Construction of mouse antibody library by phage display technology and production of scFv monoclonal antibodies against vibrio parahaemolyticus and vibrio alginolyticus

Name: Phatcharaporn Chiawwit

keyword: Vibrio parahaemolyticus

; Vibrio alginolyticus

; Single-chain variable fragment (scFv)

; Phage

; Antibody engineering

Abstract: Vibrio genus is the Gram-negative bacteria that ubiquitous in halophilic marine environments. Several Vibrio species are pathogenic causing foodborne diseases worldwide. Identification of the species using conventional culture method is often imprecise since they are genetically closely related especially Vibrio parahaemolyticus and Vibrio alginolyticus. This study aimed to produce the scFv monoclonal antibody specific to V. parahaemolyticus and V. alginolyticus. The stepwise experiment was performed to obtain these antibodies. Firstly, construction of a murine scFv antibody displayed phage library. Secondly, searching for target proteins which were unique to each species by genome comparison and then produced the recombinant proteins of those targets. Thirdly, selection of the scFv phage clones specific to the target protein of V. parahaemolyticus and V. alginolyticus. Finally, expression and purification of the particular scFv monoclonal antibodies. In this study, naïve mouse scFv antibody phage library was successfully constructed. This library contained 8.75×109 cfu of phages of which 82.9% was carrying scfv with 90.3% diversity. The scFv phages library was screened for phages binding to the peptide sequence of the ToxR target protein. Subsequently, the scfv of the bound phages were extracted and cloned into pOPE101 expression vector. Then, the scFv antibodies were produced, and eight scFv antibodies were obtained in this study. They were VP20.7, VP59.7, VP61.3 and VP62.13 which were specific to V. parahaemolyticus and VA34.1, VA74.1, VA87.1 and VA120.1 which were specific to V. alginolyticus. The binding activity of these scFv monoclonal antibodies was tested using Western blot analysis. The result revealed that each antibody could react and produce the reaction band with the correct size of its corresponding recombinant ToxR protein. Therefore, these antibodies could be further used to develop the immunoassay for detecting V. parahaemolyticus and V. alginolyticus

Thammasat University. Thammasat University Library

Address: BANGKOK

Email: preserv@tu.ac.th

Name: Pongsri Tongtawe

Role: advisor

Name: Potjanee Srimanote

Role: co-advisor

Name: Jeeraphong Thanongsaksrikul

Role: co-advisor

Name: Wanpen Chaicumpa

Role: co-advisor

Created: 2017

Modified: 2022-02-21

Issued: 2022-02-21

วิทยานิพนธ์/Thesis

application/pdf

DOI: 10.14457/TU.the.2017.1529

eng

DegreeName: Master of Science

Level: Master's Degree

Descipline: Biomedical Sciences

Grantor: Thammasat University

©copyrights Thammasat University

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