A bovine coronavirus chimeric antigen and colloidal gold immunochromatographic test paper card for detecting bovine coronavirus antibody

A chimeric antigen, coronavirus technology, applied in the directions of viruses/phages, viruses, viral peptides, etc., can solve the problems of difficult on-site large-scale application, unsuitable for grass-roots applications, and high equipment requirements, and achieve rapid diagnosis of calf diarrhea. Problems, easy to popularize and use at the grassroots level, and the effect of high practical value

Active Publication Date: 2021-07-13
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, BCoV is prevalent in some areas of my country. At the same time, it is seriously mixed with bovine rotavirus and bovine viral diarrhea virus in cattle herds, causing huge economic losses to the local cattle industry.
However, neither the inactivated vaccine nor the live attenuated vaccine is effective against BCoV. At the same time, there is still a lack of effective drugs for the treatment of BCoV. Therefore, BCoV monitoring has become an important part of BCoV control.
[0005] BCoV detection methods include: isolation and culture identification of the virus, which is not suitable for grassroots applications due to the complex nutritional requirements of BCoV and difficulty in the isolation of the first generation; electron microscope observation, BCoV is easy to be confused with fine particles in fecal samples, reducing the detection rate; Virus neutralization test (VN) is a classic method for identifying BCoV. Although it is sensitive and specific, it is complicated to operate and expensive in application. Molecular biology detection technology is a diagnostic method that has been studied more and faster in recent years. This method is sensitive High accuracy and specificity, but the operation is more complicated, and the requirements for equipment are high; the hemagglutination (HI) and hemagglutination inhibition (HA) tests are relatively simple to operate, and are suitable for epidemiological investigations, but the results are often affected by colostrum samples. IgG and IgA in feces; fluorescent antibody technology (FA) has limited application range and complicated operation, and is not suitable for rapid clinical diagnosis; enzyme-linked immunosorbent assay (ELISA) is sensitive and convenient, and has been reported to use Competitive ELISA method established by monoclonal antibody, capture ELISA method for IgM and IgA, indirect ELISA method established by using recombinant N protein as antigen, etc.
[0006] Relatively speaking, the ELISA method is currently one of the most widely used large-scale rapid diagnosis methods at the grassroots level. However, the ELISA experiment has high requirements for the accuracy of the microplate reader and the level of technical personnel, and the operation is cumbersome and time-consuming. It is not easy to apply on a large scale in the field. ; and its susceptibility to other viral or bacterial cattle diseases

Method used

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  • A bovine coronavirus chimeric antigen and colloidal gold immunochromatographic test paper card for detecting bovine coronavirus antibody
  • A bovine coronavirus chimeric antigen and colloidal gold immunochromatographic test paper card for detecting bovine coronavirus antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Prokaryotic expression of the chimeric antigen of the present invention

[0030] 1. Construction of bovine coronavirus chimeric antigen gene recombinant expression plasmid pET28a-NS

[0031]According to the conservation and antigenicity analysis of the bovine coronavirus N protein and S protein sequences on the NCBI website, select a specific region of the gene to design the bovine coronavirus chimeric antigen gene (shown in SEQ ID NO: 2), and simultaneously optimize the sequence and use chemical The synthetic method synthesizes bovine coronavirus chimeric antigen gene. Digest the pET28a(+) vector with BamHI and XhoI. The enzyme digestion system is 50uL: 1uL each of BamHI and XhoI, 20uL of the vector fragment, 5uL of 10X digestion buffer, and 3uL of ddH2O2; the digested product is subjected to 1% agarose electrophoresis Gel recovery kit recovery. Then the digested vector was ligated with the chimeric antigen gene to construct a 10 μL ligation system: 5.5 μL...

Embodiment 2

[0036] Embodiment 2: Colloidal gold immunochromatography detection test paper card for preparing bovine coronavirus antibody

[0037] 1. Preparation of gold-labeled conjugates of SPA labeled with gold particles

[0038] Take 2ml of 40nm colloidal gold particles and adjust the pH to 6.8 with 0.1mol / L potassium carbonate solution, add 20ug 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.

[0039] 2. Nitrocellulose membrane testing line and quality control line spraying and colloidal gold bonding pad pre...

Embodiment 3

[0045] Embodiment 3: colloidal gold immunochromatography test paper card specificity test

[0046] Carry out cross-tests on known bovine Brucella positive sera, bovine foot-and-mouth disease positive sera, bovine viral diarrhea virus positive sera, bovine rotavirus positive sera, bovine herpes virus positive sera and bovine coronavirus antibody positive sera, see results figure 2 .

[0047] figure 2 The results showed that only the test line and quality control line of the bovine coronavirus antibody-positive serum sample developed color, showing a positive reaction, while when reacting with other sera, the test line did not show red, showing a negative reaction, indicating that the colloidal gold immune layer The test strips are highly specific.

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Abstract

The invention relates to the technical field of immune detection, and discloses a bovine coronavirus chimeric antigen and a colloidal gold immunochromatographic test paper card for detecting bovine coronavirus antibodies. The chimeric antigen sequence of the present invention is shown in SEQ ID NO:1. The present invention provides a novel bovine coronavirus chimeric antigen, which has high specificity, repeatability and accuracy when used as a bovine coronavirus antibody detection antigen, and can be applied in related immunochromatographic detection products. The results are intuitive and can be judged by the naked eye. It can quickly and specifically detect bovine coronavirus 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 easy to promote and use at the grassroots level.

Description

technical field [0001] The invention relates to the technical field of immune detection, in particular to a bovine coronavirus chimeric antigen and a colloidal gold immunochromatographic test paper card for detecting bovine coronavirus antibodies. Background technique [0002] Bovine Coronavirus (BCoV) belongs to the Betacoronaviridae coronavirus genus group 2a, which can cause calf diarrhea, adult cattle winter scour and respiratory diseases in cattle. The milk production of infected cows decreases, the growth and development of recovered calves are affected, and the growth performance in adulthood is lower than normal, and the disease is easily mixed with other viruses and causes secondary bacterial infections, thus showing complex clinical symptoms. It brings great difficulties to diagnosis and treatment. [0003] BCoV is a non-segmented, single-stranded positive-sense RNA virus whose genome expresses five structural proteins, including nucleocapsid (N), membrane (M), sp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/165G01N33/569G01N33/558G01N33/68
CPCC07K14/005C12N2770/20022G01N33/558G01N33/56983G01N33/6854G01N2333/165
Inventor 孟赓朱文壮孟凯闻唐振庭
Owner CHINA AGRI UNIV
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