Antibody library of bacteriophages and applications in immunoassay of pesticide residue
A phage antibody library and phage antibody technology, applied in phage antibody library and its application field
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Embodiment 1
[0108] Construction of phage antibody library
[0109] Material:
[0110] Phagemid pCANTAB5E, E.coli TG1 freeze-dried strain, M13K07 helper phage are all products of Pharmacia. The kits SV Total RNA Isolation System; ReverseTranscription System; various restriction endonucleases and ligases were purchased from Promega. Immunogen milbemycin oxime (A 4 、A 3 )-BSA (MILO-BSA) laboratory preservation, female 7-week-old Balb / c mice were purchased from the Comparative Medicine Center of Yangzhou University.
[0111] Primer design
[0112] Design multiple groups of degenerate primers in the light and heavy chain framework regions FR1 and FR4 of the antibody molecule as follows: and add linking peptides (Gly 4 Ser) 3 Partial sequence and restriction site of (Linker), wherein, V L F primer contains NotI restriction site, V H Primer B contains SfiI restriction site. V H F primer contains part of Linker sequence, V L Primer B contains part of the Linker sequence, and the two Li...
Embodiment 2
[0170] Taking milbemycin pesticide as an example to screen soluble scFv
[0171] (1) Enrichment and screening of MILO-BSA phage antibody
[0172] (1) Coat the immunotube with 4ml purified MILO-BSA as the antigen (100μg / ml), the coating solution is PBS (pH7.4), coat the plate overnight at 4°C, wash the immunotube 3 times with PBS, 5min each time.
[0173] (2) Fill the immunotube with 2% MPBS and block at 37°C for 2 hours.
[0174] (3) Remove the blocking solution, add the amplified phage antibody, and incubate at 37°C for 2h.
[0175] (4) To remove unbound phage antibodies, wash the immunotube 10 times with PBST buffer (10 times in the first round, and 20 times in each subsequent round).
[0176] (5) Add 500 μl of 1 mg / ml trypsin into the immunotube, incubate at 37°C for 30 minutes, and repeat pipetting to elute the specifically bound phage antibody.
[0177] (6) Infect 3.5 ml of E.coli TG1 in the logarithmic growth phase with 500 μl of the eluate, and bathe in water at 37° ...
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