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

Antibody for resisting lumpy skin disease, test paper and kit

A technology for nodular and skin diseases, applied in the field of immunity, can solve the problems of expensive, cumbersome and time-consuming separation and culture identification methods

Active Publication Date: 2020-12-22
北京弘进久安生物科技有限公司
View PDF4 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the cumbersome and time-consuming operation of the separation and culture identification method, the PCR method, FQ-PCR and other above-mentioned methods require expensive instruments and reagents, and the detection cost is high, so it is not suitable for laboratory and on-site detection applications in grassroots units

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
  • Antibody for resisting lumpy skin disease, test paper and kit
  • Antibody for resisting lumpy skin disease, test paper and kit
  • Antibody for resisting lumpy skin disease, test paper and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034]Example 1: Prokaryotic expression and purification of P32 protein

[0035]In this example, the prokaryotic expression and purification of P32 protein were performed, and the specific steps were as follows:

[0036]1. Query the LSDV genome sequence from GeneBank, select the conservative gene LSDV-P32 and express the P32 protein through comparison and analysis. Analyzing the amino acid sequence of the P32 protein by online software TMHMM found that there is a transmembrane region at the C-terminus. Because the transmembrane region protein is insoluble, the C-terminus transmembrane region is removed during expression and codon optimization is performed. The amino acid sequence of LSDV P32 protein is shown in Table 3.

[0037]Table 3: Amino acid sequence of P32 protein

[0038]

[0039]2. Select the correctly sequenced pGEX-6P-2-N280 plasmid, transform it into BL21(DE3) competent cells, and cultivate overnight in a constant temperature incubator at 37°C. Pick a single clone and place it in 100 m...

Embodiment 2

[0041]Example 2: Preparation of P32 protein monoclonal antibody

[0042]The purified P32 protein antigen in Example 1 was used to immunize 8 female Balb / c mice aged 8-12 weeks. After immunization for 3 times, blood was collected from the orbit of the mice to obtain mouse serum, and the P32 in the mouse serum was detected by ELISA Protein antibody titer, if titer10000.

[0043]Take antibody titer>10,000 mouse spleen cells were fused with myeloma SP2 / 0 cells, and the fused cells were screened by HAT selection medium, and the fused cells were positively screened and subcloned by ELISA; the positive single clones were screened, ascites were taken, and Protein A was used / G antibody purification column to purify antibodies, ELISA titer of purified antibody>1:128000, purity>90%.

[0044]The cell line numbers corresponding to the two monoclonal antibodies are 701 and 901, respectively.

Embodiment 3

[0045]Example 3: Amplification and sequence determination of the CDR region sequence of the P32 protein monoclonal antibody

[0046]Resuscitate the hybridoma cell lines 701 and 901 and cultivate them. When the cells grow to the logarithmic growth phase, the cell count is about 8×107Cells / ml are collected. Extract the total RNA from hybridoma cells according to the TaKaRa MiniBEST Universal RNA Extraction Kit instructions. According to the reaction system shown in Table 4, keep it at 65°C for 5 minutes, then quickly cool on ice, and then store it at 42°C for 60 minutes according to the reaction system shown in Table 5; ℃ 15min; 25 ℃ 1min for 1st-Strand cDNA synthesis reaction.

[0047]Table 4: 1st-Strand cDNA synthesis reaction mixture

[0048] Reagent Usage amount Oligo dT Primer(50μM) 1μL dNTP Mixture(10mM each) 1μL Template RNA <5μg

RNase Free dH 2 O

Up to 10μL

[0049]Table 5: Reverse transcription reaction solution

[0050] Reagent Usage amount The above denatured reaction solution...

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

PropertyMeasurementUnit
Particle sizeaaaaaaaaaa
Login to View More

Abstract

The invention relates to an antibody specifically combined with lumpy skin disease (LSD) or an active fragment thereof, colloidal gold immunochromatographic test paper or kit comprising the antibody,and a preparation method and application thereof. The test paper or kit is convenient to produce, has stable effect, does not have non-specific reaction, has low coating amount, and is beneficial to mass production. The detection method for the LSD provided by the invention is simple and convenient to operate and has good reproducibility, breakthrough is achieved in the field of on-site quick detection of LSD, and existing LSD detection methods are enriched.

Description

Technical field[0001]The invention relates to the field of immunity, in particular to an antibody against bovine nodular skin disease virus, and a test paper and kit for detecting bovine nodular skin disease virus.Background technique[0002]Lumpy skin disease (LSD) is an acute and subacute contact infectious disease of cattle caused by the bovine nodular skin disease virus (LSDV). The incidence of this disease ranges from 5% to 45. %, the case fatality rate can reach 10%. LSDV belongs to the genus of goatpox virus, the subfamily of vertebrate poxviruses of the poxvirus family, and the genome homology with sheeppox virus and goatpox virus is as high as 96%. The disease is a notified disease (OIE 2014 edition list) of the World Organization for Animal Health (English: World Organization for Animal Health; French: Office international desépizooties, OIE), and it is a type of infection determined by the "List of Entry Animal Quarantine Diseases of the People's Republic of China" The occu...

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): C07K16/08G01N33/569G01N33/558G01N33/577G01N33/58
CPCC07K16/081G01N33/56983G01N33/558G01N33/577G01N33/587C07K2317/565C07K2317/56G01N2333/065G01N2469/10
Inventor 崔贝贝杨卫丽李霆段佳慧许宏瑞高宏应天翼李岩松余涛
Owner 北京弘进久安生物科技有限公司
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