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Method for rapidly and quantitatively detecting rabies virus antibody by using rabies virus G protein, coding gene of G protein and test paper

A rabies virus, quantitative detection technology, applied in the field of G protein coding gene and test paper, can solve the problems of modification, difference, and inability to carry out glycosylation, and achieve objective and accurate evaluation results

Inactive Publication Date: 2021-08-13
北京市动物疫病预防控制中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If prokaryotic expression is used to express the protein, glycosylation modification cannot be carried out, which is different from the natural state, so eukaryotic expression can only be selected for expression

Method used

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  • Method for rapidly and quantitatively detecting rabies virus antibody by using rabies virus G protein, coding gene of G protein and test paper
  • Method for rapidly and quantitatively detecting rabies virus antibody by using rabies virus G protein, coding gene of G protein and test paper
  • Method for rapidly and quantitatively detecting rabies virus antibody by using rabies virus G protein, coding gene of G protein and test paper

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Preparation and identification of rabies virus G protein:

[0037] 1.1 Materials:

[0038] Donor plasmid pcDNA3.1, Escherichia coli Mach1T1 competent cells, DL2000 DNA Marker were purchased from Takara Company; T4 DNA ligase, restriction endonuclease Hind Ⅲ, EcoRI were purchased from NEB Company ; CHO-S cells were purchased from Invitrogen; plasmid mini-extraction kits, DNA gel recovery kits, and genome extraction kits were purchased from Axygen; G protein monoclonal antibodies were purchased from Beijing Sanlian Boyue Biotechnology Co., Ltd.

[0039] 1.2 Method:

[0040] 1.2.1 Synthesis of viral G gene, construction and identification of pcDNA3.1(+)-G recombinant expression vector:

[0041] Referring to NC_001542.1 (NCBI) and P03524-strain ERA (UniProt), the gene sequence was optimized in order to improve protein stability and increase expression in eukaryotes. The gene sequence is shown in SEQ No.1 (the amino acid sequence corresponding to the sequenc...

Embodiment 2

[0054] Embodiment 2: the preparation and use of colloidal gold detection test strip:

[0055] 2.1 Preparation and inspection of colloidal gold solution: 155mL of purified water was placed on a magnetic heating stirrer, heated and stirred until boiling. Add 5mL of 1% chloroauric acid solution, after boiling, add 6mL of newly prepared 1% trisodium citrate solution under constant stirring, continue heating and boiling for 5 minutes. Stop heating, cool to room temperature (15-25°C), and store in the dark at 2-8°C. It should be a red clear liquid without turbidity and surface suspended matter;

[0056] 2.2 Labeling and inspection of recombinant G protein: Take 1mL colloidal gold solution in a centrifuge tube, transfer it to a conical flask, and add 15µL 0.2mol / L potassium carbonate. Take 10 μg of recombinant protein G purified in Example 1, add it to the colloidal gold solution, mix quickly, and place at room temperature (15-25°C) for 30 minutes. Add 10µL 20% bovine serum albumi...

Embodiment 3

[0080] Embodiment 3: the evaluation of test strip quality:

[0081] 3.1 Specificity:

[0082] Detect the specific recognition of colloidal gold test strips to common viruses, such as Japanese encephalitis virus, influenza virus, canine distemper virus, canine parvovirus, feline parvovirus, and feline calicivirus.

[0083] Materials and methods: 100 µL of serum containing positive antibodies to the following viruses were collected: Japanese encephalitis virus, H3N2, canine distemper virus, canine parvovirus, feline parvovirus, feline calicivirus, rabies virus (from dogs and cats, respectively) ), the above sera were all from the Beijing Animal Disease Prevention and Control Center, and SPF canine serum and healthy cat (healthy cats with common infection viruses detected by two methods of etiology and serology, all negative healthy cats) were used as negative controls; according to colloid Conditions of use of gold test strips, drop into the sample holes of the test strips resp...

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Abstract

The invention discloses a method for rapidly and quantitatively detecting a rabies virus antibody by using a rabies virus G protein, a coding gene of the G protein and test paper, and belongs to the technical field of biology. According to the invention, the nucleotide sequence of optimized rabies virus G protein is shown as SEQ NO.1, G protein is obtained through expression and purification, and G protein colloidal gold test paper for detecting rabies virus antibodies is prepared from the G protein. The test paper sequentially comprises a sample pad, a gold pad, a nitrocellulose membrane and a water absorption pad from one end to the other end, which are mutually overlapped and attached to the upper surface of a PVC bottom plate. The invention has the advantages that the rabies virus antibody in dog serum can be detected, the rabies virus antibody in cat serum can also be detected, and the separated serum does not need to be diluted; through quantitative detection, the concentration of the rabies virus antibody can be accurately obtained, and the evaluation on the immune effect of the vaccine is more objective and accurate.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for rapidly and quantitatively detecting rabies virus antibody using rabies virus G protein, a coding gene of the G protein and a test paper. Background technique [0002] Rabies is a disease with a fatality rate as high as 100%, and there is currently no effective treatment. The best way to prevent and control rabies so far is rabies vaccination. After vaccination, the level of antibody titer directly determines the immune effect. Therefore, how to quickly and accurately evaluate the antibody titer is a problem that researchers need to solve. [0003] At present, the commonly used rabies antibody detection methods in the world include enzyme-linked immunosorbent assay, rapid fluorescent focus inhibition test, neutralizing immunofluorescence test, and mouse neutralization test. Among them, the neutralization immunofluorescence test is a recognized gold standar...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543G01N33/558
CPCG01N33/54313G01N33/558G01N33/56983G01N2333/145
Inventor 邓柏林赵浩军郑雪莹范君文郑金来谷传慧李扬郭俊林李志军于国际王培
Owner 北京市动物疫病预防控制中心
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