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Monoclonal antibody MERS-4V2 and encoding gene and application thereof

A technology of antibody and gene, applied in application, antibody, genetic engineering, etc.

Active Publication Date: 2019-04-23
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are no specific drugs and vaccines for the treatment and prevention of this disease

Method used

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  • Monoclonal antibody MERS-4V2 and encoding gene and application thereof
  • Monoclonal antibody MERS-4V2 and encoding gene and application thereof
  • Monoclonal antibody MERS-4V2 and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1, Discovery of Monoclonal Antibody MERS-4V2

[0027] 1. Discovery of monoclonal antibody MERS-4

[0028] Through sequence and model analysis, it was found that MERS-CoV may have a potential receptor binding domain (ReceptorBinding Domain, RBD), RBD and soluble DPP4 protein were co-crystallized, the protein structure was analyzed, and the key amino acid site of RBD binding to DPP4 was found . Using purified RBD to immunize animals, it was found that it has a strong ability to induce the production of neutralizing antibodies. Using RBD to screen the human non-immune scFvs library displayed on the surface of yeast (the English full name of scFvs is "single-chain variable fragments"), and obtained several monoclonal antibodies with neutralizing activity. One of the monoclonal antibodies was named monoclonal antibody MERS-4 (belonging to IgG1 antibodies).

[0029] The heavy chain of the monoclonal antibody MERS-4 is shown in sequence 5 of the sequence listing, an...

Embodiment 2

[0037] Embodiment 2, the preparation of monoclonal antibody

[0038] 1. Construction of recombinant plasmids

[0039] 1. Synthesize double-stranded DNA fragment A, and then insert DNA fragment A into the pMD18-T vector to obtain recombinant plasmid A. It has been verified by sequencing that in the double-stranded DNA fragment A, the direction from the 5' end to the 3' end is composed of the following elements in turn: CMV promoter (as shown in sequence 7 of the sequence listing), the heavy chain of the monoclonal antibody MERS-4 The coding gene (shown in sequence 6 of the sequence listing, encoding the heavy chain shown in sequence 5 of the sequence listing), polyA (shown in sequence 8 of the sequence listing).

[0040]2. Synthesize the double-stranded DNA fragment B, and then insert the DNA fragment B into the pMD18-T vector to obtain the recombinant plasmid B. After sequencing verification, in the double-stranded DNA fragment B, the direction from the 5' end to the 3' end ...

Embodiment 3

[0053] Embodiment 3, the binding characteristic of monoclonal antibody and RBD

[0054] 1. Construction of recombinant plasmids

[0055] FastBac TM The dual carrier itself does not have a signal peptide secreted extracellularly, and RBD itself is a secreted protein, so it needs to be connected to a signal peptide. The signal peptide will be excised during the process of protein expression and secretion in insect cells, and the final protein sequence will not contain signal peptide.

[0056] Replace pFastBac with the DNA molecule shown in sequence 9 of the sequence listing TM The DNA molecule shown in sequence 10 of the sequence listing in the dual vector to obtain a recombinant plasmid. In the sequence 9 of the sequence listing, the 7th-120th nucleotides encode the gp67 signal peptide, the 130-849th nucleotides encode the RBD protein, the 880-924th nucleotides encode the AVi tag, and the 925th-942nd nuclear Nucleotide encoding His 6 Label. AVi tags can be biotinylated ...

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Abstract

The invention discloses a monoclonal antibody MERS-4V2 and an encoding gene and application thereof. The monoclonal antibody MERS-4V2 is an IgG antibody and comprises a heavy chain and a light chain;CDR1, CDR2 and CDR3 in a heavy chain variable region in the heavy chain are sequentially the 45-52nd amino acid residues, the 70-77th amino acid residues and the 116-120th amino acid residues from theN terminal in the sequence 1 in a sequence table; CDR1, CDR2 and CDR3 in a light chain variable region in the light chain are sequentially the 45-52nd amino acid residues, the 70-72nd amino acid residues and the 109-119th amino acid residues from the N terminal in the sequence 3 in the sequence table. The invention further discloses application of the IgG antibody in preparing medicine for inhibiting Middle East respiratory syndrome coronaviruses. The antibody has wide application prospects in developing medicine (vaccines) for treating and / or preventing the novel coronavirus MERS-CoV.

Description

technical field [0001] The invention relates to a monoclonal antibody MERS-4V2 and its coding gene and application. Background technique [0002] Middle East Respiratory Syndrome is a viral respiratory disease caused by a novel coronavirus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The new type of coronavirus MERS-CoV was first discovered to infect humans in the Middle East in 2012, and then this virus infection disease appeared in several European countries and regions. More than half of infected patients developed severe respiratory disease, and its clinical symptoms were very similar to the disease caused by SARS-CoV that broke out in 2003. Because the disease can be transmitted from person to person, it has attracted great attention around the world. So far, there are no specific drugs and vaccines for the treatment and prevention of this disease. [0003] MERS-CoV uses the membrane protein S on its surface to enter susceptible cells. The S protein is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14A61P11/00
CPCA61K2039/505C07K16/10C07K2317/565C07K2317/76C07K2317/92
Inventor 张林琦王新泉江力玮左腾史宣玲周盼盼
Owner TSINGHUA UNIV
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