 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 : Production of Polyclonal Antibodies Specifi c to the Recombinant Coat Protein of Blackeye cowpea mosaic virus and Its Use in Disease Detection) ผู้เขียน:Maneerat Koohapitagtam, Charassri Nualsri สื่อสิ่งพิมพ์:pdf AbstractThe coat protein gene of Blackeye cowpea mosaic virus (BICMV-CP) was amplifi ed by reverse transcription polymerase chain reaction and cloned into the expression vector pQE-80L. This plasmid was transformed into Escherichia coli DH5? competent cells. The BICMV-CP gene was expressed as a fusion protein containing a fragment of 6xHis-tag. Bacterial cells were disrupted by repeated freezethawing three times and the BICMV-CP fusion proteins were purifi ed under denaturing conditions by affi nity chromatography with Ni-NTA Agarose. A sample of 500 ?g of purifi ed protein was mixed with Freund’s complete adjuvant at a ratio of 1:1 (volume to volume). Initially, the emulsion was subcutaneously injected into a New Zealand White rabbit, followed at weekly intervals by three additional immunizations with 500 ?g of the purifi ed protein mixed with Freund’s incomplete adjuvant. Bleeding was done every week during weeks 5-12 and titers of the antisera ranging from 800-51,200 were obtained. Up to a dilution of 1:320 of the BICMV-infected yardlong bean sap could be detected by indirect enzyme-linked immunosorbent assay. The produced antiserum reacted specifi cally with the BICMV-infected plant without any cross reaction with other virus species tested. However, a weakly positive reaction with Bean common mosaic virus could be observed. |
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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