Antigen-antibody co-display method for screening antibody library of membrane antigen

A technology of antibody library and membrane antigen, which is applied in the field of membrane antigen antibody library screening and antigen-antibody co-display, which can solve the problems of inability to perform effective screening, membrane proteins cannot cause sufficient immune response, and antibody display technology cannot be used for library elutriation, etc.

Pending Publication Date: 2021-05-18
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods overcome the problem of antigen conformation, the membrane proteins expressed in these vectors cannot cause sufficient immune responses, and there are few successful reports in practical applications.
In the same way, MMPs molecules without natural conformation are used as antigens, and various antibody display technologies (including phage display, yeast display, mammalian cell display, insect virus display, etc.) cannot pan the library and effectively screen

Method used

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  • Antigen-antibody co-display method for screening antibody library of membrane antigen
  • Antigen-antibody co-display method for screening antibody library of membrane antigen
  • Antigen-antibody co-display method for screening antibody library of membrane antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1. Vector construction and expression localization of multiple transmembrane proteins-antigen proteins

[0116] Purpose: To achieve the effect of localizing in the cell membrane by fused expression of CXCR4 gene with EGFP, Cub, and GAL4 genes.

[0117] Plasmid construction:

[0118] The human CXCR4 (shown in SEQ ID NO.1, specifically:

[0119] MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS)扩增并测序,与EGFP(SEQ ID NO.2所示,具体为:

[0120] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDE...

Embodiment 2

[0127] Example 2. Vector construction and expression localization of antibody modules

[0128] Purpose: To achieve the effect of localization in the cell membrane by fused expression of At1-scFv gene with EGFP, transmembrane peptide TMpho, and NubG.

[0129] Plasmid construction:

[0130] The anti-human CXCR4 single-chain antibody At1-scFv (shown in SEQ ID NO.6, specifically:

[0131] DIVMTQSPDSLAVSLGERATINCKSSQSLFNSRTRKKYLAWYQQKPGQPPKLLIYWASKRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSRFLRAFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTSTDYYFSWVRQAPGKGLEWVGFIRTKSKGYTTEYSGSVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREPITTDPRDYWGQGTLVTVS)编码序列扩增并测序,与EGFP、泛素蛋白N端结构域NubG(SEQ ID NO.7所示,具体为:MQIFVKTLTGKTGTLEVESSDTIDNVKSKIQDKEGIP)、酵母跨膜肽TMpho(SEQ ID NO.8所示,具体 is: SWLLAFAGCKPRNVLLMAMCVVFFLSMWISNVAAPVLTYSLLSP), these four parts are fused, and the whole fusion protein is cloned into the yeast expression vector pGAD-T7 to form as Figure 8 The structure shown in A is designated as pGBK-T7-NubG-TMph...

Embodiment 3

[0136] Example 3. Interaction detection of CXCR4 single chain antibody At1-scFv with CXCR4 antigen

[0137] Objective: To observe the interaction between CXCR4 and its single-chain antibody At1-scFv.

[0138] Principle: When the CXCR4 protein interacts with the antibody, the intracellular ubiquitin Cub and Nub domains are pulled together to form an active and complete ubiquitin protein, which is then digested by the endogenous ubiquitin-specific protease of the cell to release the Cub The transcription factor GAL4 connected at the end enters the nucleus to transcribe the reporter genes His3, Ade2 and LacZ. The first two reporter genes help cells grow on SD-His-Ade-auxotrophic medium, and the latter catalyzes the development of X-a-gal substrate in the medium. Among them, antibody plasmids have two topological forms, namely Cis and Trans.

[0139] Plasmid construction:

[0140] Antigen plasmid: Human CXCR4 gene, Cub and GAL4 gene were fused to pGAD-T7 plasmid, which was desi...

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Abstract

The invention discloses an antigen-antibody co-display method for screening an antibody library of a membrane antigen. According to the method, a target antigen is membrane protein, is expressed on a cell membrane, and is coupled with an effector structural domain on the inner side of the cell membrane; a specific antibody library aiming at the target antigen is expressed on the surface of a cell membrane through transmembrane peptide, and is coupled with an intracellular effect protein structural domain through the transmembrane peptide; and if the antibody and the antigen on the cell surface can act, the effector structural domain expressed by fusion of the antibody and the antigen can be reassembled into a functional effector, and successful binding of the antibody and the antigen is reported through transcription of a reporter gene. By adopting the technical scheme provided by the invention, the specific antibody aiming at the target antigen can be quickly and effectively screened out.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an antigen-antibody co-display method for screening antibody libraries of membrane antigens. Background technique [0002] Multi-pass membrane proteins (MMPs) are an important class of membrane protein receptors, mainly including G protein coupled receptors (G-protein coupled receptors, GPCRs) and ion channel proteins (Ion channel proteins) ), the former is a seven-time transmembrane protein, which mediates the transduction of extracellular signals into the cell; the latter is a cylindrical membrane protein with an indeterminate number of transmembrane transmembranes, which mediates ion exchange inside and outside the cell. MMPs play an important role in life activities, so they become an important class of drug targets. In the early stage, small molecule drugs were used to interfere with the function of MMPs. However, because many MMPs, especially GPCR family proteins, have extreme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B30/04C12N15/12C12N15/13C12N15/81C12N15/65C12N1/19
CPCC40B30/04C07K14/705C07K16/28C12N15/81C12N15/65C07K2319/02C07K2319/03
Inventor 李京敬张荫杰
Owner SHANGHAI JIAO TONG UNIV
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