A preparation method of a polyclonal antibody for detecting rapeseed fatty acid dehydrogenase and its products and applications
A fatty acid dehydrogenase and polyclonal antibody technology, applied in the field of immunology, can solve the problems of expensive instruments, high detection and maintenance costs, and cumbersome steps, and achieve strong affinity, accurate reflection of protein content, and preparation costs low effect
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Embodiment 1
[0022] Example 1: Sequence Analysis and Prediction of Candidate Epitopes
[0023] Search the keyword: BnFAD2 in the UniProtKB database, and obtain the sequence information of rapeseed FAD2 protein.
[0024] ABCpred Prediction Server online software, analyze BnFAD2 (UniProtKB: C3W518) amino acid B cell antigen epitope, input the sequence with predicted epitope array score over 0.9 into NCBI database, and compare amino acid with rapeseed protein library to verify amino acid epitope sequence Specificity, that is, there is no homology of 5 or more consecutive amino acid sequences with other proteins in the rapeseed protein database. Select the two polypeptide sequences PA (amino acid sequence as shown in sequence 1) and PB (amino acid sequence as shown in 2) with the highest score and specificity for polypeptide synthesis, and the synthesized polypeptides are coupled to KLH respectively; The polypeptides KLH-PA and KLH-PB; the synthesized polypeptides were coupled with BSA respec...
Embodiment 2
[0026] Example 2: Preparation of polyclonal antibody serum
[0027] Animals for Immunization Select healthy male New Zealand white rabbits aged about three months and weighing about 2.5 kg, and immunize them with the special antigen prepared in Example 1. Specific steps are as follows:
[0028] Before the first immunization, blood was taken from the ear vein as a negative control. The antigen was diluted to 1 mg / mL with PBS (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4, pH7.4), and stored at -20°C after aliquoting. Take 500 μL of 1 mg / mL antigen (that is, 0.5 mg. Add 300 μL PBS to dilute again, and then add an equal volume of Freund’s complete adjuvant (first immunization) or Freund’s incomplete adjuvant (second to fourth immunization), And mix and emulsify on the vortex instrument. Rabbit is carried out limbs, armpit and back subcutaneous multi-point injection, carries out the second immunization three weeks after the first immunization, strengthens once every two weeks ...
Embodiment 3
[0029] Example 3: Preparation of Rapeseed Fatty Acid Dehydrogenase (FAD2) Polyclonal Antibody
[0030] Concentrated protein samples by ammonium sulfate precipitation method: dilute 20mL of serum with TBS buffer solution to 100mL, then add saturated ammonium sulfate solution to the solution while stirring until the concentration of ammonium sulfate solution reaches 50-60%. Stir overnight on a magnetic stirrer. The next day, the solution containing a large amount of precipitate was centrifuged, the supernatant was discarded, and the precipitate was retained. Add 10 mL of PBS buffer solution containing sodium azide to dissolve the precipitate, and perform dialysis treatment with a dialysis bag (change the dialysate every 4 hours, a total of six times), so as to remove ammonium sulfate. After dialysis, the solution in the dialysis bag was centrifuged to collect the supernatant.
[0031] Purification of anti-polypeptide antibodies by affinity chromatography: add the above-mention...
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