A FAP nanobody nb67
A nanobody, DNA molecule technology, applied in the field of biomedicine or biopharmaceuticals, to achieve the effect of good specificity and high affinity
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Embodiment 1
[0026] Example 1: Construction of FAP-specific Nanobody library:
[0027] (1) First, synthesize FAP polypeptide, mix 1 mg FAP and Freund's adjuvant in equal volumes, immunize a Xinjiang dromedary camel, once a week, a total of 7 immunizations, to stimulate B cells to express antigen-specific Nanobodies;
[0028] (2) After 7 immunizations, extract 100 mL of camel peripheral blood lymphocytes and extract total RNA;
[0029] (3) Synthesize cDNA and amplify VHH by nested PCR;
[0030] (4) Use restriction enzymes PstI and NotI to cut 20ug pComb3 phage display vector (supplied by Biovector China Plasmid Vector Strain Cell Gene Collection) and 10ug VHH and ligate the two fragments; (5) Transform the ligated product into electrotransduction In the cell TG1, construct a FAP Nanobody library and determine the storage capacity, the storage capacity is 1.85×10 8 .
Embodiment 2
[0031] Example 2: Nanobody screening process for FAP:
[0032] (1) Will be dissolved in 100mM NaHCO 3 , 20ug FAP in pH 8.2 is coupled to the NUNC plate, placed overnight at 4°C;
[0033] (2) Add 100uL 0.1% casein on the next day, and block for 1-3h at room temperature;
[0034] (3) After 1-3h, add 100uL phage (5×10 11 tfu immunized camel nanobody phage display gene library), at room temperature for 1-2h;
[0035] (4) Wash 4-6 times with 0.05% PBS+Tween-20 to wash off unbound phages;
[0036] (5) Dissociate the phage that specifically binds to FAP with 100mM TEA (triethylamine), and infect E. coli TG1 growing in the logarithmic phase, culture for 1h at 37°C, produce and purify the phage for the next round Screening, the same screening process is repeated 3-5 rounds, and enrichment is gradually obtained.
Embodiment 3
[0037] Example 3: Using phage enzyme-linked immunoassay (ELISA) to screen specific single positive clones:
[0038] (1) From the cell culture dishes containing phage after the above 3-5 rounds of selection, select 96 single colonies and inoculate them in TB medium containing 100 micrograms per milliliter of ampicillin (1 liter of TB medium containing 2.3 grams of phosphoric acid Potassium dihydrogen, 12.52 g of dipotassium hydrogen phosphate, 12 g of peptone, 24 g of yeast extract, 4 ml of glycerol), after growth to the logarithmic phase, add a final concentration of 1 millimolar isopropyl thiogalactoside ( IPTG), cultivate overnight at 30-35°C.
[0039] (2) Use the permeation method to obtain the crude antibody, and transfer the antibody to the antigen-coated ELISA plate, and place it at room temperature for 1-1.5 hours.
[0040] (3) Wash the unbound antibody with PBST, add a mouse anti-HA tagantibody (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.), and place it at...
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