Bovine herpes virus type I antibody blocking ELISA detection method

A bovine herpes virus and detection method technology, which is applied in the directions of viral peptides, chemical instruments and methods, botanical equipment and methods, etc., can solve the problem that the bovine herpes virus type I detection method cannot perform fast, high-throughput, detection, and inability to detect virus particles and other problems, to achieve the effect of high specificity, low emission and time saving

Inactive Publication Date: 2021-01-05
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Claims
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AI Technical Summary

Problems solved by technology

[0003] The present invention provides a bovine herpes virus type I antibody blocking ELISA detection method to solve the problem that the existing bovine herpes virus type I detection method cannot perform rapid and high-throughput detection or cannot detect low-emission virus particles in the incubation period

Method used

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  • Bovine herpes virus type I antibody blocking ELISA detection method
  • Bovine herpes virus type I antibody blocking ELISA detection method
  • Bovine herpes virus type I antibody blocking ELISA detection method

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specific Embodiment approach 1

[0026] Specific embodiment one: the detection method of bovine herpes virus type I antibody blocking ELISA of the present embodiment is carried out according to the following steps:

[0027] 1. The antigen recombinant gD protein coated by the ELISA plate is coated with the reaction plate, and BSA is blocked;

[0028] 2. After the serum to be tested and the negative and positive control serum are processed, add the reaction plate to block;

[0029] 3. Horseradish peroxidase-labeled mAb 2B4 binds to the unblocked antigen gD protein, and develops color;

[0030] 4. Terminate the reaction, measure OD450nm, and calculate the inhibition rate; that is, the detection is completed.

[0031] In the first step of this embodiment, the preparation method of the BSA blocking solution is as follows:

[0032] 1. First prepare PBS with pH 7.4: weigh 8g NaCl, 0.2g KCl, 1.42g Na 2 HPO 4 ·12H 2 O and 0.27gKH 2 PO 4 Add 800mL ddH 2 Stir until all dissolved, adjust pH to 7.4, dilute to 1L, ...

specific Embodiment approach 2

[0053] Embodiment 2: The difference between this embodiment and Embodiment 1 is that the antigenic recombinant gD protein in step 1 is prepared according to the following method:

[0054] 1. Extracting bovine herpesvirus type I genomic DNA;

[0055] 2. Recovery and purification of amplified PCR products, primer sequences are as follows:

[0056] gD-T1: 5'-G GAATTC ATGCAAGGGCCGACATTGG-3’

[0057] gD-T2: 5'-CC CTCGAG CAGCGCGCTGTAGTTGACGTT-3’

[0058] 3. The PCR product is ligated with the T vector, and the ligated product is transferred into competent cells, identified by enzyme digestion, screened for positive clones, and sequenced;

[0059] Fourth, Eco RI-HF and Xho I double digestion of positive cloned plasmids and pGEX-6P-1, construction of pGEX-6P-1-gD expression vector, transfer into competent cells, pick positive clones after culture, double digestion identification;

[0060] 5. The culture medium of positive clones was induced to express gD recombinant protein wit...

specific Embodiment approach 3

[0064] Embodiment 3: The difference between this embodiment and Embodiment 2 is that: in step 2, the PCR amplification conditions are: 98°C pre-denaturation for 30s; 98°C denaturation for 10s, 63°C annealing for 30s, 72°C extension for 45s, setting 25 cycle, with a final extension at 72°C for 10 min. Other steps and parameters are the same as in the second embodiment.

[0065] In this embodiment, the temperature and time of pre-denaturation, denaturation, annealing, and extension are set according to the high GC content PCR enzyme instructions of Beijing Sizhengbai Biotechnology Co., Ltd.

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Abstract

The invention discloses a bovine herpes virus type I antibody blocking ELISA detection method, belongs to a serological detection method for antibody detection, and is mainly used for detecting bovineherpes virus type I. The detection method is characterized in that the antigen coated by the elisa plate in the ELISA detection method is jointly completed by herpes virus type I gD recombinant protein and horseradish peroxidase labeled monoclonal antibody 2B4. Specifically, the blocking ELISA is established by bovine herpes virus type I recombinant gD protein and horse radish peroxidase labeledmonoclonal antibody 2B4. The detection method is strong in specificity and high in sensitivity, and can directly detect bovine herpes virus type I infected people in an incubation period.

Description

technical field [0001] The invention relates to an ELISA detection method. Background technique [0002] Bovine infectious rhinotracheitis, also known as necrotizing rhinitis and red nose disease, is a contact infectious disease caused by bovine herpesvirus 1 (Bovineherpesvirus 1, BoHV-1). The establishment of it will help to take early prevention and control measures against the spread of the virus. At present, the commonly used detection methods, such as the isolation and identification of bovine herpes virus type I virus, electron microscopy, histopathology, polymerase chain reaction and serological detection, have their own advantages and disadvantages, of which the first three cannot be used for rapid and high-throughput detection. , while the polymerase chain reaction cannot detect viral antigens in the latent period. SUMMARY OF THE INVENTION [0003] The present invention provides a bovine herpes virus type I antibody blocking ELISA detection method in order to so...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/543G01N33/535C12N15/70C12N15/38C07K14/06
CPCG01N33/577G01N33/56994G01N33/54306G01N33/535C07K14/005C12N15/70G01N2333/06G01N2469/20
Inventor 宋佰芬翟璐宋丽马金柱佟春玉于永忠崔玉东
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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