This document discusses methods for constructing antibody scFv fragment libraries displayed on phage (phage display scFv libraries) for stability selection. It describes several strategies for PCR amplification and assembly of variable heavy (VH) and light (VL) chain domains connected by a linker to produce single-chain Fv fragments (scFvs). These include normal PCR, pull-through PCR, overlap extension PCR, and RACE PCR strategies. It then provides a basic protocol for constructing naive scFv libraries using peripheral blood mononuclear cells, including steps for VH and VL repertoire construction and assembly into a phagemid vector for phage display.

1. scfv phage library
• Antibody scFv fragments (single-chain Fv) are popular recombinant antibody formats and
the smallest antibody fragments capable of recognizing an antigen, but often suffer from
limited stability. Phage display is a powerful tool in antibody engineering and is also
applicable for stability selection. ScFv is well displayed on filamentous phage and is often
the preferred antibody format in antibody engineering. ScFv variants with improved
stability can be selected from large randomly mutated phage displayed libraries with a
specific antigen after the unstable variants have been inactivated by heat or chaotropic
agents. ScFv molecules combine the coding sequence of the variable heavy (VH) and
sequence of the variable light chain (VL) domains of an antibody in a single-gene encoded
format. The resulting polypeptides, with the variable light (VH ) and heavy chain (VH )
domains connected by a flexible peptide linker, were assemble into functional antigen-
binding sites.
• The orientation of ligation of VL and VH is VL-linker- VH or VH-linker- VL. The antigen
specificity of the two construction types is similar but they can lead to secretory
expression in E.coli in different level. So you can choose any of the two construction
orientations and then design primer by PCR method and sequence assembly type.
Several PCR strategies are listed below:

2. • Normal PCR strategy: Design primer (introduce restriction enzyme sites) for amplifying VH and VL gene based
on antibody CDR sequence. Link C-terminal of VH and N- terminal of VL, or C-terminal of VL and N- terminal
of VH by linker (design primer also based on sequence of VH, VL and linker. Choose appropriate restriction
enzyme to gain the goal of sequence assembly. You can choose any assembly fragment types to clone, eg,. a.
Design primers for amplifying VH gene and linker-VL. b. Design primers for amplifying VH-linker gene and VL)
to construct antibody fragment or gene fragment. The suitable that the length of linker is 15-20 amino acids.
• Pull-through PCR strategy: To avoid non-specific binding or reducing PCR efficiency. You can choose this
method to introduce restriction sites on the end of a PCR product. The pull-through method performs a PCR
like normal and you purify the PCR product from a gel. From this PCR product, you then run a few additional
PCR steps with primers that have your restriction sites on the ends. If there is restriction enzyme site in your
target VH or VL gene, it is suggested to use this method for getting ligation PCR product and digested vector.
• Slicing by overlap extension PCR strategy: Don’t need to introduce restriction enzyme site into primers for
ligation of VH, linker and VL. ScFv fragments were assembled by this method. After assembly, the scFv
fragments (VL- linker - VH or VH - linker - VL ) were further amplified and, concurrently, longer tails were
added to the ends in order to facilitate the subsequent digestion of the fragments by restriction enzymes.
Then digest phagemid vectors by the same enzyme for combining them with scFv fragment .
• RACE (rapid amplication cDNA) PCR strategy: This technique can provide the sequence of an RNA transcript
from a small known sequence within the transcript to the 5' end (5' RACE-PCR) or 3' end (3' RACE-PCR) of
the RNA. Add universal linker primers in mRNA transcription, gain unknown sequence of cDNA by gene
specific primer.

3. • Here, we describe a basic protocol concluded by Simon Lennard for
the construction of scFv gene libraries (naïve libraries) by pull-through
PCR strategy and phage display. The basic protocol flow chart is
shown in Fig.1.

5. • Materials
• Fresh source of lymphoid tissue from which mRNA can be isolated.
• Kit for the preparation of mRNA (e.g., an oligo (dT)-purification system).
• Kit for the synthesis of cDNA (e.g.,“First-strand cDNA synthesis kit”).
• PCR reagents: Taq DNA polymerase with 10x Taq DNA polymerase buffer, a stock of deoxyribonucleoside triphosphates (dNTPs). PCR H2O [ACS] reagent;
• Oligonucleotide primers (design based on your requirement) at 10 μM.
• Kit for the isolation of DNA from gels.
• Phage display vector(s) and appropriate restriction enzymes for cloning.(Here we represent for vector 1 and vector 2,enzyme A, B, C, D,E)
• Kit for medium-scale isolation of plasmid DNA.
• Ligation kit, 5x ligation buffer (500 mM Tris-HCl, 25 mM MgCl2, pH7.4).
• Electrocompetent Escherichia coli strain(e.g., TG1).
• Gene Pulser and suitable electroporation cuvets.
• 2TY as liquid and solid media.
• Glucose 20% (w/v) sterile-filtered.
• Filtersterilized antibiotic for screening positive clones (e.g., Ampicillin (100 mg/mL stock) and kanamycin (50 mg/mL stock)) .
• Kit for the purification of PCR products.
• 20% (w/v) Polyethylene glycol (PEG) 8000, 2.5 M NaCl.
• TE buffer (10 mM Tris-HCl, 1 mM ethylene diamine tetraacetic acid, pH 8.0).
• Cesium chloride (CsCl).

6. • Methods
• First strand cDNA synthesis from PBLC mRNA
• PBLC (peripheral blood lymphocytes cell) isolation and synthesis of primary
cDNA template. Total RNA was prepared from PBMC by using a RNA
Purification kit. First strand cDNA was synthesized from total RNA by using
a First-Strand cDNA Synthesis kit. Follow the manufacturer’s instructions.
(Note: Rapid processing of fresh tissue samples is essential if the full
diversity of the Ab repertoire is to be recovered. Tissue, mRNA, or cDNA
product can be stored at -70°C, if some loss of diversity is acceptable
(perhaps libraries from infected or immunized individuals, rather than a
comprehensive naive library). 50 mL of blood should yield approx 1 x
107cells, which in turn yield about 10 μg total RNA, of which 1–5% is mRNA.
It is important to ensure that there is enough cDNA for all the VH and
VL PCR reactions planned, each of which requires 0.5 ng cDNA.)

7. • Amplification of Antibody Variable Region Genes: The goal of this process is gain VH and linker-VL gene sequence.
• VH Repertoire Construction
• VH gene repertoires were PCR amplified by using the cDNA prepared from above procedure. To amplify the VHgenes from the cDNA and plasmid template, the primers were
designed based on what described on pull-through PCR strategy. Perform PCR reactions for each VH primers using the optimizing cycling parameters by PCR reagents.
• Digest the purified VH DNA and vector 1 with two required enzymes (enzyme A and enzyme B) respectively using the reaction buffer provided by the providers. After digestion,
heat-inactivate the enzyme at proper temperature.
• Purify digesting product of VH DNA and vector 1 respectively by Kit for the purification of PCR products..
• Ligate purified product of VH DNA and vector 1 by Ligation kit.
(Note: Set up a series of trial ligations covering a range of molar ratios of insert vector (e.g., 1:1, 2:1, 4:1, 1:2) and a fixed concentration of vector 1 within the range specified by
the manufacturer of the ligation kit. A “vector only” ligation control should be included to determine the background caused by nonrecombinants.)
• Transformation: Electroporate the DNA ligations into competent E. coli. Add 2TY containing 2% glucose (2TYG) and allow the cells to recover for 1 h with shaking at 200 rpm in a
37°C incubator.
• Plate aliquots from each transformation to 2TYG plates containing antibiotic (e.g., ampicillin 100 μg/mL; 2TYAG) and incubate overnight at 30°C.
(Note: Confirm that background levels of vector self-ligation are minimal and which ligation ratio yields the highest numbers of transformants.)
• From the planned size of the VH library and the recovery of transformants from the optimal ligation, calculate the number of ligation reactions required for library construction.
Set up ligations and precipitate the products as before (steps 4). Go on performing electroporations recovery and determine the library size by serial dilutions.
• Perform at least 40 electroporations with electrocompetent E. coli cells (step 5), and let the cells recover for 1 h in 2TYG with shaking at 200 rpm at 37°C. Pool the cells from all
electroporations, and take a small aliquot for plating at 10-fold serial dilutions onto 2TYAG plates to determine the size of the library. Scrape cells from the large plates and
prepare glycerol stocks for storage at -70°C.
•

8. • VL Repertoire Construction (Note: VL kappa and VL lamda gene fragments are amplified separately using each back primer in
combination with the appropriate equimolar mixture of the J kappa or J lamda forward primers. After recovery of the combined
VL repertoire, the next stage is to clone in the (Gly4Ser)3 scFv linker from an existing scFv, together with a dummy VH, recovered by
PCR from an irrelevant clone.)
• Perform PCR reactions using the same cycling parameters as outlined for recovery of VH products (see VH repertoire construction
step 1).
• Digest the purified VL DNA and vector 2 respectively by two required enzymes (C and D restriction enzymes).
• Perform ligation reactions and subsequent electroporations as described for the VH repertoire (see VH repertoire construction).
The size of the VH and VL repertoires can be estimated.
• To introduce the scFv-linker (e.g., (Gly4Ser)3) into the finished VL repertoire, PCR amplify the linker from an existing scFv, together
with an irrelevant (dummy) VH fragment. Digest linker single product with enzyme E. Get Purified product described previously
(see VH repertoire construction). And then estimate its concentration by comparison with DNA markers.
• Prepare the vector 2 containing the VL repertoire on mini prep scale and sequentially digest with enzyme D and enzyme E.
• Perform sufficient ligation reactions to dummy VH-linker DNA fragment into a pool of the VH and VL libraries.
• Transformation: Electroporate into E. coli cells, and plate out as described previously (see VH repertoire construction).

9. • Construction of The ScFv Library: The amplified VH and linker-VL
genes were gel-purified on agarose, the scFv assembly and expression
vector were digested and ligated to gain scFv library.