Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

Hybridoma cell strain and rabies virus phosphoprotein monoclonal antibody generated by same

A hybridoma cell line and monoclonal antibody technology, applied in the field of cell engineering, can solve problems such as harming human health, and achieve the effects of high sensitivity, good specificity and high specificity

Active Publication Date: 2017-05-24
KUNMING UNIV OF SCI & TECH
View PDF0 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a monoclonal antibody that can specifically bind to the phosphoprotein in rabies virus and the hybridization method for producing the antibody against the current situation that rabies seriously endangers human health and there are still various difficulties in the prevention and treatment process. Tumor cells

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
  • Hybridoma cell strain and rabies virus phosphoprotein monoclonal antibody generated by same
  • Hybridoma cell strain and rabies virus phosphoprotein monoclonal antibody generated by same
  • Hybridoma cell strain and rabies virus phosphoprotein monoclonal antibody generated by same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Expression and purification of rabies virus phosphoprotein

[0021] 1. Gene cloning and expression vector construction

[0022] Rabies virus Flury-HEP strain is preserved by our laboratory, and primers are designed to amplify the target fragment as follows: upstream primer RV-PF: 5'-CGCGAATTCATGAGCAAGATCTTTGTTAATCCGAG-3'; downstream primer RV-PR: 5'-GTCGACGCCGCTTAGCATGATGTGTAGCGATCCAAGT-3'; EcoRI and SalI restriction sites were added to both ends of the primers; the target fragment size was 894bp; after 48 hours of infection of N2a cells with rabies virus, RNA was extracted and reverse transcribed to obtain cDNA, which was used as a template for gene cloning. The PCR product was purified with a kit, cloned into pMD19-T, and positive clones were selected and sequenced. The positive clone with the correct sequence was digested with double enzymes, cloned into the pET-32a expression vector, and transformed into Rosetta (DE3) competent.

[0023] 2. Expressio...

Embodiment 2

[0025] Example 2: Establishment of hybridoma cell lines

[0026] 1. Experimental materials

[0027] 1. Immunogen: the recombinant phosphoprotein of rabies virus is used as the immunogen;

[0028] 2. Medium: RPMI-1640 medium was purchased from HyClone; HAT was purchased from Gibco; HT was purchased from Gibco;

[0029] 3. Experimental animals: Balb / c mice, 8-12 weeks old, female, SPF grade animal culture;

[0030] 4. Other experimental materials: Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from SIGMA Company; PEG4000 was purchased from SIGMA Company; HRP-goat anti-mouse IgG antibody was purchased from abcam Company; the rest of the reagents were domestic analytically pure products .

[0031] 2. Establishment of hybridoma cell lines

[0032] 1. Animal immunity

[0033] (1) Basic immunization: mix the immunogen with Freund's complete adjuvant in equal volumes and fully emulsify it, inject it subcutaneously at multiple points on the back of the ...

Embodiment 3

[0048] Example 3: Mass preparation and identification of anti-rabies virus phosphoprotein monoclonal antibody

[0049] 1. Antibody preparation

[0050] Choose 6-8 week old Balb / c mice, and inject 0.5mL liquid paraffin intraperitoneally, 0.5mL per mouse. One week later, hybridoma cells were inoculated intraperitoneally, and the number of inoculated cells per mouse was 1×10 5 -1×10 6 indivual. After an interval of 5 days, when the abdomen is obviously enlarged and the skin feels tense when touched with hands, the ascites can be collected with a No. 9 needle.

[0051] Centrifuge the ascitic fluid (13000r / min for 30 minutes), remove cell components and other precipitates, and collect the supernatant. Purify with Protein-Sepharose CL-4B, the upper column solution is 20mM PBS buffer solution, the column chromatography eluent is pH2.7, 20mM glycine buffer solution, and obtain the monoclonal antibody against rabies virus recombinant phosphoprotein.

[0052] 2. Antibody Identifica...

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 provides a hybridoma cell strain 1A4. The hybridoma cell strain provided by the invention can be used for secreting and preparing a monoclonal antibody capable of effectively identifying rabies virus phosphoprotein; the titer of a mouse ascites antibody is 2*10<5> measured by indirect enzyme-linked immunosorbent assay; the monoclonal antibody does not conduct cross reaction with other proteins of rabies virus, other antigens and pathogens, and has the advantages of high specificity and high sensitivity and can be applied to the biological diagnosis of rabies virus by ELISA, Western-blot and immunofluorescence and has good application prospect.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to a hybridoma cell line 1A4 and a rabies virus phosphoprotein monoclonal antibody produced therefrom. Background technique [0002] Rabies is a zoonotic disease caused by rabies virus, and the mortality rate is almost 100%. 60,000 people die of rabies every year in the world. my country is one of the countries where rabies is more prevalent. Rabies is a Class B infectious disease under the statutory reporting management in my country, and its pathogen is Rabies virus. Phosphoproteins play certain roles in virus escape, virus transcription and replication, and intracellular movement, and may also be used as clinical serum detection antigens for virus infection. Although human-derived anti-rabies virus drugs have been greatly developed in recent years, very effective therapeutic drugs have not yet been developed. Therefore, rabies vaccine is still the most effec...

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): C12N5/20C07K16/10G01N33/577G01N33/569C12R1/91
CPCG01N33/56983G01N33/577C07K16/10C07K2317/56
Inventor 张金阳胡娟夏雪山宋玉竹韩芹芹陈强
Owner KUNMING UNIV OF SCI & TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
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