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Infectious bovine rhinotracheitis virus gD protein and application thereof

A technology for rhinotracheitis virus and rhinotracheitis, which is applied in the directions of viruses, viral peptides, viruses/phages, etc., can solve the problems of inability to develop grassroots pastures, time-consuming and labor-intensive, and high cost, and achieves convenient promotion and use, and rapid detection. Convenient, highly sensitive effect

Inactive Publication Date: 2016-07-13
NBGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Conventional IBRV diagnostic tests mainly include classic virus isolation and culture, immunofluorescence, ELISA and molecular biological detection methods such as PCR, etc., all of which rely on instruments and equipment and the delicate operation of professional laboratory personnel, which are either costly or time-consuming It is labor-intensive and cannot be carried out on grass-roots pastures

Method used

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  • Infectious bovine rhinotracheitis virus gD protein and application thereof
  • Infectious bovine rhinotracheitis virus gD protein and application thereof
  • Infectious bovine rhinotracheitis virus gD protein and application thereof

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Effect test

Embodiment 1

[0026] Embodiment 1: Cloning expression and purification of gD protein of bovine infectious rhinotracheitis virus

[0027] (1) Cloning, expression and identification of gD protein:

[0028] Design primers with reference to the IBRV gene sequence registered in GenBank, its upstream primer gD-F is the sequence listed in SEQIDNo.3, and its downstream primer gD-R is the sequence listed in SEQIDNo.4, which is used to clone the gD gene fragment. , EcoRI and HindIII restriction sites were introduced into the downstream primers, and the primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The gD gene fragment was amplified by PCR using the pUC-gD plasmid as a template. Reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 55.6°C for 45 s, extension at 72°C for 2 min, after 30 cycles, extension at 72°C for 7 min, and termination at 4°C. The purified PCR product was digested with the expression vector pF...

Embodiment 2

[0032] Embodiment 2: colloidal gold labeling of bovine infectious rhinotracheitis virus gD protein

[0033] According to conventional means, a 40nm colloidal gold particle solution was prepared by trisodium citrate reduction method, and the pH value was adjusted to 9.0 for later use. Take a certain amount of colloidal gold particle solution, place it on a magnetic stirrer, then dilute the purified gD protein antigen with PBS at a ratio of 1:2000, add it to the colloidal gold solution and stir for 60 minutes, then add the PEG20000 solution to mix In the reaction solution, the final concentration of PEG was about 1%, placed in a refrigerated centrifuge (4°C) and centrifuged at a high speed (11000-13000rpm) for 10 minutes, then discarded the supernatant. The lower precipitate was repeatedly washed with PBS and centrifuged to discard excess supernatant. Finally, the precipitate was resuspended in PBS buffer to make the final protein concentration about 50 μg / mL, and stored at 4°C...

Embodiment 3

[0034] Embodiment 3: colloidal gold test strip assembly

[0035] With the PVC backboard (1) as the support, nitrocellulose membrane (4), gold standard pad (2), sample pad (3), and water-absorbing pad (5) are pasted on it. Among them, the gold label pad (2) is made of polyester film. After being treated with PBS containing 1% BSA and 1% Tween-20, the prepared colloidal gold-labeled gD antigen is sprayed in 40 μL, and dried at 37oC for 2 hours for later use. .

[0036] The sample pad (3) is a polyester fiber pad, which is soaked in a self-made treatment solution containing a surfactant and dried for later use.

[0037] The nitrocellulose membrane (4) is coated with two lines: the quality control line (6) and the detection line (7), in which the detection line is coated with the detection antigen gD, and the quality control line is coated with rabbit anti-bovine Infectious rhinotracheitis virus gD protein antibody (self-made), the detection antigen and antibody were diluted wit...

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Abstract

The invention discloses a test strip for detecting infectious bovine rhinotracheitis, which comprises a PVC backboard, a sample pad, a gold mark pad, a cellulose membrane and a water absorbing pad. The test strip is characterized in that infectious bovine rhinotracheitis virus gD protein according to the claim 1, marked by colloidal gold, is coated on the gold mark pad; and the infectious bovine rhinotracheitis virus gD protein and a rabbit anti-infectious bovine rhinotracheitis virus gD protein antibody are separately coated on a detection line and a quality control line of the cellulose membrane. The test strip for detecting the infectious bovine rhinotracheitis virus antibody in a bovine serum sample is high in sensitivity, is good in specificity, is quick and convenient to detect, is easy to use, and is free of special apparatus equipment, can detect results within 10 minutes, and is very convenient for being popularized and used in the basic-level breeding unit.

Description

technical field [0001] The invention relates to the field of biotechnology, and relates to a bovine infectious rhinotracheitis virus gD protein and application thereof. Background technique [0002] Infectious bovine rhinotracheitis (IBR) is an acute contagious disease of cattle caused by bovine herpesvirus type 1 (BHV-1). Clinically, it is divided into respiratory tract, reproductive tract, brain, conjunctiva and other infection types, which harms calves, cows and bulls. The mortality rate of the disease is low, and it is mostly subclinical, but it is prone to secondary bacterial infection, leading to bronchial pneumonia, etc. Severe respiratory disease, which has great harm to cattle. According to Wang Zhiliang et al. (2011), 996 serum samples from 11 dairy farms in Beijing, Tianjin, Shaanxi, Shanxi, Shandong, Xinjiang, Henan, and Hebei were tested for IBRV antibodies, and a total of 120 positive sera were detected. The group positive rate was 77.8%, the highest positive...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/06G01N33/569G01N33/558G01N33/532
CPCC07K14/005C12N2710/16022G01N33/532G01N33/558G01N33/56994G01N2333/06
Inventor 韦海涛宋彦军冯小宇杨春江赵荣茂王林马孝斌梅力于在江雷琪莉莫勋李刚高云玲
Owner NBGEN
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