Bromadiolone nanobody and its application
A technology of nanobody and bromdiolone, which is applied in the field of bromidilone nanobody, can solve the problems of high production cost, limited application, complex structure, etc., and achieves the effects of acid and alkali resistance production, simple structure and easy production.
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Embodiment 1
[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions. Embodiment 1 Construction of anti-bromadiolone nanobody immune library
[0034] The modified bromadiolone hapten ( figure 1 ) and bovine serum albumin (Bovine Serum Albumin, BSA) covalently coupled to obtain bromadiolone artificial antigen BRD-BSA, take 300 μg of BRD-BSA and Freund's complete adjuvant mixed emulsification, alpaca (Alpaca) Multiple subcutaneous injections for immunization. For booster immunization, 150 μg of BRD-BSA was mixed and emulsified with Freund's incomplete adjuvant at intervals of three weeks, ...
Embodiment 2
[0040] Example 2 Panning and identification of anti-bromadiolone heavy chain single domain antibody
[0041] The nanobody of bromadiolone was panned from the anti-bromadiolone nanobody library prepared in Example 1 by solid-phase affinity panning.
[0042] Bromadiolone hapten was covalently coupled with ovalbumin (OVA) to prepare the original coating BRD-OVA; the original coating was diluted with coating buffer (0.05M, pH 9.6, carbonate buffer) , add 100 μL per well into the microwell plate, and coat overnight at 4°C (decrease the coating concentration round by round, the coating concentration of the first to fourth rounds is 1000 μg / mL, 100 μg / mL, 10 μg / mL, 1 μg / mL respectively) Discard the liquid in the well, wash with PBST (phosphate buffered solution containing 0.5% v / v Tween-20) for 3 times, and pat dry; add 3% skim milk to each well at 300 μL, block for 1.5 h at 37°C; discard the liquid in the well, PBST Wash 3 times and pat dry; add 100 μL / well phage antibody library (...
Embodiment 3
[0046] Example 3 Expression and purification of anti-bromadiolone nanobody
[0047] Restriction endonuclease reagent sfiI was used to digest recombinant phagemid pAK100-BRD and expression vector pJB33, and 1% agarose gel electrophoresis recovered anti-bromadiolone nanobody gene and pJB33 carrier gene, and T4 DNA ligase The obtained anti-bromadiolone nanobody gene fragment was ligated with the expression vector pJB33, and then the recombinant expression plasmid was transformed into 100 μL RV308 competent cells. Immediately after transformation, the transformation product was added to 900 μL of medium containing 2×YTG, and incubated at 37° C. with shaking at 250 rpm for 1 hour. Take 100 μL of the bacterial solution and spread it on a 2×YTG plate containing 30 μg / mL chloramphenicol, and culture it upside down at 37°C overnight. Pick a single colony for bacterial liquid PCR identification, extract the plasmid with the correct strain and send it for sequencing, and save the strain...
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