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| Construction and Panning of Canine Phage scFv Library for Dog (Canis lupus familiaris) Erythrocyte Antigen 1.1 |
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Abstract Abstract The canine blood typing reagents are lack of use in pet diagnosis and treatment in China. Our purpose is to develop canine DEA1.1 blood typing antibodies and provide materials for blood typing of pet dogs (Canis lupus familiaris) before blood transfusion. In this study, Beagle dog was taken as experimental animals, and antibody gene was derived from peripheral blood lymphocytes. Degenerate primers were designed for the variable region gene of canine antibody. Complementary DNA fragments of variable heavy (VH) and variable light (VL) chains of the antibodies were synthesized by reverse transcription RT-PCR, and were assembled into single-chain antibody variable fragment (scFv) by splice overlap extension PCR. Both scFv and the pCANTAB5E vector were double-digested with restriction endonuclease Sfi Ⅰ and Not Ⅰ, respectively, and they were connected with T4 ligase. Single chain antibody library was constructed. Dot enzyme-linked immunosorbent assay (Dot-ELISA) method was used to screen single-chain antibody for anti-DEA1.1 blood group. The positive clone of blood group antibody was identified, and the prokaryotic expression of recombinant scFv protein was constructed. Clinically samples tested validate specific antibodies with DEA1.1 blood can be used to pre-clinically test transfusion of dog DEA1.1. The results showed that the degenerate region of canine antibody variable region was available, and the canine scFv library was successfully constructed. The constructed immune library had an actual capacity of 7×105, being a diverse and large enough storage capacity. Finally, 3 strains of canine DEA1.1 blood group antigen with the single chain antibody were screened. Clone16 protein was identified as a soluble protein, being derived from prokaryotic expression protein. Clone16 single chain antibody was obtained by affinity purification, and further clinical samples showed strong binding characteristics. This study provides a theoretical basis for the preparation of canine DEA1.1 blood group identification reagents.
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Received: 22 December 2016
Published: 01 June 2017
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