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首页> 外文期刊>The Kasetsart Journal >Construction of Single-Chain Variable Fragment (scFv) Specific to Cucumber Mosaic Virus by Phage Display Technology
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Construction of Single-Chain Variable Fragment (scFv) Specific to Cucumber Mosaic Virus by Phage Display Technology

机译：噬菌体展示技术构建黄瓜花叶病毒特异性单链可变片段（scFv）

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摘要

Cucumber mosaic virus (CMV) causes serious problems in economically important crops, especially members of the Solanaceae and Cucurbitaceae families. Serological detection of this virus by specific antibodies is required as a control measure as well as for quarantine investigation to ensure any components used for agricultural propagation, especially commercial seeds, are disease free. However, the selection of recombinant antibodies by phage display nowadays presents a real challenge to provide the antibodies that are urgently needed. In this research, an anti-CMV single-chain variable fragment (scFv) was constructed using a phage display system. Both heavy (V_h) and kappa light chain variable (V_k) genes were amplified by RT-PCR from the hybridoma cell line CM2, secreting a monoclonal antibody (MAb) specific to both serogroup Ⅰ and Ⅱ of CMV. The V_h and V_k amplified products, approximately 400 bp in length, were joined by a PCR overlapping extension method to generate the scFv gene. A recombinant phagemid pCANTAB5E harboring the scFv gene was constructed and transformed into Escherichia coli TG1. The bacterial transformants were rescued by helper phage M13 to produce phage-displayd scFv and the screening for CMV-specific scFv was carried out by ELISA. Three positive, recombinant clones (2C1, 6A1 and 1D4) which gave high signal-to-noise in ELISA were utilized in order to produce soluble antibodies. Western blotting and DNA sequencing were performed to characterize the scFv products. The result showed that all clones were identical and able to bind CMV of both subgroups. DNA comparisons showed that all the V_h belonged to the J558.32 subgroup and JH2, while V_k belonged to V_k genes and JK2.
机译：黄瓜花叶病毒（CMV）在具有重要经济意义的农作物中尤其是茄科和葫芦科成员中引起严重问题。作为控制措施以及隔离检查，需要通过特异性抗体对该病毒进行血清学检测，以确保用于农业繁殖的任何成分（尤其是商业种子）均无病。然而，如今通过噬菌体展示选择重组抗体提出了提供迫切需要的抗体的真正挑战。在这项研究中，使用噬菌体展示系统构建了抗CMV单链可变片段（scFv）。通过RT-PCR从杂交瘤细胞系CM2中扩增出重链（V_h）和κ轻链可变（V_k）基因，并分泌了对CMV的血清群Ⅰ和Ⅱ具有特异性的单克隆抗体（MAb）。通过PCR重叠延伸方法将长度约为400bp的V_h和V_k扩增产物连接，以产生scFv基因。构建了具有scFv基因的重组噬菌粒pCANTAB5E，并将其转化到大肠杆菌TG1中。通过辅助噬菌体M13拯救细菌转化体，以产生噬菌体展示的scFv，并且通过ELISA进行CMV特异性scFv的筛选。为了产生可溶性抗体，使用了在ELISA中具有高信噪比的三个阳性重组克隆（2C1、6A1和1D4）。进行蛋白质印迹和DNA测序以表征scFv产物。结果表明，所有克隆都是相同的，并且能够结合两个亚组的CMV。 DNA比较表明，所有的V_h都属于J558.32亚组和JH2，而V_k则属于V_k基因和JK2。

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