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

Fully human anti-staphylococcus aureus alpha-hemolysin recombinant antibody

A staphylococcus, recombinant antibody technology, applied in recombinant DNA technology, antibacterial immunoglobulin, immunoglobulin and other directions, can solve the problem of lack of fully human antibody, achieve strong penetration, eliminate human anti-mouse antibody reaction , the effect of easy operation

Active Publication Date: 2019-11-12
SOUTHWEST MEDICAL UNIVERISTY
View PDF4 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still a lack of self-developed fully human antibodies against Staphylococcus aureus α-HL in this field in China

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
  • Fully human anti-staphylococcus aureus alpha-hemolysin recombinant antibody
  • Fully human anti-staphylococcus aureus alpha-hemolysin recombinant antibody
  • Fully human anti-staphylococcus aureus alpha-hemolysin recombinant antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation of Staphylococcus aureus recombinant protein antigen

[0029] A large amount of antigen protein is required in the process of preparing fully human antibodies against Staphylococcus aureus α-HL. In order to promote the soluble expression of the antigen protein, the p-ColdTF fusion expression vector is used for expression, which carries the TF molecular chaperone (48KD), which is conducive to the co-translational folding of the newly expressed polypeptide. In addition, the carrier carries a His-Tag, which is conducive to protein purification.

[0030] Extract total RNA from Staphylococcus aureus by boiling method, using total RNA as template, Oliga dT 15 Reverse transcription for primers to obtain cDNA. Obtain the α-HL gene sequence of Staphylococcus aureus in NCBI gene database, design primers according to the gene sequence, and use Nest PCR to amplify α-HL. The synthesized recombinant protein gene was cloned into the p-Cold TF fusion expression vecto...

Embodiment 2

[0032] Example 2 Screening of anti-Staphylococcus aureus α-HL single chain antibodies using phage display antibody library technology

[0033] The laboratory has constructed a natural fully human scFv antibody library in the early stage, with a library capacity of 2.5×10 8 , Good diversity. Using α-HL biotinylated protein as the antigen, the natural fully human scFv antibody library was enriched by phage display using the immunomagnetic bead method, clones were randomly selected from the enriched scFv antibody library, and monoclonal sub-phage amplification was performed. To increase the expression, the expressed scFv was detected by phage ELISA with anti-M13-HRP monoclonal antibody. For the specific experimental procedures of this example, see: Master's thesis "Wu Tong. Cloning and expression of Staphylococcus aureus α-hemolysin and screening of anti-HLA-α fully human single-chain antibodies [D]. Sichuan: Sichuan Medical University, 2016".

[0034] The results showed that after ...

Embodiment 3

[0035] Example 3 Preliminary identification of anti-α-HL single chain antibody

[0036] The OD obtained from the natural fully human scFv antibody library in Example 2 450 The single-chain antibody with the highest value was expressed in large quantities and then the plasmid was extracted and sequenced according to the instructions. The 15 anti-α-HL single-chain antibodies with correct sequencing results were inserted into the prokaryotic expression vector pLZ16 for soluble expression verification. The pLZ16 vector was constructed by our laboratory based on the pUC plasmid, and contains FLAG and His-tag tags. It has been reported in some documents published by our laboratory, such as "Wang Xu, Yuan Qing, Ye Yingchun, etc. Anti-IL-33 Whole People Soluble expression and identification of the source scFv-Fc antibody[J] Modern Immunology, 2016; 36(6):462-465".

[0037] 1. PCR amplification of scFv target gene and verification

[0038] (1) Preparation of reaction solution (20μl system): ...

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 discloses an anti-staphylococcus aureus alpha-hemolysin single-chain antibody scFv2 or scFv46, further discloses an anti-staphylococcus aureus alpha-hemolysin recombinant antibody scFv2-Fc or scFv46-Fc containing the scFv2 or scFv46 and a human antibody constant region Fc segment amino acid sequence, and further discloses a universal expression vector sp-Fc / pcDNA3.1 of the fully human anti-staphylococcus aureus alpha-hemolysin recombinant antibody. The nucleic acid sequence of an encoding gene of the universal expression vector is shown in SEQ ID No:13. The invention further discloses a eukaryotic expression vector of the fully human anti-staphylococcus aureus alpha-hemolysin recombinant antibody. The eukaryotic expression vector is obtained by inserting the single-chain antibody into the sp-Fc / pcDNA3.1. The invention further discloses a recombinant expression vector sp-scFv2-Fc / pMH3 or sp-scFv46-Fc / pMH3.

Description

Technical field [0001] The present invention relates to the technical field of antibody engineering, in particular to a fully human anti-staphylococcus aureus alpha-hemolysin recombinant antibody. Background technique [0002] Staphylococcus aureus is a major gram-positive pathogen. Its infection can cause serious life-threatening diseases, such as pneumonia, bacteremia, and endocarditis. Antibiotics have long been the main effective way to treat Staphylococcus aureus infections. However, as the abuse of antibiotics and disinfectants worldwide and the release of antibiotic residues to the environment become more and more serious, methicillin-resistant Staphylococcus aureus (MRSA), which is highly pathogenic and has a wide range of transmission routes, has become a global nosocomial infection The primary pathogen. The emergence of vancomycin intermediate Staphylococcus aureus (VISA) strains and highly vancomycin-resistant Staphylococcus aureus (VRSA) strains has brought a more s...

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): C07K16/12C07K16/46C12N15/70
CPCC07K16/1271C07K16/46C07K2317/24C07K2317/622C12N15/70
Inventor 袁青年四季李春于红叶迎春
Owner SOUTHWEST MEDICAL UNIVERISTY
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