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

摘要:

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。

关键词:关键词：黄曲霉毒素;单链抗体;表达; BL21(DE3);Origami (DE3);pET22b

Abstract:

Abstract: [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 BL21(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.

Key words:Keywords: aflatoxin; scFv; expression; BL21(DE3); pET