一．Nanoantibody Immune Library

The most special and important step in constructing an immune library is animal immunization, which causes the V, D and J gene fragments of the camel heavy chain V region to undergo combinatorial rearrangement at random sites in the CDR3 domain. The method of inducing the camel's body to produce specific heavy chain antibodies is similar to obtaining traditional antibodies from other animals. It mainly involves periodically and continuously injecting the animal with antigens coupled with adjuvants for a period of time, so that the body can produce a sufficient immune response to the antigen to generate heavy chain antibodies with strong specificity and high affinity. After the immunization, lymphocytes are separated from the camel's blood, and total RNA is extracted. Then, RNA is used as a template for reverse transcription to obtain a cDNA library; primers are designed according to the conserved region of the heavy chain antibody, and the VHH gene fragment is amplified by 2-3 rounds of PCR using nested PCR technology, and appropriate restriction sites (such as PstI and NotI) are introduced at the same time. Next, the VHH gene is cloned into a phagemid vector (such as pHEN4 and pMECS), fused with the phage gene II protein (gIIp) for expression, and then displayed on the phage surface for specific antigen recognition and screening. The recombinant phagemid vector is then transformed into E. coli cells and the library is amplified to form an antigen-biased immune library knife. The quality of the library can generally be evaluated from aspects such as library capacity and diversity.

二．Construction of Nanoantibody Phage Display Library

1. Preparation of TG1 Electroporation Competent Cells

(1) Streak TG1 cells on LB plates and invert at 37 °C overnight;

(2) Pick a single clone and inoculate it in 5 mL 2xTY medium and incubate at 37 °C overnight;

(3) Inoculate 2 mL of overnight cultured bacteria into 300 mL 2xTY medium and incubate at 37 °C, 220 rpm until OD600 is about 1;

(4) Place the bacterial solution on ice for 1 h, transfer the bacterial solution to a centrifuge tube after 1 h, and centrifuge at 4 °C, 4000 rpm for 15 min;

(5) Discard the supernatant, add an equal volume of 1 mM, pH 7.0 Hepes solution (pre-cooled on ice) to resuspend the bacteria, and centrifuge at 4 °C, 4000 rpm for 15 min;

(6) Add 1/2 volume of 10% glycerol to resuspend the bacteria, and centrifuge at 4 °C, 4 °C, 4000 rpm centrifugation for 15 min;

(7) Discard the supernatant, add 10 mL 10% glycerol to each tube to resuspend the bacteria, centrifuge at 4 °C, 4000 rpm for 15 min, discard the supernatant, remove the residual liquid with a gun, and add 10% glycerol to a final volume of 1 mL.

2. TG1 Competent Cell Transformation Efficiency Detection

(1) Take 2 μL pMECS plasmid (10 ng/μL) and add it to 48 μL prepared TG1 competent cells, flick to mix, and place on ice for 1 min.

(2) Transfer to an electroporation cup (pre-cooled at -20 °C), set the voltage to 1700 V, and perform electroporation. Immediately after electroporation, mix with 800 μL of SOC medium pre-heated at 37 °C, aspirate, and transfer to a test tube;

3) Incubate at 37 °C, 220 rpm on a shaker for 1 h, remove the bacterial solution, perform gradient dilution, and finally take 100 μL to apply on LA+GLU plate, incubate at 37 C overnight, and calculate the transformation efficiency;

3. Formation of Phage Nanoantibody Library

(1) After the competent state transformation efficiency test is completed, add 100 μL of the ligation product to 1 mL of electroporation TG1 competent state and mix;

(2) Aliquot 40 μL of competent state into each electroporation cup (pre-cooled at -20 °C), place the electroporation cup into the electroporator, and perform electroporation at 1700V to allow the recombinant phage to enter the competent cells;

(3) After the electroporation, add SOC medium 1 mL, blow the electric shock cup, transfer to a conical flask preheated at 37 °C, and finally wash all the electric shock cups once with 2 mL SOC medium, transfer to a conical flask, and culture at 37 °C on a shaker for 1 h;

(4) After 1 h, collect the bacterial liquid into a 50 mL centrifuge tube, centrifuge at 25 °C, 4000 rpm for 10 min, resuspend the bacteria with 5 mL SOC, take out 50 μL for gradient dilution to determine the storage capacity. The remaining bacterial liquid is evenly spread on 10 LA+GLU plates. Culture at 37 °C for 16 h;

(5) Take out the cultured plates, add 5 mL of liquid LB medium to each plate, scrape the bacteria with a coating stick, pour into a conical flask, and then rinse the plate with 3 mL LB, and merge into the conical flask;

(6) Transfer the bacterial liquid into a 50 mL centrifuge tube, centrifuge at 25 °C, 4000 rpm for 10 min, and discard the supernatant. Add 30 mL of 15% glycerol + LB liquid medium, vortex to resuspend, dispense into 1.5 mL EP tubes, and store at -80 °C for later use.

Figure 1 Library Construction Process

三．Phage Construction Skills

The library capacity and diversity of phage libraries are one of the important criteria for measuring library quality. The larger the capacity and the better the diversity of the library, the more effective the guarantee for the successful screening of nanoantibodies;

The key points affecting the library capacity and diversity include: RNA degradation during RNA extraction, templates and primers during reverse transcription, primer selection and template dosage during amplification, PCR amplification rounds, conversion efficiency of competent bacteria, size of the connection system and other factors.

Tek Biotech can provide customers with yeast display library construction and screening services for camel-derived VHH nanoantibody libraries and scFv antibodies of other species. VHH nanoantibody library, also known as camel-derived heavy chain antibody library, has an antibody molecular weight of 15kDa and is a unique antibody produced by camel-derived animals. The yeast antibody library display system is limited by its own physical properties, and the physical library titer is generally <10^7. Therefore, it is mainly used for antibody discovery projects where customers do not have high requirements for project library capacity, PBMC cells are sorted after animal immunization, and disulfide bond formation is avoided. At the same time, compared with phage display library technology, the yeast display library has higher antibody activity after trace antibody expression than the latter. Thanks to this basis, the affinity of antibodies can be distinguished during flow cytometry screening.