Abstract：
Small single-chain variable fragments (scFv) are promising biomolecules to inhibit and neutralize toxins and to act as antivenoms. In this work, we aimed to produce a functional scFv-6009FV in the yeast Pichia pastoris, which inhibits the pure Cn2 neurotoxin and the whole venom of Centruroides noxius. We were able to achieve yields of up to 31.6 ± 2 mg/L in flasks. Furthermore, the protein showed a structure of 6.1 % α-helix, 49.1 % β-sheet, and 44.8 % of random coil by CD. Mass spectrometry confirmed the amino acid sequence and showed no glycosylation profile for this molecule. Purified scFv-6009FV allowed us to develop anti-scFvs in rabbits, which were then used in affinity columns to purify other scFvs. Determination of its half-maximal inhibitory concentration value (IC50) was 40 % better than the scFvs produced by E. coli as a control. Finally, we found that scFv-6009FV was able to inhibit ex vivo the pure Cn2 toxin and the whole venom from C. noxius in murine rescue experiments. These results demonstrated that under the conditions assayed here, P. pastoris is suited to produce scFv-6009FV that, compared to scFvs produced by E. coli, maintains the characteristics of an antibody and neutralizes the Cn2 toxin more effectively.
摘要：
小的单链可变片段（scFv）是有前途的生物分子，可以抑制和中和毒素并充当抗蛇毒血清。在这项工作中,我们的目标是在巴斯德毕赤酵母中产生功能性scFv-6009FV，抑制纯Cn2神经毒素和Centruroidesnoxius的整个毒液。我们能够在烧瓶中获得高达31.6±2mg/L的产量。此外，蛋白质显示6.1%的α-螺旋结构,49.1%β-折叠，和44.8%的无规卷曲由CD。质谱证实了氨基酸序列,并且没有显示该分子的糖基化谱。纯化的scFv-6009FV允许我们在兔子中开发抗scFv，然后将其用于亲和柱中以纯化其他scFvs。测定其半最大抑制浓度值(IC50)比作为对照的由大肠杆菌产生的scFvs好40%。最后,我们发现scFv-6009FV能够体外抑制纯Cn2毒素和小鼠解救实验中来自C.noxius的整个毒液。这些结果表明，在这里分析的条件下，巴斯德毕赤酵母适合生产scFv-6009FV，与大肠杆菌产生的scFvs相比，保持抗体的特性并更有效地中和Cn2毒素。
