Selisko Barbara, Cosío Gabriela, García Consuelo, Becerril Baltazar, Possani Lourival D, Horjales Eduardo
Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology/National Autonomous University of Mexico (UNAM), Av. Universidad 2001, Apartado Postal 510-3, Cuernavaca, Morelos 62210, Mexico.
Toxicon. 2004 Jan;43(1):43-51. doi: 10.1016/j.toxicon.2003.10.015.
The murine monoclonal antibody BCF2 is able to neutralize the venom of the scorpion Centruroides noxius Hoffmann. A chimeric Fab of BCF2 (chFab-BCF2) comprising the variable regions of murine BCF2 and human constant regions was assembled. chFab-BCF2 was expressed as a soluble and functional protein in the periplasmic space of Escherichia coli. An expression yield of 1 mg/l was reached by combination of late-log-phase induction, rich culture medium, low expression temperature and addition of sucrose (0.3 M) to the culture medium. The addition of sucrose induced secretion of 60% of the protein into the medium. After expression for 23 h, a novel process was used to release the remaining periplasmic protein in situ consisting in the addition of lysozyme and sucrose up to 0.6 M (20%) directly to the culture medium. chFab-BCF2 was recovered by ammonium sulfate precipitation and purified in a single step by affinity chromatography using anti-human anti-F(ab')(2) IgG coupled to Sepharose-proteinG. Pure chFab-BCF2 maintained a similar nanomolar affinity as BCF2 to its cognate antigen, the Na(+)-channel-affecting toxin Cn2. Recombinant chFab-BCF2 was able to neutralize Cn2 in vivo even at a molar ratio of 1:1, as well as the whole venom of C. noxius. Thus, it is a promising candidate to be used as a specific and efficient recombinant antidote against scorpion stings.
鼠单克隆抗体BCF2能够中和墨西哥毒蝎霍夫曼氏毒蝎（Centruroides noxius Hoffmann）的毒液。构建了一种嵌合Fab片段（chFab-BCF2），其包含鼠BCF2的可变区和人恒定区。chFab-BCF2在大肠杆菌的周质空间中表达为可溶性功能蛋白。通过对数后期诱导、丰富培养基、低表达温度以及向培养基中添加蔗糖（0.3 M）的组合，实现了1 mg/l的表达产量。添加蔗糖可使60%的蛋白分泌到培养基中。表达23小时后，采用一种新方法原位释放剩余的周质蛋白，即直接向培养基中添加溶菌酶和蔗糖至0.6 M（20%）。通过硫酸铵沉淀回收chFab-BCF2，并使用偶联到琼脂糖蛋白G的抗人抗F(ab')(2) IgG通过亲和色谱一步纯化。纯的chFab-BCF2与其同源抗原、影响钠通道的毒素Cn2保持与BCF2相似的纳摩尔亲和力。重组chFab-BCF2即使在1:1的摩尔比下也能够在体内中和Cn2以及墨西哥毒蝎的全毒液。因此，它是一种有前景的候选物，可作为针对蝎蜇伤的特异性高效重组解毒剂。
