Anti-human EGFR nano antibody and application
A nanobody and antibody drug technology, applied in the direction of antibody, application, anti-animal/human immunoglobulin, etc., can solve the problems of poor penetration ability, large molecular weight, unable to effectively remove small lesions, etc., to achieve good stability, promote Applied, easy-to-modify effects
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Embodiment 1
[0047] Example 1 preparation of helper phage
[0048] (1) Take out the glycerol-preserved TG1 Escherichia coli clone strain from the -80°C refrigerator, spread it on a TYE non-resistant plate using the four-section method, and incubate it upside down at 37°C for 13 hours.
[0049] (2) Pick a TG1 monoclonal strain from the plate and inoculate it into 5 mL of 2×TY non-resistance liquid medium, and culture it at 37° C. and 230 rpm for 13 hours.
[0050] (3) Transfer the TG1 bacterial solution to 5mL 2×TY non-resistant liquid medium at a volume ratio of 1:100, and culture it at 37°C and 230rpm for 2h (bacterial solution OD 600 = around 0.5).
[0051] (4) Take 200 μL TG1 bacterial solution (OD 600 =0.5 or so) into a 1.5mL centrifuge tube, add 10μL of KM13 helper phage (1×10 13 pfu / mL), placed in preheated 37°C water bath for 30min.
[0052] (5) Prepare soft agar, let it cool to about 40°C, pour the TG1 bacterial solution treated in step (4), mix, and then pour the pre-prepared ...
Embodiment 2
[0057] Example 2 Amplification of Phage Nanobody Library
[0058] (1) Thaw the TG1 bacteria containing the antibody plasmid (i.e., the human nanobody phage library, SourceBioscience, London, UK) on ice, and transfer it to 500mL 2×TY liquid medium (containing 0.1% ampicillin by mass volume ratio) and mass volume ratio of 1% glucose), 37 ° C, 230rpm shaker culture 2.5h (OD 600 =0.5), and then add 2×10 12 The helper phage, 37 ℃ water bath for 30min.
[0059] (2) Divide the bacterial solution into 250 mL / bottle, centrifuge at 3200 g for 10 min, remove the supernatant, and use 500 mL of 2×TY medium (containing 0.1% ampicillin by mass volume ratio and 0.05% kanamycin by mass volume ratio) The pellet was resuspended, and cultured with shaking at 25°C and 220rpm for 20h.
[0060] (3) The bacterial liquid was centrifuged at 3200 g for 20 min, and the supernatant was collected, and the phage was purified by the same method as steps (8)-(9) in Example 1, and the prepared library phage...
Embodiment 3
[0062] Example 3 Screening Nanobodies Against EGFR Fragments from a Phage Nanobody Library
[0063] (1) Screening of anti-EGFR nanobody phage
[0064] 1) Coat the EGFR polypeptide (purchased from Shanghai Botai Biological Co., Ltd., with the amino acid sequence shown in SEQ ID NO.15) in the immunotube (nunc) (the first round: 0.1 mg / mL; the second and third rounds : 0.05mg / mL; the fourth and fifth rounds: 0.025mg / mL;), set the PBS (control) tube. overnight at 4°C.
[0065] 2) Pour out the coating solution, wash each tube with 4.5mL PBS three times (after adding the liquid each time, discard without stopping), add 4.5mL of BSA blocking solution with a concentration of 2% by mass volume ratio to each tube, and place at room temperature 2h.
[0066] 3) Pour off the BSA blocking solution, and wash each tube with 4.5mL PBS three times (after adding the liquid each time, discard it directly without stopping).
[0067] 4) Add 4mL to each tube containing 5×10 12 2% BSA solution o...
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