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首页> 外文期刊>Cell Research >Generation and selection of immunized Fab phage display library against human B cell lymphoma
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Generation and selection of immunized Fab phage display library against human B cell lymphoma

机译：针对人B细胞淋巴瘤的免疫Fab噬菌体展示文库的产生和选择

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The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production of monoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody (HAMA) response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the k light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94 × 10~7. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains (V_H) and variable light domains (V_L) were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.
机译：在B细胞淋巴瘤患者的治疗中使用单克隆抗体作为靶向治疗的批准为该组患者带来了新的治疗选择。通过传统的杂交瘤技术生产单克隆抗体是昂贵的，并且所得的鼠抗体通常具有触发人抗小鼠抗体（HAMA）应答的缺点。因此，通过噬菌体展示技术产生的重组Fab抗体可以作为治疗B细胞淋巴瘤的合适替代品。在这项研究中，我们从用人B淋巴瘤细胞免疫的BALB / c小鼠的脾细胞中提取了总RNA，并使用RT-PCR扩增了编码k轻链和Fd重链片段的cDNA。在适当的限制性消化后，将这些cDNA片段依次插入噬菌粒载体pComb3H-SS中，以构建免疫的Fab噬菌体展示文库。所构建文库的多样性约为1.94×10〜7。经过五轮生物淘选后，通过ELISA鉴定出的阳性克隆产生了可溶性Fab抗体。从八个阳性克隆中，选择FabC06，FabC21，FabC43和FabC59进行序列分析。在氨基酸序列水平上，发现可变重域（V_H）和可变轻域（V_L）分别与相应鼠种系基因编码的序列具有88-92％和89-94％的同源性。此外，通过免疫组织化学和蛋白质印迹证实了与B细胞淋巴瘤的膜蛋白的反应性。这些免疫的Fab抗体可能为进一步研究B细胞淋巴瘤提供有价值的工具，也可能有助于改善疾病治疗。

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