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Bovine rotavirus chimeric antigen and colloidal gold immunochromatography test paper card for detecting bovine rotavirus antibody

An immunochromatographic test strip, bovine rotavirus technology, applied in the direction of virus/phage, virus, virus peptide, etc., can solve the problems of complicated operation and long time, achieve high specificity, high practical value, and solve calf diarrhea The effect of quickly diagnosing problems

Active Publication Date: 2020-03-24
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ELISA method still has the disadvantages of complicated operation, long time consumption, and the need for certain equipment.

Method used

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  • Bovine rotavirus chimeric antigen and colloidal gold immunochromatography test paper card for detecting bovine rotavirus antibody
  • Bovine rotavirus chimeric antigen and colloidal gold immunochromatography test paper card for detecting bovine rotavirus antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Prokaryotic expression of the chimeric antigen of the present invention

[0027] 1. Construction of bovine rotavirus chimeric antigen gene recombinant expression plasmid pET28a-BRV-VP

[0028] According to the analysis and research of bovine rotavirus VP4 protein (accession number: AFA41728.1), VP6 protein (accession number: AFA41729.1), and VP7 protein (accession number: AFC41005.1) on the NCBI website, select special regions The bovine rotavirus chimeric antigen gene (shown in SEQ ID NO: 2) is genetically designed, and the sequence is optimized at the same time, and the bovine rotavirus chimeric antigen gene is synthesized by a chemical synthesis method. Digest the pET28a(+) vector with BamHI and XhoI. The digestion system is 50uL: 1uL each of BamHI and XhoI, 20uL of the vector fragment, 5uL of 10X digestion buffer, and 23uL of ddH2O; the digested product is subjected to 1% agarose electrophoresis Post-gel recovery kit recovery. Then the digested vector w...

Embodiment 2

[0033] Embodiment 2: Colloidal gold immunochromatography detection test paper card for preparing bovine rotavirus antibody

[0034] 1. Preparation of gold-labeled conjugates of SPA labeled with gold particles

[0035] Take 2ml of 40nm colloidal gold particles and use 0.1mol / L potassium carbonate to adjust the pH to 6.8, add 10ug Staphylococcus aureus protein A (SPA), mix quickly and mix at room temperature on a 3D rotator for 30min, then add the final concentration 1% BSA and mix on a 3D rotary mixer for 30min, centrifuge the gold standard solution at 12000r / min at 4°C for 10min, carefully discard the supernatant, wash the precipitate twice with 0.01M PBS buffer, and then centrifuge the resulting precipitate It is the purified SPA gold-labeled complex. The prepared colloidal gold-labeled SPA is resuspended in 0.01M PBS and stored at 4°C for later use.

[0036] 2. Nitrocellulose membrane testing line and quality control line spraying and colloidal gold bonding pad preparation ...

Embodiment 3

[0042] Embodiment 3: colloidal gold immunochromatography test paper card specificity test

[0043] A cross-test was carried out on the known bovine Brucella antibody-positive serum, bovine foot-and-mouth disease O-type antibody-positive serum, bovine viral diarrhea virus antibody-positive serum, bovine coronavirus antibody-positive serum and bovine rotavirus antibody-positive serum. The results are shown in figure 2 .

[0044] figure 2 The results showed that only the bovine rotavirus antibody-positive serum samples had positive reactions, both the detection line and the quality control line were colored, and when they reacted with other sera, the detection lines were not red, and they were negative reactions, indicating that the method is effective. Highly specific.

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Abstract

The invention, which relates to the technical field of immunodetection, discloses a bovine rotavirus chimeric antigen and a colloidal gold immunochromatography test paper card for detecting a bovine rotavirus antibody. The sequence of the chimeric antigen is shown as SEQ ID NO: 1. The invention provides a novel bovine rotavirus chimeric antigen. When being used as a detection antigen of a bovine rotavirus antibody, the antigen gas the high specificity, repeatability and accuracy and can be applied to related immunochromatographic detection products; the result is visual and can be judged by naked eyes; the bovine rotavirus antibody in serum can be detected rapidly and specifically; and a problem of rapid diagnosis of calf diarrhea in large-scale dairy farms can be solved. The bovine rotavirus chimeric antigen has the great practical value and is convenient to popularize and use in grassroots.

Description

technical field [0001] The invention relates to the technical field of immune detection, in particular to a bovine rotavirus chimeric antigen and a colloidal gold immunochromatographic test paper card for detecting bovine rotavirus antibodies. Background technique [0002] Rotavirus (RV) belongs to the genus Rotavirus of the Reoviridae family and is divided into seven serogroups A to G, among which serogroups A to C can infect cattle and cause bovine rotavirus diarrhea, while Group A rotavirus is the most harmful to newborn calves of 1 to 7 days of age. Nearly 50% of neonatal calf diarrhea symptoms are related to bovine rotavirus infection. Infected calves clinically manifest symptoms such as mental depression, watery diarrhea, acidosis, etc., and are prone to secondary bacterial infections. When mixed with rotavirus infection When it is more severe, the mortality rate of sick calves can be as high as 50% to 100%. [0003] Bovine rotavirus diarrhea is one of the important ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00G01N33/68G01N33/569G01N33/558
CPCC07K14/005C07K2319/00C12N2720/12322G01N33/558G01N33/56983G01N33/68
Inventor 孟赓朱文壮孟凯闻唐振庭
Owner CHINA AGRI UNIV
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