Kaplan D, Baldi C, Chiaramonte M G, Fernandez M M, Levin M J, Malchiodi E, Baldi A
Institute of Biology and Experimental Medicine (IBYME), Buenos Aires, Argentina.
Biochem Biophys Res Commun. 1998 Dec 9;253(1):53-8. doi: 10.1006/bbrc.1998.9654.
Cruzipain, the major proteinase of Trypanosoma cruzi, plays an important role in the biology of this parasite. This study reports the development of a recombinant Fab antibody, using RNA isolated from the anti-Ag163B6 hybridoma against cruzipain. This procedure involves the use of cDNAs obtained with the aid of a specific set of primers complementary to the complete light kappa chain (L kappa) and the first two domains of the IgG1 heavy chain (VH/CH1). These products were subsequently cloned in the pComb3 system, from which the gIII gene had been removed, and expressed in Escherichia coli cells. The recombinant Fab molecule recognized cruzipain by ELISA, in a fashion similar to the original mAb anti-Ag163B6. Nucleotide sequence analysis of the recombinant molecule, together with its immunological recognition by specific anti-mouse IgG (Fab)2, indicated the immunoglobulin nature of the recombinant product. Moreover, both the mAb anti-Ag163B6 and the soluble Fab fragment described here react similarly with the intact parasite surface, as observed in an indirect immunofluorescence assay. In conclusion, our recombinant Fab anti-Ag 163B6 allows the possible use of this molecule for diagnosis, antigen purification, and eventually treatment of Chagas-afflicted individuals.
克氏锥虫主要蛋白酶克鲁兹蛋白酶在该寄生虫的生物学特性中发挥着重要作用。本研究报告了一种重组Fab抗体的研发过程，该抗体利用从抗Ag163B6杂交瘤中分离的RNA针对克鲁兹蛋白酶制备。此过程涉及使用借助一组与完整κ轻链（Lκ）和IgG1重链的前两个结构域（VH/CH1）互补的特异性引物获得的cDNA。这些产物随后被克隆到已去除gIII基因的pComb3系统中，并在大肠杆菌细胞中表达。重组Fab分子通过ELISA识别克鲁兹蛋白酶，其方式与原始单克隆抗体抗Ag163B6相似。重组分子的核苷酸序列分析及其被特异性抗小鼠IgG（Fab）2的免疫识别表明了重组产物的免疫球蛋白性质。此外，如间接免疫荧光试验中所观察到的，抗Ag163B6单克隆抗体和此处描述的可溶性Fab片段与完整寄生虫表面的反应相似。总之，我们的重组Fab抗Ag 163B6使得该分子有可能用于诊断、抗原纯化以及最终治疗恰加斯病患者。
