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Rabies virus recombinant antigen and preparation method and application thereof

A rabies virus, recombinant antigen technology, applied in the directions of virus antigen components, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of low sensitivity, poor specificity and high preparation cost of the method

Active Publication Date: 2020-07-31
北京世纪元亨动物防疫技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the whole virus antigen detection kit has the advantage of high specificity in detecting rabies antibodies, there is a great risk of infection for the staff during the preparation and later purification of the whole virus antigen, and the preparation cost is high and the amount obtained is small
However, when using nucleoprotein antigen or glycoprotein antigen detection kits alone to detect rabies antibodies, there are deficiencies such as missed detection, poor specificity, and low sensitivity of the method.

Method used

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  • Rabies virus recombinant antigen and preparation method and application thereof
  • Rabies virus recombinant antigen and preparation method and application thereof
  • Rabies virus recombinant antigen and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] The preparation of embodiment 1 rabies virus recombinant antigen

[0086] (1) Primer design

[0087] Use DNAman and DNAstar to analyze the gene of amino acid sequence fragment N (as shown in SEQ ID NO.4), the gene of amino acid sequence fragment G (as shown in SEQ ID NO.5) and the gene of amino acid sequence fragment P (as shown in SEQ ID NO. 6), design 3 pairs of primers (primer 1F and primer 1R, primer 2F and primer 2R, primer 3F and primer 3R), respectively amplify the genes of amino acid sequence fragments N, P and G. In order to facilitate the connection of the three amplified gene fragments in series, and to facilitate the formation of each gene's own specific epitope after the series connection, the nucleotide sequence of the flexible peptide and the amino acid of the flexible peptide are added to the primers between the gene connections. The sequence is GGGSSS, GGSGGS or GGGGS, and the flexible peptide GGGGS is selected in this example, see the double underline...

Embodiment 2

[0120] Embodiment 2 A kind of rabies antibody detection test strip and its preparation method

[0121] This embodiment provides a rabies antibody detection test strip, such as Figure 6 As shown, it includes: a base plate 7 and a sample pad 1, a gold standard pad 2, a nitrocellulose membrane 3 and an absorbent pad 6 adhered to the base plate in sequence along the length direction of the base plate 7, and the sample pad 1. Between the gold standard pad 2, the nitrocellulose membrane 3 and the water-absorbing pad 6, they are sequentially in contact with and only partially overlap with adjacent parts; the nitrocellulose membrane 3 is adhered to the middle part of the bottom plate 7 , which are provided with spaced detection lines 4 and quality control lines 5, the detection line 4 is close to the gold standard pad 2, and the quality control line 5 is close to the water-absorbing pad 6;

[0122] Colloidal gold-labeled rabbit anti-dog IgG is coated on the gold label pad 2;

[012...

Embodiment 3

[0145] Embodiment 3 A kind of rabies antibody detection kit and detection method

[0146] This embodiment provides a rabies antibody detection kit, comprising: the recombinant rabies virus antigen purified in embodiment 1, at a concentration of 0.5 μg / mL.

[0147] Further, the enzyme-labeled secondary antibody is also included, which is HRPO (horseradish peroxidase)-labeled SPA (staphylococcus protein A) at a dilution of 1:10000.

[0148] Further, it also includes blocking solution, which is 100g Tris, 50g casein, 500g sucrose, add water to 30L, pH7.5.

[0149] Further, sample diluent, PBS buffer solution containing 10% bovine serum is also included.

[0150] Further, TMB substrate solution is also included.

[0151] The present embodiment provides the indirect ELISA method utilizing above-mentioned kit to detect rabies antibody, comprises the following steps:

[0152] Coating: Use the purified rabies virus recombinant antigen sample (1.0mg / mL~2mg / ml) in Example 1 as the co...

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Abstract

The invention relates to the technical field of rabies application, in particular to a rabies virus recombinant antigen and a preparation method and application thereof. The rabies virus recombinant antigen comprises one of the following parts (a) to (c): (a) amino acid sequence fragments N, G and P are sequentially included in the direction from an end N to an end C; (b) the rabies virus recombinant antigen is obtained by substituting, deleting or adding at least one or more amino acids to the amino acid sequence fragments N, G and / or P in (a), and an immune reaction of mammals to the rabiesvirus recombinant antigen can be induced; or (c) an amino acid sequence having homology with the amino acid sequence of the rabies virus recombinant antigen in (a) is obtained, and an immune reactionof the mammals to the rabies virus recombinant antigen can be induced. The rabies virus recombinant antigen has an antigen epitope with good antigenicity and small non-specificity. Compared with a whole-virus antigen, the rabies virus recombinant antigen has the advantages of stability, safety, low culture cost and high efficiency.

Description

technical field [0001] The invention relates to the technical field of rabies application, in particular to a rabies virus recombinant antigen, a preparation method and use thereof. Background technique [0002] Rabies is a deadly, highly neurotropic zoonotic infectious disease caused by Rabies virus (RV), which spreads all over the world. Rabies virus can cause fatal central nervous system infections in humans and a variety of animals. Rabies The main clinical manifestations are hydrophobia, photophobia, dysphagia, mania, etc. The fatality rate is almost 100%. According to a report released by the World Health Organization in June 2018, rabies kills 59,000 people each year (nearly half of them are children), 59.6% in Asia, 36.4% in Africa, and 99% of human rabies is transmitted by dogs and cats. Since there is no effective treatment for rabies at present, rabies is mainly prevented and controlled through vaccine immunization, and it is particularly important to monitor the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C07K16/10C07K16/06G01N33/569G01N33/558A61K39/205A61P31/14
CPCC07K14/005C07K16/10C07K16/06G01N33/56983G01N33/558A61K39/12A61P31/14C12N2760/20122C12N2760/20134C07K2319/40G01N2333/145G01N2469/20A61K2039/552Y02A50/30
Inventor 刘巧荣陈西钊马君
Owner 北京世纪元亨动物防疫技术有限公司
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