Authors: Maneerat Koohapitagtam Suang Rungpragayphan Ratchanee Hongprayoon Wichai Kositratana Theerapol Sirinarumitr
Publish Date: 2009/06/25
Volume: 37, Issue: 4, Pages: 1677-1683
Abstract
Nonimmune phage scFv library is one of the most attractive resources for therapeutics diagnostics and basic research As a matter of fact quality of the library is limited by inefficient PCR cloning of antibody genes using degenerated primers PCR using this type of primers is difficult to optimize conditions for efficient amplification and therefore causes loss of antibody diversities To overcome this problem we described a novel twostep amplification of Vκ and VH genes with newly designed primer sets Initially we amplified Vκ and VH genes from their signal sequences to the joining region to keep antibody diversity as large as possible Thereafter highly degenerated primers were used to amplify the Vκ and VH genes from the framework region 1 to the joining region The Vκ and VH genes from the second PCR then were linked by PCR overlapping extension to generate the scFv library Fifteen clones from the library were randomly picked and sequenced and the diversity of fulllength scFvs was confirmed Expression capability of clones in the library was 80 after confirmation using colony hybridization The results demonstrated the efficiency of this strategy and the primer sets for construction of the scFv libraryWe wish to thank the Center for Agricultural Biotechnology Postgraduate Education Research Development Office Commission on Higher Education Ministry of Education and the Graduate School Kasetsart University for their financial support This work was also partially supported by Faculty of Pharmacy Silpakorn University Sanamchandra Palace Nakhon Pathom
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