Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Serological detection method of bovine brucellosis A19-delta VirB12 vaccine

An A19-, detection method technology, which is applied in the field of serological detection of bovine brucellosis A19-ΔVirB12 vaccine, can solve the problems of endangering human and animal health, strong virulence, strong virulence, etc., and achieves the goal of reducing huge economic losses. Effect

Pending Publication Date: 2020-07-10
VETERINARY INST XINJINAG ACADEMY OF ANIMAL SCI CLINIC MEDICAL SCI RES CENT XINJIANG ACADEMY OF ANIMAL HUSBANDRY SCI
View PDF6 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First of all, the A19 vaccine is a live attenuated vaccine with strong virulence, and there is a problem of strong virulence in production and application, which poses a threat to human health
Secondly, the A19 vaccine lacks differential diagnostic markers. Current conventional serological methods cannot distinguish vaccinated animals from naturally diseased animals, causing diseased animals to exist in natural populations for a long time, seriously endangering the health of humans and animals, and hindering the development of brucellosis in animals. Eradication and decontamination of mycosis

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Serological detection method of bovine brucellosis A19-delta VirB12 vaccine
  • Serological detection method of bovine brucellosis A19-delta VirB12 vaccine
  • Serological detection method of bovine brucellosis A19-delta VirB12 vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Preparation of VirB12 antigen protein

[0027] Strain and vector: The A19 molecular marker vaccine was developed by the Brucellosis research group of the Institute of Veterinary Medicine, Xinjiang Academy of Animal Science; Brucella strain 2308 was purchased from the China Institute of Veterinary Medicine, and the cloned pGEM-T vector was a commercial reagent, purchased from Promega; Prokaryotic The expression vector pET-28a(+) is preserved by the Institute of Veterinary Medicine, Xinjiang Academy of Animal Science.

[0028] Reagents: restriction endonucleases were purchased from Fermentas; plasmid DNA extraction kits and DNA gel recovery kits were purchased from Promega; T4DNA ligase, DNA Marker, and DH5α were products of Beijing Dingguo Biotech. The enzyme-labeled secondary antibody was rabbit anti-bovine IgG horseradish peroxidase (Bethyl Company, USA); the His-trap column for protein purification was a product of GE Company, USA; and the sequence determination ...

Embodiment 2V

[0032] Example 2 Identification of immune activity of VirB12 recombinant protein

[0033] The refolded protein in Example 1 was subjected to SDS-PAGE electrophoresis, transferred to the membrane, blocked, and washed, and then the membrane was soaked in Brucella positive serum at room temperature for 1 hour. After washing the membrane with phosphate buffer, rabbit anti- Cattle secondary antibody, incubated for 1h at room temperature, 4-chloro-1-naphthol developed until the band was clear, and the reaction was terminated by washing with distilled water. The purified VirB12 protein was used as the antigen, and the A19 vaccine-immunized bovine serum, A19-ΔVirB12-immunized bovine serum, non-immune brucellosis-negative bovine serum, and natural brucellosis-positive bovine serum were used as antibodies.

[0034] Western-blot experiment results show that VirB12 protein has immunological activity. See the identification results figure 1 ,among them figure 1 Among them, M is a low molecu...

Embodiment 3A19

[0035] Example 3 IELISA Identification of A19-ΔVirB12 Vaccine Immune Antibody and Natural Infection Antibody

[0036] Experimental materials and equipment: Denmark NUNC enzyme-labeled 96-well reaction plate, rabbit anti-bovine IgG horseradish peroxidase (U.S., Bethyl company), microplate reader (Bio-Rad 680), micropipette (10-1000μL) .

[0037] Reagent and solution formula:

[0038] Microplate pretreatment solution (PBST10): 1×PBS (pH7.4) 1000mL, Tween205mL.

[0039] Coating diluent (0.05mol / L sodium carbonate-sodium bicarbonate buffer, pH 9.6): Na 2 CO 3 1.5g, NaHCO 3 2.9g, Na 2 N 3 0.2g, add deionized water to 1000ml, adjust to pH=9.6.

[0040] Washing solution (PBST, pH 7.4): NaCl 8.0g, KH 2 PO 4 0.2g, NaHPO 4 ·12H 2 O 2.9g, KCl 0.2g, Tween 200.5ml, add deionized water to 1000ml, and adjust to pH 7.4.

[0041] Sample diluent: 1×PBS1000mL, Tween205mL, (pH 10.8).

[0042] Enzyme-labeled secondary antibody: rabbit anti-bovine IgG horseradish peroxidase.

[0043] Substrate color developing...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a serological detection method of a bovine brucellosis A19-delta VirB12 vaccine. An indirect enzyme-linked immunosorbent assay (iELISA) method for identifying and distinguishing a bovine brucellosis immune antibody and a natural infection antibody is established by taking a brucellosis VirB12 protein as a labeled antigen. According to the iELISA method provided by the invention, a problem that bovine brucellosis immune animals and clinical diseased animals are difficult to identify is solved, and the method has practical application value in prevention, control, eradication and purification of bovine brucellosis.

Description

Technical field [0001] The present invention belongs to the technical field of veterinary vaccine detection, and relates to a serological detection method for bovine brucellosis A19-ΔVirB12 vaccine, specifically a differential diagnosis detection technology for bovine brucellosis A19-ΔVirB12 vaccine immune antibody and natural infection antibody. Background technique [0002] Brucellosis (Brucellosis) is a zoonotic disease caused by Brucella (Brucella), which is widespread in all parts of the world, referred to as brucellosis. The prevention and eradication of livestock brucellosis has become the main task of the public health security defense system of many countries and regions. In most countries and regions where brucellosis is endemic, vaccination is one of the main measures and the most effective means to prevent and control brucellosis among animals. At present, the attenuated brucella live vaccines widely used in the world mainly include bovine S19, bovine RB51 and sheep ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): G01N33/68G01N33/58G01N33/569G01N33/543C12N15/70C12N15/31C07K14/23C07K1/16C12N15/11
CPCG01N33/6854G01N33/581G01N33/56911G01N33/54306C12N15/70C07K14/23G01N2333/23G01N2469/20
Inventor 易新萍叶锋马晓菁钟旗刘丽娅谷文喜谢彩云张旭薛晶李静古努尔·吐尔逊李岩
Owner VETERINARY INST XINJINAG ACADEMY OF ANIMAL SCI CLINIC MEDICAL SCI RES CENT XINJIANG ACADEMY OF ANIMAL HUSBANDRY SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com