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How to construct a Fab antibody phage display combinatorial library using pComb3 phagemid vector?
1. Cloning of immunoglobulin genes
1) Extraction of total RNA.
2) Synthesis of first-strand cDNA.
3) PCR amplifications of the heavy chain (HC) Fd region and whole κ light chain (LC).
4) Purification and double-digestion of HC PCR products using SpeI/XhoI restriction enzymes and LC PCR products using SacI/XbaI restriction enzymes.
5) Ligation of the purified HC and LC PCR products into the SpeI/XhoI -linearized and SacI/XbaI linearized pComb3 vector, respectively.
6) Transfection or electroporation of the constructed combinatorial library into XL1-Blue cells, and screening on LB agar plates containing 100 μg/ml of carbenicillin and 100 mM glucose.
7) Selection of single colonies and culture in Super broth (SB) with 50 μg/ml of carbenicillin.
8) Infection with VCSM13 helper phage for overnight culture to obtain the primer Fab phage display libraries.
9) Panning of the primer Fab phage display libraries.
10) Examination of the binding activity of Fab-phage clones to the corresponding antigen using indirect immunofluorescence assay.
11) The identity of the positive clones was determined by sequencing variable heavy (VH) and variable light (VL) domains, respectively.
2. Expression and purification of rFab fragment.
3. Determination of the reactivity and specificity of rFab fragment using immunological assay.
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