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2012•Biotechnology(Faisalabad)Requires access

Construction and Characterization of Native Rabbit ScFv Phage Display Library

Sen Li

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Abstract

Objective:To construct a native rabbit phage display library of single chain Fv(scFv) antibodies.Method:Rabbit antibody variable heavy(VH) and variable light(VL) genes were amplified by RT-PCR from non-immunized rabbit's spleen.Using over-lap PCR obtained the scFv fragments and the products were ligated into the phagemid vector pComb3XSS.The recombinant phagemid was then electroporated into competent E.coli XL1-Blue to construct a rabbit scFv library,then using this library to screen antibodies with affinity for creatine kinase.Result:The results showed that the library contained 4×108 clones,the gene recombination rate was 95% and the library exhibited high diversity by DNA fingerprinting.Three scFv antibodies against creatine kinase were successfully selected from the library by panning.Conclusion:The constructed native rabbit phage scFv library has a high quality and can be used for generating scFv antibodies for antigens.

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What this paper is about

Objective:To construct a native rabbit phage display library of single chain Fv(scFv) antibodies.Method:Rabbit antibody variable heavy(VH) and variable light(VL) genes were amplified by RT-PCR from non-immunized rabbit's spleen.Using over-lap PCR obtained the scFv fragments and the products were ligated into the phagemid vector pComb3XSS.The recombinant phagemid was then electroporated into competent E.coli XL1-Blue to construct a rabbit scFv library,then using this library to screen antibodies with affinity for creatine kinase.Result:The results showed that the library contained 4×108 clones,the gene recombination rate was 95% and the library exhibited high diversity by DNA fingerprinting.Three scFv antibodies against creatine kinase were successfully selected from the library by panning.Conclusion:The constructed native rabbit phage scFv library has a high quality and can be used for generating scFv antibodies for antigens.

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Available abstract

Objective:To construct a native rabbit phage display library of single chain Fv(scFv) antibodies.Method:Rabbit antibody variable heavy(VH) and variable light(VL) genes were amplified by RT-PCR from non-immunized rabbit's spleen.Using over-lap PCR obtained the scFv fragments and the products were ligated into the phagemid vector pComb3XSS.The recombinant phagemid was then electroporated into competent E.coli XL1-Blue to construct a rabbit scFv library,then using this library to screen antibodies with affinity for creatine kinase.Result:The results showed that the library contained 4×108 clones,the gene recombination rate was 95% and the library exhibited high diversity by DNA fingerprinting.Three scFv antibodies against creatine kinase were successfully selected from the library by panning.Conclusion:The constructed native rabbit phage scFv library has a high quality and can be used for generating scFv antibodies for antigens.

Key concepts: Phage display, Phagemid, Panning (audio), Molecular biology, Recombinant DNA, Antibody, Single-chain variable fragment, Immunoglobulin light chain

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