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

FIPV N recombinant protein and obtained colloidal gold test strip for rapidly detecting FIPV infection

A technology of colloidal gold test paper and recombinant protein, applied in biological testing, recombinant DNA technology, DNA / RNA fragments, etc., can solve the problems of high variability and undetectability of S protein, and achieve short cycle, cost saving and low cost detection way effect

Pending Publication Date: 2022-02-25
深圳赫兹生命科学技术有限公司
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the S protein has high variability and contains ADE epitopes, so it cannot be used for detection

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
  • FIPV N recombinant protein and obtained colloidal gold test strip for rapidly detecting FIPV infection
  • FIPV N recombinant protein and obtained colloidal gold test strip for rapidly detecting FIPV infection
  • FIPV N recombinant protein and obtained colloidal gold test strip for rapidly detecting FIPV infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1FI

[0037]Construction and prokaryotic expression of embodiment 1 FIPV N protein recombinant plasmid

[0038] The protein secondary structure prediction software and protein nuclear localization signal prediction software in Novopro online tool were used to analyze the secondary structure and nuclear localization signal (NLS) of the FIPV N protein sequence of 79-1146 strain (GeneBank ID: DQ 010921.1). Combined with the high antigenic index and α-helix, β-sheet, β-turn enriched regions, the FIPV N expression gene 151-1131bp was selected for codon optimization, and a gene fragment with a size of 1134bp was synthesized. Synthesize his tag, TrxA tag, BamHI and XhoI restriction sites, and add a TEV restriction site after the upstream BamHI site, such as figure 1 As shown in A. The synthetic N gene was digested with BamHI and XhoI and then ligated into the pET32a vector which was digested with the same enzymes. After the pET32a-FIPV N recombinant plasmid expressed in prokaryotic is di...

Embodiment 2F

[0040] Embodiment 2 FIPV N protein purification and identification

[0041] Add the supernatant protein sample collected in Example 1 to the nickel column equilibrated with 50mM Tris-HCl in advance, incubate for 1h to make the protein fully bind to the nickel column, then let the sample flow out slowly, collect the effluent, and load the sample again . Use 5 times the column volume of 50mM and 100mM imidazole to elute and collect the protein; transfer the collected protein to an ultrafiltration tube, 4°C, 3000r / min ultrafiltration concentration, take 8μl of the concentrated protein, add 2μL 5 ×The protein loading buffer was mixed and denatured for 10 minutes, and 8 μl samples were taken for SDS-PAGE identification.

[0042] SDS-PAGE electrophoresis showed that in the inclusion body, 50 and 100mM imidazole elution concentrate, the target band appeared at about 55kDa ( figure 2 A). Moreover, a relatively pure FIPV N protein was obtained after 100 mM elution of the concentrat...

Embodiment 3F

[0043] Example 3 FIPV N protein quantification

[0044] Take 25 μL of 8 serially diluted BSA protein standard samples, respectively named A, B, C, D, E, F, G, H, and take 25 μL of FIPV N protein samples to be tested, and add them to the microwell plate in turn. Add 200 μL of working solution to each well, and shake on the shaker at room temperature for 30 seconds to make it fully mixed. Seal the microwell plate with a ziplock bag and place it at 37°C for 30 minutes. Cool the 96-well plate to room temperature, read the absorbance value of the sample at 562 nm with a microplate reader, and calculate the concentration of the protein to be tested according to the obtained linear equation. The concentrations of N protein and its two serial dilutions were 27.27, 13.56, 5.65, 3.83, 2.46, 0.63 and 0.31 μg / mL, respectively.

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
Diameteraaaaaaaaaa
Login to View More

Abstract

The invention provides an FIPV N recombinant protein and an obtained colloidal gold test strip for rapidly detecting FIPV infection, belonging to the technical field of biological detection. The amino acid sequence of the FIPV N recombinant protein provided by the invention is as shown in Seq ID No. 1. Based on high conservative property and excellent immunity of N protein, the FIPV N protein is expressed by using a prokaryotic expression system, and a colloidal gold test strip is researched and developed by using the protein and a polyclonal antibody obtained after a kitten is immunized by the protein and is used for detecting negative and positive serum of the FIPV, so a foundation is laid for establishing a rapid diagnosis method of the FIPV.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a FIPV N recombinant protein and the obtained colloidal gold test strip for rapidly detecting FIPV infection. Background technique [0002] Feline coronavirus (FIPV) is a disease with a high fatality rate in cats. It occurs in cats all over the world. It is mainly transmitted among cats through contact or through the digestive tract and respiratory tract. The main target cells of FIPV are cats. Monocytes and macrophages, and the ability to efficiently infect and activate and replicate in monocytes / macrophages, can lead to inflammatory lesions including vasculitis, ascites, and fibrous and granulomatous lesions. [0003] FIPV has four main structural proteins, including spike glycoprotein (spike, S), membrane (membrane, M) protein, small membrane (small envelope, E) protein, nucleocapsid (nucleocapsid, N) protein. Although the S protein is a FIPV neutralizing antigen...

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): C07K14/165C12N15/50C12N15/70C12N15/11G01N33/569G01N33/558G01N33/58G01N33/543G01N33/52
CPCC07K14/005C12N15/70G01N33/56983G01N33/558G01N33/587G01N33/54346G01N33/523G01N2333/165G01N2469/20C12N2770/20022C12N2800/101Y02A50/30
Inventor 查丽莎周宇杭郑琪周婕洪亮汪石鹏傅舟宇王磊黄茜
Owner 深圳赫兹生命科学技术有限公司
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