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

Human anti-human tumor necrosis factor-alpha single chain antibody

A fully human antibody and antigen technology, applied in the field of bioengineering

Inactive Publication Date: 2009-04-15
CHINA PHARM UNIV
View PDF0 Cites 12 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But Cimzia is a humanized antibody and still retains some mouse sequences

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
  • Human anti-human tumor necrosis factor-alpha single chain antibody
  • Human anti-human tumor necrosis factor-alpha single chain antibody
  • Human anti-human tumor necrosis factor-alpha single chain antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1 Screening of fully human anti-hTNF-α single-chain antibody

[0016] Coating buffer (50mM NaHCO 3 , pH9.6) to dilute hTNF-α to 100μg / ml; take 4ml and add it to an immune tube, and coat overnight at 4°C; the next day, discard the supernatant, and wash the tube 3 times quickly with PBS; 2% MPBS (containing 2% degreased Milk PBS), blocked at 37°C for 2 hours; discarded the blocking solution, and washed the tube 3 times with PBS quickly; the phage antibody library (10 12 ~10 13 p.f.u) was suspended in 4ml 2% MPBS and added to the immunotube. After repeated inversion at room temperature for 30 minutes, it was left to stand at room temperature for more than 90 minutes; the tube was washed 10 times with PBS containing 0.1% Tween-20, and then washed 10 times with PBS to remove Remove detergent; add 1ml 100mM triethylamine (700μl 7.18mol / L triethylamine added to 50ml water), and incubate repeatedly at room temperature for 10min for specific elution; 0.5ml 1M Tris (pH7...

Embodiment 2

[0017] Example 2 Construction of prokaryotic expression plasmid pET22b-F6

[0018] The F6 gene or pET22b vector was double digested with Nco I and Not I (purchased from Takara), and the reaction system was as follows:

[0019]

[0020] The above reaction system was reacted at 37°C for 2h, electrophoresed on 1.0% agarose, recovered by cutting the gel, dissolved in TE of the sample, and stored at -20°C for future use.

[0021] The double digested F6 gene and pET22b vector were ligated with T4 ligase (purchased from Takara), and the reaction system was as follows:

[0022]

[0023] The above reaction system was reacted at 16°C for 12h.

[0024] Take 10 μl of the constructed plasmid pET22b-F6, add it to 90 μl of competent cell (BL21) suspension, and mix thoroughly; after placing it in an ice bath for 30 minutes, immediately place it in a 42°C water bath for heat shock for 45 seconds, and then place it in an ice bath for 2 minutes; Add 900μl fresh LB liquid medium, shake an...

Embodiment 3

[0025] Example 3 Soluble expression and isolation and purification of anti-hTNF-α single-chain antibody

[0026] Select the strain with correct sequencing and culture to OD 600 ≈0.6, add IPTG with a final concentration of 1mM, induce overnight at 37°C, and detect the result of induced expression by 12% SDS-PAGE ( image 3 ). The band indicated by the arrow is the target protein induced to express, and the band size is about 28kDa, which is consistent with the theoretical value.

[0027] 6000rpm / min, centrifuge at 4°C for 5min, collect the cells; resuspend the cells in PBS, sonicate (sonication 3s, interval 3s, 10min in total); 10000rpm / min, centrifuge at 4°C for 20min, keep the supernatant; pass the supernatant over nickel column (Novagen product), elution with different concentrations of imidazole; 12% SDS-PAGE detection of purified results ( Figure 4 ), store the target protein at -20°C. Figure 4 The size of the target band indicated by the middle arrow is about 28kDa,...

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 whole human source single chain antibody combined with specificities of human tumor necrosis factor-Alpha (hTNF-Alpha). The antibody of the invention is formed by connecting the first end and the tail end of the variable area of a light chain and a heavy chain of the antibody by a flexible connecting peptide. The antibody can neutralize the activity of hTNF-Alpha in extracorporeal tests, and can be used for detecting the hTNF-Alpha or can be applicable to treating related diseases of the hTNF-Alpha, such as rheumatoid arthritis and Crohn's disease. The invention also comprises nucleogelase, carrier and host cell that express the whole human source single chain antibody of the invention.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a single-chain antibody capable of specifically binding to human TNF-α. Background technique [0002] Tumor necrosis factor-α (TNF-α) is mainly a cytokine produced by activated monocytes / macrophages. It is initially produced as a precursor protein with a relative molecular weight of about 26kDa consisting of 233 amino acids. In the cell, the C-terminus is outside the cell, called transmembrane TNF-α (TM-TNF-α). Under the action of metalloprotease, the extracellular part is hydrolyzed into a polypeptide composed of 157 amino acids with a molecular weight of about 17kDa, which is called soluble secreted TNF-α (S-TNF-α). Mature TNF-α (namely S-TNF-α) binds to the TNF receptor (TNFR) on the cell surface in the form of trimer and mediates various biological activities. This is a cytokine with a variety of biological effects. Early studies found that TNF-α can induce tumor ce...

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): C07K16/24C12N15/13C12N15/70C12N1/21A61K39/395A61P19/02A61P29/00A61P43/00
Inventor 王旻陈卫张娟李海鑫张弢
Owner CHINA PHARM UNIV
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