抗黄曲霉毒素B1单链抗体在大肠杆菌中的表达及优化  

Expression and optimization of anti-AFB1 scFv in Escherichia coli

作　　者：杨炼[1] 张艳红[1] 丁虎生[1] 王丽云[1] 陈卫[1] 张灏[1] 

机构地区：[1]江南大学食品学院,食品科学与技术国家重点实验室,无锡214122

出　　处：《微生物学报》2009年第7期880-888,共9页Acta Microbiologica Sinica

基　　金：教育部新世纪优秀人才支持计划资助项目(NCET-06-0482)~~

摘　　要：【目的】继杂交瘤技术后,重组抗体技术是新一代的抗体制备技术。然而如何用原核系统中较多地表达具有生物活性的单链抗体,避免包涵体形成仍是一个需要探讨的问题。【方法】将目的基因scFv-H4克隆到载体pET22b上,分别转入大肠杆菌BL21(DE3)和Origami(DE3)中,通过改变诱导温度和IPTG浓度,比较具有生物活性的蛋白量以及包涵体的量。【结果】在BL21(DE3)中,pET22b能产生大量表达scFv-H4,而BL21(DE3)的含有trxA和gor双突变的衍生菌Origami(DE3)表达的scFv-H4的总量较少,但是具有生物活性的蛋白量较多(35 mg/L培养物),具有生物活性的蛋白比例也较BL21(DE3)高。另外IPTG的浓度对scFv-H4表达没有显著影响,而较高的诱导温度会促使表达的蛋白形成包涵体。【结论】在较低的温度下,pET22b能在Origami(DE3)能较好地表达具有生物活性的scFv-H4,减少包涵体的比例,为后续的抗体性质研究和改造奠定了基础。[Objective] A drawback of the expression of single chain antibody fragment （scFv） in prokaryotic system is the protein accumulation in the cytoplasm as inclusion body. We aimed at high-level production of an anti-aflatoxin B1 scFv in functional form. [Methods]The gene of scFv-H4 was cloned into pET22b vector and transformed into E. coli BL21（DE3） and Origami （DE3）, respectively. The amount of functional scFv-H4 was optimized in terms of IPTG concentration and induction temperature. [ Results]scFv-H4 could be expressed in both B[21 （DE3） and Origami （ DE3）. Compared with BL21 （DE3）, Origami（DE3） could express multifunctional scFv-H4 （35 mg/mL） and less in inclusion body （11% of the total expression）. The expression of scFv-H4 was significantly affected by induction temperature rather than IPTG concentration. [ Conclusion] The pET22b could be used for high-level expression of the functional scFv-H4 in Origami （DE3）, which has an oxidative cytoplasm. In addition, the induction at low temperature avoided the formation of inclusion body.

关 键 词：黄曲霉毒素 单链抗体 表达 B121(DE3) Origami(DE3) pET22b 

分 类 号：Q786[生物学—分子生物学]