A bovine rotavirus chimeric antigen and a colloidal gold immunochromatographic test paper card for detecting bovine rotavirus antibodies

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

Active Publication Date: 2021-07-13
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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  • A bovine rotavirus chimeric antigen and a colloidal gold immunochromatographic test paper card for detecting bovine rotavirus antibodies
  • A bovine rotavirus chimeric antigen and a colloidal gold immunochromatographic test paper card for detecting bovine rotavirus antibodies

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 chemical synthesis. 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 was ligate...

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 relates to the technical field of immune detection, and discloses a bovine rotavirus chimeric antigen and a colloidal gold immunochromatographic test paper card for detecting bovine rotavirus antibodies. The chimeric antigen sequence of the present invention is shown in SEQ ID NO:1. The invention provides a novel bovine rotavirus chimeric antigen, which has high specificity, repeatability and accuracy when used as a detection antigen for bovine rotavirus antibodies, and can be applied to related immunochromatographic detection products Among them, the results are intuitive and can be judged by the naked eye. It can quickly and specifically detect bovine rotavirus antibodies in serum, and solve the problem of rapid diagnosis of calf diarrhea in large-scale dairy farms. It has high practical value and is convenient for grass-roots promotion and use.

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, of 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 epi...

Claims

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Application Information

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Patent Type & Authority Patents(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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