Synthetic phage display nano antibody library and application thereof
A phage display and nanobody technology, applied in the fields of biomedicine and molecular biology, can solve the problems of limited prealbumin detection, poor antibody stability and low sensitivity, etc.
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Embodiment 1
[0023] Example 1: Construction of a phage display nanobody library based on the amino acid sequence of the CDR3 region of the nanobody conservative framework cAbBCII10, which is randomly liberalized but excludes cysteine:
[0024](1) Ask the bio-company to synthesize the entire DNA sequence corresponding to the FR1 region to the FR3 region of the nanobody conservative framework cAbBCII10, namely SEQ ID NO: 15, 5'-CAA GTT CAA TTG GTT GAA TCT GGT GGT GGT TCT GTT CAA GCT GG TGG TTC TTT GAG ATT GTC TTG TAC TGC TTC TGG TGG TTC TGA ATAT TCT TAT TCT ACT TTT TCT TTG GGT TGG TTT AGA CAA GCT CCA GGT CAA GAA AGA GAA GCT GTT GCT GCT ATT GCT TCT ATG GGT GGT TTG ACT TAT TAT GCT GAT TCT GTT AAA GGT AGA TTT ACT ATT TCT AGA GAT AAT GCT AAA AAT ACT GTT ACT TTG CAA ATG AAT AAT TTG AAA CCA GAA GAT ACT GCT ATT TAT TAT TGT GCT GCT GCT-3', used as PCR template 1; synthesized using three bases The base codon makes the CDR3 region contain 16 amino acids and the amino acids are randomly arranged random...
Embodiment 2
[0025] Example 2: Screening process for Nanobodies against prealbumin PA:
[0026] (1) Add 200 microliters of transformed TG1 cells to 100 milliliters of 2×TY medium and incubate at 37°C for 3 hours. (2) Add 40 microliters of VCSM13 helper phage and let stand at room temperature for 30 minutes. (3) Centrifuge for 10 minutes, resuspend the centrifuged cells into 250 ml of 2×TY medium, and incubate overnight at 37°C; (4) Centrifuge the culture solution at 8000 rpm for 30 minutes, take the supernatant, and use PEG / The amplified phages were precipitated with NaCl, and the precipitated phages were dissolved in PBS solution. (5) A total of 10 micrograms of prealbumin PA dissolved in 100 millimolar pH 8.2 NaHCO3 was coupled to a NUNC microtiter plate, and placed overnight at 4°C, and a negative control was set up at the same time. (26) On the second day, add 100 microliters of 0.1% casein to the two wells respectively, and block for 2 hours at room temperature. (7) After 2 hours,...
Embodiment 3
[0027] Example 3: Using phage enzyme-linked immunosorbent method (ELISA) to screen specific single positive clones:
[0028] (1) From the cell culture dish containing phage after the above 6 rounds of selection, pick 96 single colonies and inoculate them in TB medium containing 100 micrograms of ampicillin per milliliter (1 liter of TB medium contains 2.3 grams of potassium dihydrogen phosphate , 12.52 grams of dipotassium hydrogen phosphate, 12 grams of peptone, 24 grams of yeast extract, 4 milliliters of glycerol), after growing to the logarithmic phase, adding IPTG with a final concentration of 1 mmol, and culturing overnight at 28°C. (2) Use the infiltration method to obtain the crudely extracted antibody, and transfer the antibody to an antigen-coated ELISA plate, and place it at room temperature for 1 hour. (3) Wash off unbound antibodies with PBST, add primary mouse anti-HA tag antibody (mouse anti-HA antibody, purchased from Beijing Kangwei Century Biotechnology Co., L...
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