Monoclonal antibody MERS-4, as well as coding gene and application thereof

A technology of antibody and coding, applied in the fields of application, antibody, genetic engineering, etc., to achieve the effect of extensive theoretical guidance value

Active Publication Date: 2015-05-20
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-4, as well as coding gene and application thereof
  • Monoclonal antibody MERS-4, as well as coding gene and application thereof
  • Monoclonal antibody MERS-4, as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, discovery of monoclonal antibody

[0023] Through sequence and model analysis, it was found that MERS-CoV may have a potential receptor binding domain (Receptor Binding Domain, RBD). The RBD and soluble DPP4 protein were co-crystallized, and the protein structure was analyzed. It was found that there are key amino acids in the RBD that bind to DPP4 location. 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).

Embodiment 2

[0024] Embodiment 2, the preparation of monoclonal antibody MERS-4

[0025] 1. Synthesize the double-stranded DNA fragment A shown in Sequence 2 of the sequence listing, and then insert the DNA fragment A into the pMD18-T vector to obtain recombinant plasmid A.

[0026] In Sequence 2 of the sequence listing, nucleotides 1-888 from the 5' end are the CMV promoter, nucleotides 889-2274 are the coding gene for the heavy chain of the monoclonal antibody MERS-4, and nucleotides 2275-2402 for ployA. The double-stranded DNA molecule shown in sequence 2 of the sequence listing encodes the protein shown in sequence 1 of the sequence listing.

[0027] 2. Synthesize the double-stranded DNA fragment B shown in sequence 4 of the sequence listing, and then insert the DNA fragment B into the pMD18-T vector to obtain the recombinant plasmid B.

[0028] In sequence 4 of the sequence listing, nucleotides 1-888 from the 5' end are the CMV promoter, nucleotides 889-1596 are the coding gene for ...

Embodiment 3

[0032] Example 3, Application of Monoclonal Antibody MERS-4

[0033] 1. Detection of neutralizing activity of monoclonal antibody MERS-4 against MERS-CoV pseudovirus

[0034] A plasmid expressing the full-length membrane protein of MERS-CoV (named MERS-CoV membrane protein plasmid) and the backbone plasmid pNL4-3R-E-luciferase were co-transfected into 293T cells, and after incubation, infectious but non-replicating MERS- CoV pseudotyped virus, its infectivity is similar to live virus.

[0035] Insert the double-stranded DNA molecule (the gene encoding the full-length membrane protein of MERS-CoV) shown in Sequence 5 of the sequence listing between the HindIII and XhoI restriction sites of the pcDNA3.1(+) vector to obtain the MERS-CoV membrane protein grain.

[0036] 1. Preparation of MERS-CoV pseudovirus

[0037] Co-transfect 293T cells with MERS-CoV membrane protein plasmid and backbone plasmid pNL4-3R-E-luciferase, and incubate at 37°C. Collect the cell culture supernatan...

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Abstract

The invention discloses a monoclonal antibody MERS-4, as well as a coding gene and an application thereof. The antibody provided by the invention is named monoclonal antibody MERS-4, comprises a chain A and a chain B, wherein the chain A is (a) protein consisting of an amino acid sequence as shown in a sequence 1 or (b) protein which is derived from the sequence 1 by substituting and / or deleting and / or adding one or more amino acid residues on the sequence 1 and has the same function; and the chain B is (c) protein comprising an amino acid sequence as shown in a sequence 3 or (d) which is derived from the sequence 3 by substituting and / or deleting and / or adding one or more amino acid residues on the sequence 3 and has the same function. The monoclonal antibody can be used for effectively inhibiting MERS-CoV invasion cells, can be used as medicines for preventing or treating MERS-CoV, and has an important theoretical guiding value and a wide application prospect for prevention and treatment of MERS-CoV.

Description

technical field [0001] The invention relates to a monoclonal antibody MERS-4, its coding gene and application. Background technique [0002] The new type of coronavirus (MERS-CoV) was first found to infect humans in the Middle East in 2012, and then the diseases caused by this virus infection appeared in several European countries and regions. More than half of infected patients will develop severe respiratory disease, and its clinical symptoms are very similar to those caused by SARS-CoV that broke out in 2003. Because the disease can be transmitted from person to person, it has attracted great attention all over the world. So far, there are no specific drugs and vaccines for the treatment and prevention of this disease. [0003] MERS-CoV uses a membrane protein (S protein) on its surface to enter susceptible cells. The S protein is composed of the S1 domain at the N-terminus, the S2 domain and the transmembrane domain at the near-membrane end, and the susceptibility of ...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10A61K39/42A61P31/14
Inventor 张林琦王新泉江力玮王年爽史宣玲左腾
Owner TSINGHUA UNIV
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