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

Monoclonal antibody against natural cow gamma-interferon, hybridoma cell strain secreting antibody and application

A hybridoma cell line and monoclonal antibody technology, applied in the field of biological cytology, can solve the problems of limiting antibody affinity and application value

Active Publication Date: 2015-04-29
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
View PDF1 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problem of preparing monoclonal antibodies obtained from eukaryotically expressed glycosylated proteins in the prior art, most of the obtained antibodies can only react with prokaryotic expression products and cannot react with natural glycosylated proteins, which limits the obtained In order to solve problems such as antibody affinity and application value, the present invention provides a method for preparing monoclonal antibodies after immunizing animals with eukaryotic expression products, and provides a monoclonal antibody against natural bovine γ-interferon, secreting the antibody hybridoma cell line

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
  • Monoclonal antibody against natural cow gamma-interferon, hybridoma cell strain secreting antibody and application
  • Monoclonal antibody against natural cow gamma-interferon, hybridoma cell strain secreting antibody and application
  • Monoclonal antibody against natural cow gamma-interferon, hybridoma cell strain secreting antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Construction of prokaryotic expression plasmid and expression and purification of protein

[0030] 1. Isolation and activation of bovine peripheral blood lymphocytes

[0031] Aseptically collect anticoagulant blood from bovine jugular vein, add one volume of Hank’s solution, mix gently, carefully and slowly add to equal volume of bovine lymphocyte separation medium. Centrifuge at 2000r / min for 20min, absorb the white cell layer on the interface, collect it in another centrifuge tube, wash twice with RPMI-1640, and centrifuge at 1500r / min for 10min. The collected cells were suspended in RPMI-1640 culture medium containing 10 μg / ml Concanavalin A (ConA, product of Sigma Company), and the cell density was about 10 7 pcs, ml, at 37°C, CO 2 Stimulate culture in the incubator for 8h. The stimulated lymphocytes were collected by centrifugation in a centrifuge tube, washed three times with PBS, and used to extract total RNA.

[0032] 2. Extraction of total RNA fr...

Embodiment 2

[0042] Embodiment 2: Eukaryotic expression plasmid construction

[0043] Extraction of mouse cell genome: take the spleen of the mouse, use the blood / cell / tissue genomic DNA extraction kit (product of Tiangen Biochemical Technology Co., Ltd.) to extract genomic DNA, and operate according to the instructions.

[0044] Using the mouse cell genome as a template, the signal peptide sequence of IFN-γ (named MIE) was amplified, and the primers were: EXIF-U: 5'-gatGGTACCatgaacgctacacactgcatc; EXIF-OR: 5'- ttcttcaaATGATGATGATGATGATG aatgactgtgccgtggcag (the underlined part is the complementary sequence of overlapping PCR), using Pfu high-fidelity DNA polymerase (product of Fermentas). The amplification system is: sterilized water 26μl, 5×Buffer 5μl, primer EXIF-U 2μl (20umol), primer EXIF-OR 2μl (20umol), dNTP (2.5mM) 4μl, DNA polymerase 1μl, genomic DNA 5μl. The amplification program was as follows: 95°C pre-denaturation for 5 minutes, followed by cycling (95°C 30sec; 56°C 30sec; 7...

Embodiment 3

[0049] Example 3: Animal Immunization

[0050] Before immunization, the blood was collected by docking the tail of the mice, and the serum was separated as a negative control. Recombinant plasmid p3.1-MbIFN for immunization was prepared with an endotoxin-removing plasmid kit (product of Omega Company). The immunization dose was 100 μg of plasmid DNA per mouse, and 50 μg was injected into each left and right thigh. The specific operation is as follows: first, wet the hair of the inner thigh muscle of the mouse with alcohol, and draw an appropriate volume of endotoxin-free plasmid solution (containing 100 μg plasmid ), inject at two points respectively in the inner muscles of the left and right legs of the mouse (insertion depth is 2 mm to 2.5 mm, and attention should be paid to avoid muscle piercing or insufficient depth). Inject slowly, stop for a few seconds after injection and then remove the needle to prevent leakage. After the injection, wet the hair on the outside of th...

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
molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention discloses a monoclonal antibody against natural cow gamma-interferon, a hybridoma cell strain secreting the antibody and application. The hybridoma cell strain capable of stably secreting the monoclonal antibody against the natural cow gamma-interferon is named as 3D7 and is preserved in China General Microbiological Culture Collection Center, the strain preservation number is CGMCC No.9329, and the preservation data is June 25, 2014. According to the invention, a eukaryotic expression vector of the cow gamma-interferon is used for immunizing a mouse to screen the hybridoma cell strain capable of generating the antibody against the natural cow 1FN gamma-interferon by means of indirect immunofluorescence assay technology, the problem that the traditionally prepared IFN gamma-interferon can only react with a prokaryotic expression product and cannot react with natural IFN gamma is solved successfully, and a new technological means is provided for the detection of the natural cow gamma-interferon.

Description

technical field [0001] The present invention relates to an anti-bovine γ-interferon monoclonal antibody, a hybridoma cell line and its application for secreting the antibody, in particular to an anti-natural bovine γ-interferon monoclonal antibody and a hybridoma cell for secreting the antibody The strain also relates to the application of the monoclonal antibody in the detection of natural bovine gamma-interferon (IFN-gamma). The invention belongs to the field of biological cytology. Background technique [0002] Interferon (IFN) is a type of glycoprotein secreted by cells under the action of a specific inducer and has biological activities such as anti-virus, anti-tumor and immune regulation functions. It consists of 1 amino acid and contains more than 17 kinds of amino acids, among which the content of aspartic acid, glutamic acid and leucine is relatively high (Iiaacs et al., 1957). According to the difference of gene sequence and receptor, IFN can be divided into thre...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/24G01N33/68G01N33/577C12R1/91
Inventor 陈利苹刘思国朱海波于申业宋宁宁
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI 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