Special detection kit for acute hepatopancreatic necrosis syndrome and preparation method thereof
A kit and hepatopancreas technology are applied to the special detection kit for acute hepatopancreatic necrosis syndrome and the field of preparation thereof, which can solve the problems of low detection efficiency of the kit, inability to diagnose toxin expression, unfavorable rapid diagnosis of epidemic situation, etc. The effect of reducing the incidence of false positives, strong resistance and high sensitivity
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Embodiment 1
[0067] Embodiment 1: Preparation of pirA and pirB proteins
[0068] (1) Cloning of Vibrio parahaemolyticus pirA and pirB genes, construction and identification of expression vectors
[0069] (a) primers are designed according to the sequence of Vibrio parahaemolyticus pirA and pirB genes:
[0070] The PCR amplification primers of the pirA gene sequence are:
[0071] Upstream primer: 5'- CGCGGATCC ATGAGTAACAATATAAAACATGAAAC-3', wherein the underlined part is the BamH I restriction site;
[0072] Downstream primer: 5'- CCCGCGGCCGC TTAGTGGTAATAGATTGTACAGAAA-3', where the underlined part is the Not I restriction site.
[0073] The primers for PCR amplification of the pirB gene sequence are:
[0074] Upstream primer: 5'- CGCGGATCC ATGACTAACGAATACGTTGTAACAA-3', wherein the underlined part is the BamH I restriction site;
[0075] Downstream primer: 5'- CCCGCGGCCGC CTACTTTTTCTGTACCAAATTCATCG-3', where the underlined part is the Not I restriction site.
[0076] The plasm...
Embodiment 2
[0106] Example 2: Anti-acute hepatopancreatic necrosis syndrome toxin protein egg yolk antibody extracted after immunization of laying hens
[0107] For immunization, the pirA and pirB proteins were mixed as antigens with an equal volume of Fischer's adjuvant, and multi-point subcutaneous injection was used to immunize laying hens;
[0108] Specifically, measure the concentration of the purified toxin protein, adjust the concentration of the purified recombinant protein to 0.01-0.1 mg / mL with PBS, mix the adjusted recombinant protein with the adjuvant at a ratio of 1:1 and follow the company's special Laying hens were immunized with five times of two-way immunization. For the first immunization, 1 mL of recombinant protein was fully mixed with 1 mL of Freund's complete adjuvant and injected into the chest muscle at four points. Two weeks later, 0.5 mL of recombinant protein was fully mixed with 0.5 mL of Freund's incomplete adjuvant and then injected into the chest muscle at f...
Embodiment 3
[0111] Embodiment 3: the assembly of kit
[0112] (1) Coating microtiter plate: Dilute the purified specific egg yolk antibody against pir toxin protein to a concentration of 5 μg / mL, take 100 μL of the diluted specific egg yolk antibody solution and coat a 96-well microtiter plate, and wrap at 4°C. be overnight. After taking it out the next day, wash the plate 3 times with PBST, 3 minutes each time. 1% BSA prepared in PBST solution was used as a blocking solution, 100 μL of blocking solution was added to each well, and blocked at 37° C. for 1 h. After sealing, the plate was washed 3 times with PBST, 3 min each time. Put it into a special packaging bag for 96-well microplate plate, seal it with a sealing machine and store it at 4°C.
[0113] (2) Enzyme-labeled detection antibody: the enzyme-labeled polyclonal antibody against pir toxin protein can be obtained or purchased commercially through technical service outsourcing, and can also be prepared by conventional methods in...
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