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Single-domain heavy-chain antibody, encoding gene, preparation method and use thereof and pharmaceutical composition

A single-domain heavy chain antibody and composition technology, applied in the biological field, can solve the problems of difficulty and lack of MERS vaccine development, and achieve good neutralizing activity, strong reactivity, and simple preparation methods

Active Publication Date: 2022-01-11
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a single-domain heavy chain antibody and its encoding gene, preparation method, application and pharmaceutical composition in order to overcome the lack of vaccine products against MERS-CoV in the prior art and the difficulties in the development of MERS vaccines.

Method used

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  • Single-domain heavy-chain antibody, encoding gene, preparation method and use thereof and pharmaceutical composition
  • Single-domain heavy-chain antibody, encoding gene, preparation method and use thereof and pharmaceutical composition
  • Single-domain heavy-chain antibody, encoding gene, preparation method and use thereof and pharmaceutical composition

Examples

Experimental program
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Effect test

Embodiment 1

[0080] This example is used to illustrate the expression and identification of the single domain heavy chain antibody Nano-Anti-MRBD.

[0081] (1) Expression vector construction

[0082] The inventor designed the nucleotide sequence shown in SEQ ID NO: 2 as the coding gene of the single domain heavy chain antibody Nano-Anti-MRBD (amino acid sequence shown in SEQ ID NO: 1), wherein the 5' end is the first -6 is the recognition site of BamH I, the 7th-363rd (i.e. the nucleotide sequence shown in SEQ ID NO:3) is the Nano-Anti-MRBD coding sequence, and the 364th-366th is a stop codon, Positions 367-372 are the recognition sites of Xba I.

[0083] The coding gene of the single domain heavy chain antibody Nano-Anti-MRBD and the pCold I vector (purchased from TAKARA Company, the brand is D3361, which The expressed protein carries a Poly-His tag (the amino acid sequence of the tag is shown in SEQ ID NO: 6) for double enzyme digestion, and the digested product is ligated to obtain th...

Embodiment 2

[0108] This example is used to illustrate the binding properties of the single domain heavy chain antibody Nano-Anti-MRBD to MERS-CoV.

[0109] The indirect ELISA method was used to detect the reactivity of Nano-Anti-MRBD and MERS-CoV RBD, the specific method is as follows:

[0110] Use the MERS-CoV RBD-Fc protein (the fusion protein of the RBD functional region of the MERS-CoV S protein and the human IgG Fc fragment, the amino acid sequence of which is shown in SEQ ID NO: 4) to coat a 96-well microtiter plate at a coating concentration of 1 μg / mL, 50 μL per well. The concentrated Nano-Anti-MRBD protein sample obtained in Example 1 was prepared into protein solutions with different concentrations as the primary antibody in the experimental group using blocking solution (PBS solution containing 3% by weight BSA), and HRP-His mouse monoclonal antibody (purchased Zikangwei Century Company (brand name: CW0285M) was used as the secondary antibody. In the control group, SARS-CoV a...

Embodiment 3

[0113] This example is used to illustrate the neutralizing activity of single domain heavy chain antibody Nano-Anti-MRBD to MERS-CoV pseudovirus.

[0114] The Huh-7 cells used in this example were purchased from ATCC, and the preparation method of the MERS-CoV pseudovirus (containing firefly luciferase reporter gene) used was referred to: A safe and convenient pseudovirus-basedinhibition assay to detect neutralizing antibodies and screen for Viral entry inhibitors against the novel human coronavirus MERS-CoV. Zhao G, Du L, Ma C, et al. Virol J. 2013;10:266. doi:10.1186 / 1743-422X-10-266.

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Abstract

The invention relates to the technical field of biology and discloses a single-domain heavy-chain antibody, an encoding gene, a preparation method and use thereof and a pharmaceutical composition. The single-domain heavy-chain antibody can be specifically combined with a receptor binding region of the Middle East Respiratory Syndrome Coronavirus, has good neutralizing activity on the Middle East Respiratory Syndrome Coronavirus, and has the potential as an active component in an antiviral product for preventing and treating the Middle East Respiratory Syndrome.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a single domain heavy chain antibody and its encoding gene, preparation method, application and pharmaceutical composition. Background technique [0002] Coronaviruses were first isolated from chickens in 1937. Studies have found that coronaviruses can only infect vertebrates. Among them, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was first identified in September 2012. It was discovered that it is one of the seven known coronaviruses that can infect humans. MERS-CoV has the characteristics of strong pathogenicity, high lethality, and the ability of human-to-human transmission. It is another coronavirus that poses a serious threat to human health and development after severe acute respiratory syndrome coronavirus (SARS-CoV). Virus. Although there has not been a large-scale infection of MERS-CoV in my country, there is still a risk of a serious epidemic that wil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14C12N15/70C12N15/11C12R1/19
CPCC07K16/10A61P31/14C12N15/70C07K2317/569C07K2317/76A61K2039/505Y02A50/30
Inventor 李江凡赵光宇邓永强邱洪杰秦成峰
Owner ACADEMY OF MILITARY MEDICAL SCI
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