The cDNA library (complementary DNA library) uses mRNA in tissues as a template, reverse transcribes and synthesizes double-stranded cDNA, and each cDNA molecule is inserted into a vector to form a recombinant, which is then introduced into the host cell for cloning and amplification. The collection of cDNAs in the recombinant is the cDNA library. It represents the library of mRNA in a specific cell or tissue.

The cDNA library is the premise and basis for studying the gene expression of a specific organ, specific tissue, and specific developmental period of a certain organism at the genomic level. The traditional screening method is to copy the clones onto a nylon membrane at a high density for colony in situ hybridization screening. This method is labor-intensive and must use radioactive isotopes. Nowadays, as a powerful tool for selecting specific binding peptides or proteins in large-capacity libraries (≥10^8 clones), the arrival of phage display technology provides opportunities for the development of cDNA clone gene expression.

一．Advantages of cDNA Library

1. The library can be established if the genetic material is RNA virus;

2. Because the number of clones is much smaller than that of genomic library, it is easy to screen;

3. Specific expression genes can be isolated from the cDNA library of differentiated cells.

4. Other RNAs have been excluded when the library is built, which reduces the false positive rate.

5. cDNA library can be expressed in bacteria and can be screened by various strategies.

二．The Difference between cDNA Library and Genomic Library

三．Vectors for Phage cDNA Library Display

1. Filamentous Phage

Because the full-length cDNA contains a translation terminator at the 3' end and lacks a suitable ribosome binding site when expressed in prokaryotes (if the cDNA is derived from eukaryotic cells), the most practical way to ensure cDNA expression in phage is to fuse the 5' end to the vector gene. The traditional construction method is to fuse the target gene to the N-terminus of pill or pVIII, and it is generally believed that direct fusion to the C-terminus of either capsid protein is incompatible with phage assembly. Therefore, three specialized vectors were designed around these restrictions: (1) pill was fused to the leucine zipper of cJun, and the translated cDNA product was fused to the C-terminus of the leucine zipper of cFos (pJufo). During phage assembly, the zipper is fused to the end of the phage, so the capsid-enclosed cDNA is connected to the product it encodes. (2) Direct mutual acquisition method: utilizing the functional modularity of the small capsid protein pill, that is, the eDNA is expressed by fusion with the C-terminus of the infectious domain of pill, and the protein probe is placed in the C-terminus of pill and embedded in the core of the phage. Only those phages that can establish a stable interaction between the probe and the specific eDNA-encoded product can restore their infectivity and be used for amplification in E. coli. (3) The C-terminus of the small capsid protein pVI is expressed on the surface, so a glycine-rich hexapeptide gene VI (gVI)-cDNA fusion gene can be displayed on the phage surface. Protein pVI stabilizes the structure of the phage by providing a link between pill and the phage core.

2. Soluble Phage

In the filamentous phage system, the capsid proteins are first assembled into phage particles and then localized in the periplasm. This approach is suitable for proteins with disulfide bonds (such as secreted proteins or extracellular domains of receptors), but it is incompatible with eDNA-encoded products that can fold normally under a reducing environment. In fact, the internal structure of most phage particles is similar. For example, in λ phage, C-terminal fusion with capsid protein or tail protein has been reported. Capsid protein is used to display and select cDNA fragments, which are derived from the hepatitis C virus genome, human brain and mouse embryo. In T4 phage, the solubility of the C-terminus of capsid protein WAC and SOC.

四．Construction Process of Phage Display cDNA Library (taking λ phage as an example)

1. Total RNA extraction: Add Trizol reagent to the culture dish to ensure that all cells are lysed; add chloroform and place at room temperature; centrifuge at 12000g for 15min, and draw the upper colorless aqueous phase containing total RNA into a new centrifuge tube; add isopropanol and precipitate at room temperature for 10min. Centrifuge at 12000g for 10min, RNA precipitate can be seen at the bottom of the tube, and the supernatant is discarded; add 75% ethanol, mix and centrifuge, and discard the supernatant; after the RNA is dried, add DEPC water to dissolve. Take a small amount to measure A260/280, and use agarose gel electrophoresis to identify the extraction effect. Treat the total RNA with DNase I to remove impurity DNA, and observe the removal effect by agarose gel electrophoresis.

2. Reverse transcription synthesis of cDNA: synthesis of the first strand of cDNA; synthesis of the second strand of cDNA; modification of double-stranded ends.

3. Obtaining the primary library: digestion with proteinase K; SfiⅠ enzyme digestion; connection of cDNA with λTTrriippllEExx 2 vector; in vitro packaging of λ phage.

4. Titration of primary library: (1) Pick a single clone and transfer it to LB (containing maltose/MgSO₄) medium, shake it at 37℃ and 140 rpm overnight, and make OD600 reach 2.0. Centrifuge at 5000 rpm for 5 min, discard the supernatant, and resuspend the precipitate with 7.5 mL 10 mM MgSO₄; (2) Preheat LB/MgSO₄ solid medium at 37℃; (3) Dilute the bacteria with λ dilution buffer (dilution range 1:5-1:20); (4) Add 1 μL of diluted phage to 200 μL XL1-Blue overnight culture, and allow the phage to adsorb at 37℃ for 10-15 min; (5) Add 2 mL of melted LB/MgSO₄ top agar, mix well, and immediately pour into LB solid medium preheated at 37℃; (6) Cool at room temperature for 10 min, and place the culture dish at 37℃ for 6-18 h.

5. Amplification of the primary library: (1) Pick a single clone from the primary library and add it to 15 mL LB (containing maltose/MgSO₄) medium. Shake it at 140 rpm at 37°C overnight until the OD600 reaches 2.0. Centrifuge at 5,000 rpm for 5 min, discard the supernatant, and resuspend the precipitate in 7.5 mL 10 mM MgSO₄. (2) Preheat LB/MgSO₄ solid medium at 37°C. (3) Mix 500 μL of bacterial culture and diluted phage in a 4 mL centrifuge tube (so that each plate contains 1×10^5 plaques); incubate in a 37°C water bath for 15 min; add 4.5 mL of melted LB/MgSO₄ top agar to each tube; mix quickly and pour the mixture of bacteria and phage into the LB/MgSO₄ agar plate and rotate to make it evenly distributed; (4) Incubate at room temperature for 10 min to allow the agar to solidify, and place the plate in an incubator at 37°C for 6 to 18 h until the phage converges; (5) Add 12 mL of 1×λ dilution buffer to each plate and incubate at 4°C overnight; the plaques are merged to form an amplified library; shake the plate on a horizontal shaker at 50 rpm for 1 h at room temperature; (6) Collect the λ phage lysate into a sterile beaker, mix thoroughly, and divide evenly into 50 mL centrifuge tubes; add 10 mL of chloroform to each tube and vortex for 2 min; centrifuge at 5,000 g for 10 min, collect the supernatant into a new tube, cover tightly and place at 4°C.

Tek Biotech has M13, T4 and T7 phage display systems. According to the customer's project requirements (parameters such as the size of the displayed protein or antibody fragment), different phages will be used to display proteins or antibodies. Phage display technology displays exogenous peptides and phage capsid proteins on the phage surface for high-throughput screening and enrichment, and qualitatively analyzes the clones of the required functions. The technology displays antibodies, antibody fragments, peptides, cDNA, etc. In the completed cases, the M13 phage pIII gene display system is more commonly used, and the constructed library capacity can reach 10^11, of which the effective library capacity is 10^9. At the same time, customers can also provide antigens for in vitro peripheral blood lymphocyte sensitization to obtain monoclonal antibodies with higher affinity and specificity. Usually, the antibody affinity prepared based on this method can reach 10^-13 M level.