Construction Of Avian Single-chain Antibody (SCFV) Library Using Phage Display Technique And Screening Of Specific Antibodies Against Porcine CD46

Phage display technology is a powerful method for the selection of monoclonalantibodies against a target antigen; in recent years, it has been applied in increasingratio for monoclonal antibody screening. The CD46receptor is also called asmembrane cofactor protein (MCP) found in all nucleated cells and relativelyconserved in mammals. CD46acts as a key regulator in the classical and alternativecomplement activation cascades of the innate immune system; besides, CD46isused as a cellular receptor by several pathogens. The present study aimed toconstruct chicken sourced single chain antibody (scFv) library using phage displaytechnique and screening specific antibodies against porcine CD46in order toprovide technical support for the preparation of monoclone IgY antibodies with highaffinity and specificity.The specific pathogen free (SPF) cockerel was immunized with recombinanteukaryotic expression vector of CD46followed by CD46fusion protein expressedusing E. coli BL21. Splenic B-Lymphocytes were collected after forth immunizationand total RNA was extracted in order to synthesis cDNA. The primers to amplifyVH and VL genes were selected as described in the previous literatures and a seriesof overlapping PCR reactions were performed to amplify the scFvs with VH and VLas the templates together. Then scFv was inserted into pCANTAB5E by T4DNAligation enzyme after digested with Sfi I and Not I enzymes together. Subsequently,the newly constructed vector was transfected into competent cell E.coli TG1strainby chemical method. The size of the generated library was calculated by the phageplaque and it was found to be6.0×105. Ten plaques were randomly selected for thePCR testing of the recombinant gene and the correct recombinant rate was100%.Eight plaques were used for BstO I digest, in which two clones shown the sameresult, indicating that the phage library has high quality and diversity.After infecting TG1cells by M13KO7, scFvs were displayed on the surface ofphage. Different concentrations of CD46antigen (10,5,2.5,2μg/mL, respectively)was coated in the immune tube in order to pan the specific scFvs and10pfu phages were used for every round of panning analysis. The generated library sizes were3.9×106,1.81×107,5.84×108and4.11×109pfu/mL. The increasing binding activityof phages against CD46proteins in the panning process indicated that, the specificphage has been effectively enriched. Plaques were randomly selected from the4thround of eluted phages and23positive clones were obtained through PCR analysis.Three recombinant phage clones with higher reactivity against CD46were furtheridentified by phage ELISA analysis.In this study, a chicken sourced scFv phage display antibody library wasgenerated and identified; and three chicken sourced scFv with good reactivityagainst CD46(expressed using prokaryotic system) were obtained; these threeantibodies could pave a platform for the establishment of CD46detection kit usingavian sourced scFv antibody and formed a foundation for immunology andpathology associated with CD46researches.