Fully human anti-Staphylococcus aureus α-hemolysin recombinant antibody

A Staphylococcus, recombinant antibody technology, applied in the direction of recombinant DNA technology, anti-bacterial immunoglobulin, immunoglobulin, etc., can solve the problem of lack of fully human antibodies, achieve strong penetration, small molecular weight, and retain biological Effects of Activity and Specificity

Active Publication Date: 2020-12-01
SOUTHWEST MEDICAL UNIVERISTY
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  • 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

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  • Fully human anti-Staphylococcus aureus α-hemolysin recombinant antibody
  • Fully human anti-Staphylococcus aureus α-hemolysin recombinant antibody
  • Fully human anti-Staphylococcus aureus α-hemolysin recombinant antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The preparation of embodiment 1 Staphylococcus aureus recombinant protein antigen

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

[0030] Boiling method to extract total RNA of Staphylococcus aureus, using total RNA as template, Oliga dT 15 cDNA was obtained by reverse transcription with primers. The α-HL gene sequence of Staphylococcus aureus was obtained from the NCBI gene database, primers were designed according to the gene sequence, and α-HL was amplified by nested PCR. The synthetic recombinant protein gen...

Embodiment 2

[0032] Example 2 Using phage display antibody library technology to screen anti-Staphylococcus aureus α-HL single chain antibody

[0033] Our 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 antigen, the natural fully human scFv antibody library was enriched by phage display by immunomagnetic bead method, clones were randomly selected from the enriched scFv antibody library, and monoclonal phage amplification was carried out. The expressed scFv was detected by phage ELISA with anti-M13-HRP monoclonal antibody. For the specific experimental steps of this example, see: Master's degree thesis "Wu Tong. Cloning and expression of Staphylococcus aureus α-hemolysin and screening of anti-HLA-α fully human single-chain antibody [D]. Sichuan: Sichuan Medical University, 2016".

[0034] The results showed that after four rounds of phage display screening, ...

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 the plasmid was extracted and sequenced according to the instructions. The 15 strains of anti-α-HL single-chain antibody 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, containing FLAG and His-tag tags, which have been reported in some documents published by our laboratory, such as "Wang Xu, Yuan Qing, Ye Yingchun, etc. Anti-IL-33 whole human Soluble expression and identification of 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 (20...

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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 invention relates to the technical field of antibody engineering, in particular to a fully human anti-Staphylococcus aureus α-hemolysin recombinant antibody. Background technique [0002] Staphylococcus aureus is a major Gram-positive pathogen, and its infection can cause life-threatening serious diseases such as pneumonia, bacteremia, endocarditis, etc. Antibiotics have long been the mainstay of effective treatment for S. aureus infections. However, with the worldwide abuse of antibiotics, disinfectants and the release of antibiotic residues to the environment, the situation of methicillin-resistant Staphylococcus aureus (MRSA) with strong pathogenicity and wide transmission route has become the leading cause of nosocomial infection worldwide. primary pathogens. The emergence of vancomycin intermediate Staphylococcus aureus (VISA) strains and highly vancomycin-resistant Staphylococcus aureus (VRSA) strains has brought more severe challenges to t...

Claims

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Application Information

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Patent Type & Authority Patents(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
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