体(CBL scFv)的毕赤酵母菌株,筛选高拷贝转化子并诱导CBL scFv在毕赤酵母中的分泌表达。将构建的含有CBL scFv基因的重组真核表达载体pPICZαA-CBL经BstX I酶切线性化后,利用电转化方法转化毕赤酵母菌GS115感受态细胞,通过Zeocin抗性平板筛选高拷贝转化子,以甲醇诱导蛋白表达,对表达产物进行SDS-PAGE、Western blotting鉴定及ELISA活性分析。结果显示,通过筛选出的重组毕赤酵母菌株分泌表达了1个分子量约为41kDa的蛋白,该蛋白可与鼠抗His标签单克隆抗体发生特异性结合,且可被游离的CBL竞争性抑制,IC50值为5.1ng/mL。经检测,重组抗体表达量为0.095g/L。这说明分泌表达CBL scFv的毕赤酵母菌株构建成功,为后期CBL scFv发酵与制备奠定了基础。A Pichia pastoris strain expressing recombinant anti-clenbutero （CBL） single chain Fv （scFv） was constructed.The recombinant plasmid, pPICZαA-CBL,was linearized by endonuclease BstX I and transformed into Pichia pastoris GS115 by electroporation.The multicopy transformants were obtained by Zeocin hyper-resistance screening and were induced to express by methanol.The induced culture supernatant was collected and detected by SDS-PAGE, western bloting and indirect competitive ELISA.The results showed that CBL scFv gene was inserted into the chromosome of GS115 successfully. The CBL scFv expressed from the pPICZocA- CBL recombinants showed 41kDa and anti-His-tag mAb recognized-activity by SDS-PAGE and Western blotting respectively,and could competitively combine with CBL, the IC50 was 5.1ng/mLThe yield of the CBL scFv was 0. 095g/L.It suggested that a Pichia pastoris strain expressing CBL scFv had been constructed successfully.Further large scale production of CBL scFv could be continued on the basis of our study.

关 键 词：克伦特罗 单链抗体 毕赤酵母 蛋白表达 

分 类 号：TS201.3[轻工技术与工程—食品科学]