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Antibody joint detection kit containing porcine pseudorabies virus gD and gE proteins and preparation method and application thereof

A porcine pseudorabies and kit technology, which is applied in the field of biomedicine, can solve the problems of inaccurate quantification of detection results, time-consuming operation and calculation, and increased labor intensity of breeding, so as to improve the convenience and accuracy of operation, easy operation, and avoid Effects of data processing and mass data analysis

Pending Publication Date: 2021-11-19
洛阳中科生物芯片技术有限公司
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Chinese patents CN103792373A and CN106405082A use colloidal gold detection technology to prepare colloidal gold detection test strips for simultaneous detection of PRVgD and gE antibodies, but the detection results cannot be accurately quantified. The level of PRVgD antibody and PRVgE antibody in the test results can be comprehensively judged whether the pig is or not Whether the pig herd is in a healthy state or in the early, middle or late stage of infection, so as to guide the clinical formulation of immunization strategies and decontamination measures
The daily prevention of two diseases requires secondary immunization. Considering the actual production, multiple immunizations will stimulate the animal body and affect its growth, and will increase the labor intensity and labor cost in breeding. For this reason, the dual vaccine was developed , but He Weijie (Study on the Immune Interference Phenomenon of Swine Fever and Porcine Pseudorabies Dual Vaccine, Master Thesis of Hebei Agricultural University, 2016) found that swine fever and porcine pseudorabies dual vaccine can interfere with swine fever Early antibody production
The results of these experiments need to be monitored with corresponding kits to avoid disease or death of pigs caused by false immune effects. Currently, the commercially available kits are all independent kits, and the operation and calculation during monitoring are time-consuming and labor-intensive.

Method used

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  • Antibody joint detection kit containing porcine pseudorabies virus gD and gE proteins and preparation method and application thereof
  • Antibody joint detection kit containing porcine pseudorabies virus gD and gE proteins and preparation method and application thereof
  • Antibody joint detection kit containing porcine pseudorabies virus gD and gE proteins and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1 Preparation of raw materials of porcine pseudorabies virus gD, gE protein antibody-linked detection kit

[0074] 1.1 Preparation and identification of porcine pseudorabies virus gD protein

[0075] According to CN105251000A patent, the PRVgD protein was prepared, and the recombinant PRVgD protein was obtained after purification with Ni affinity chromatographic column. The obvious protein band was identified at about 50KDa by SDS-PAGE, and the purity was 94% after analysis; it was determined by BCA kit Protein content, the result is 5.6mg / ml.

[0076] 1.2 Preparation and identification of porcine pseudorabies virus gE protein

[0077] 1.2.1 Amplification of porcine pseudorabies virus gE gene

[0078] Inoculate PRV HN1201 strain virus (porcine pseudorabies virus strain is HN1201 strain (Pseudorabies virus, strain HN1201)) on the well-growing PK15 cells, and the preservation number is CCTCC NO.V201311, preserved in the China Type Culture Collection Center, a...

Embodiment 2

[0109] Example 2 Preparation of porcine pseudorabies virus gD, gE protein antibody dual detection kit

[0110] 2.1 Preparation of sample solution

[0111] Preparation of 5% glycerin solution: Accurately weigh 5.00g of glycerin, put it in a 100ml volumetric flask, add a small amount of purified water and rotate gently to dissolve fully, avoid excessive air bubbles, add purified water to the scale line, turn up and down and shake for 10 time, spare;

[0112] Preparation of 5% sorbitol solution: Accurately weigh 5.00g of sorbitol, put it in a 250ml beaker, add an appropriate amount of purified water and stir to dissolve it completely, then completely transfer it to a 100ml volumetric flask, add purified water to the scale line, turn it up and down Shake 10 times, set aside;

[0113] Preparation of 0.05% Triton solution: Measure 50 μl Triton with a pipette gun and put it in a 100ml volumetric flask, add an appropriate amount of purified water to dissolve it completely, add purif...

Embodiment 3

[0174] Example 3 Preparation of Porcine Pseudorabies Virus gD, gE Protein Antibody, Hog Fever Virus E2 Protein Antibody Triple Detection Kit

[0175] 3.1 Selection of swine fever virus E2 protein coating amount

[0176]As shown in Example 2.1, the classical swine fever virus E2 protein of Example 1 was diluted to an appropriate concentration with the spotting solution, and the spotting volume was 20 nl, so that the final contents of the classical swine fever virus E2 protein spotted on the membrane were as shown in Table 3 respectively. , everything else remains unchanged. Use P3, N serum to detect according to the method of embodiment 2.2, the result (see Table 9): PRVgD, PRVgE under the situation that the coating amount is constant detects all negative (table 9 does not reflect); N / P3 is in CSFV E2 bag When the coating amount is low, the value is small, and when the coating amount of CSFV E2 reaches 0.4ng / point or more, the data is significantly improved, and the size of th...

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Abstract

The invention relates to an antibody joint detection kit containing porcine pseudorabies virus gD and gE protein antibodies. The joint detection kit comprises one or more porcine pseudorabies virus gD and gE protein antibody detection chips and an enzyme-labeled reagent, the detection chips are coated with porcine pseudorabies virus gD protein, gE protein, a quality control product and a blank control point, the enzyme-labeled reagent is a solution containing an enzyme-labeled porcine pseudorabies virus gD and gE protein monoclonal antibody, the quality control product is a goat anti-mouse polyclonal antibody, and the coating amount is 1-4 ng / dot. According to the kit prepared by the invention, the quality control product is arranged on the reaction carrier, negative control does not need to be arranged in the kit, the detection is rapid, the operation is simple and convenient, the integration is high, one-button intelligent data processing is realized, the kit can be used for detecting the infection state of the porcine pseudorabies virus in a swinery, evaluating the immune effect of the porcine pseudorabies virus vaccine in the swinery and identifying the infection condition of the newly introduced porcine pseudorabies virus, and is convenient for formulating an immune program and carrying out early purification.

Description

technical field [0001] The invention relates to a dual detection kit and a triple detection kit for antibodies to porcine pseudorabies virus gD and gE proteins, a preparation method and application thereof, and belongs to the field of biomedicine. Background technique [0002] Pseudorabies (Pseudorabies, PR; also known as Aujeszky's disease, Aujeszky's disease), is caused by porcine herpesvirus type I (Suid herpes virus I) in the α subfamily of the Herpesviridae family (Herpesviridae). An acute infectious disease of various livestock and wild animals such as sheep, with fever, severe itching (except pigs), and encephalomyelitis as the main symptoms. Pigs are the main storage host and source of infection of the disease. Porcine pseudorabies virus can infect pigs of different ages, but pregnant sows and suckling piglets are the most serious infection: it leads to abortion, stillbirth and mummified fetuses in pregnant sows; when fattening pigs are infected with porcine pseudor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/543G01N33/535
CPCG01N33/56983G01N33/54306G01N33/535G01N2333/03
Inventor 田克恭昌静峰李玉芳张许科
Owner 洛阳中科生物芯片技术有限公司
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