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ผลงานตีพิมพ์ในวารสารวิชาการEfficient amplification of light and heavy chain variable regions and construction of a non-immune phage scFv libraryผู้แต่ง: Koohapitagtam, M, Rungpragayphan, S,  Dr.Ratchanee Hongprayoon, Associate Professor , Dr.Wichai Kositratana, Associate Professor , Dr.Theerapol Sirinarumitr, Associate Professor , วารสาร:  
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 หัวเรื่อง:ไม่มีชื่อไทย (ชื่ออังกฤษ : Construction of Single-Chain Variable Fragment (scFv) Specific to Cucumber Mosaic Virus by Phage Display Technology) ผู้เขียน:Maneerat Koohapitagtam, Suang Rungpragayphan, ดร.รัชนี ฮงประยูร, รองศาสตราจารย์ สื่อสิ่งพิมพ์:pdf Abstract 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 (VH) and kappa light chain variable (Vk) 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 VH and Vk 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 VH belonged to the J558.32 subgroup and JH2, while Vk belonged to Vk genes and JK2. |
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