Shark single-domain antibody targeting new coronavirus N protein as well as preparation method and application of shark single-domain antibody

A single-domain antibody, protein technology, applied in antiviral immunoglobulin, botanical equipment and methods, biochemical equipment and methods, etc., to achieve the effect of reducing production costs

Active Publication Date: 2021-09-03
MINJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the problems in the prior art that need to further improve the high-sensitivity detection of the new crown antigen and the development and application of antibodies, the present invention creatively proposes a shark single-domain antibody targeting the N-protein of the new coronavirus and its preparation method and application, prepared by the present invention The obtained shark single-domain antibody can specifically recognize the endogenously produced N protein of the new coronavirus, and has good sensitivity and accuracy when applied to the detection of the new coronavirus, and has good potential in the development of the new coronavirus detection kit. Application prospect

Method used

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  • Shark single-domain antibody targeting new coronavirus N protein as well as preparation method and application of shark single-domain antibody
  • Shark single-domain antibody targeting new coronavirus N protein as well as preparation method and application of shark single-domain antibody
  • Shark single-domain antibody targeting new coronavirus N protein as well as preparation method and application of shark single-domain antibody

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Embodiment 1

[0036] A shark single-domain antibody targeting the N protein of the new coronavirus, named N05 antibody, its amino acid sequence is shown in SEQ ID NO:1, and its nucleotide sequence is shown in SEQ ID NO:3.

[0037] A shark single-domain antibody targeting the N protein of the new coronavirus, named N38 antibody, its amino acid sequence is SEQ ID NO:2, and its nucleotide sequence is SEQ ID NO:4.

Embodiment 2

[0039] A method for preparing a shark single-domain antibody targeting the N protein of the new coronavirus, specifically comprising the following steps:

[0040] (1) Isolate the peripheral blood lymphocytes of the immune striped bamboo shark, extract the total RNA, and then reverse transcribe it into cDNA;

[0041] Immunization of the recombinant new coronavirus N protein of the striped bamboo shark: The striped bamboo shark was immunized with the purified prokaryotic recombinant new coronavirus N protein, and the specific method was as follows: according to the dose of 5nM / kg, the antigen was emulsified with Freund's adjuvant Afterwards, multi-point subcutaneous injections were carried out to the Bamboo Shark for immunization, and the interval between each immunization was 14 days. After 14 days after completing the sixth immunization, 5 mL of blood was collected from the tail vein of the Bamboo Shark into a sodium heparin anticoagulant tube, and the immunization was complete...

Embodiment 3

[0089] Nucleic acid, vector, composition or complex

[0090] The present invention relates to a nucleic acid molecule encoding the shark single domain antibody of the present invention, and the nucleic acid of the present invention can be RNA, DNA or cDNA.

[0091] A nucleic acid of the invention may also be in the form of, may be present in and / or may be part of a vector, such as a plasmid, cosmid or YAC. The vector may especially be an expression vector, ie a vector providing for expression of a shark single domain antibody in vitro and / or in vivo (ie in a suitable host cell, host organism and / or expression system). The expression vector typically comprises at least one nucleic acid molecule of the invention operably linked to one or more suitable expression control elements (eg, promoters, enhancers, terminators, etc.). Selection of such regulatory elements and their sequences for expression in a particular host is well known to those skilled in the art.

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Abstract

The invention discloses a shark single-domain antibody targeting new coronavirus N protein as well as a preparation method and application thereof, and belongs to the technical field of biology. The amino acid sequence is as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the nucleotide sequence is as shown in SEQ ID NO: 3 or SEQ ID NO: 4; meanwhile, the invention also provides a preparation method of the shark single-domain antibody targeting the new coronavirus N protein, and the preparation method comprises the following steps: separating peripheral blood lymphocytes of immune stripe bamboo sharks, extracting total RNA, and carrying out reverse transcription to obtain cDNA; taking cDNA as a template, amplifying a vNAR fragment of mottled bamboo shark, and connecting with a vector to construct a phage library; screening positive clones for identifying the new coronavirus N protein from the phage library; and constructing an expression vector, and inducing to express the new coronavirus N protein single-domain antibody to finally obtain the shark single-domain antibody targeting the new coronavirus N protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a shark single-domain antibody targeting the N protein of the new coronavirus and its preparation method and application. Background technique [0002] Coronaviruses belong to the Coronaviridae family and the genus Coronavirus, and are a class of single-stranded positive-sense RNA viruses; since the first virus was discovered in 1937, a variety of coronaviruses have been identified, belonging to the genus α, β, γ, and δ, among which Seven coronaviruses can infect humans, namely HCoV-229E, HCoV-NL63 of the genus α, HCoV-OC43, HCoV-HKU1, SARS-CoV, MERS-CoV of the genus β, and the new coronavirus SARS-CoV identified at the end of 2019 -2. [0003] SARS-CoV-2 is a respiratory virus that can be carried by droplets. It is different from any of the six known coronaviruses that can infect humans. The virus has at least 17 sites on different proteins that involve amino acid chang...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/70G01N33/569A61K39/42A61P31/14
CPCC07K16/10C12N15/70G01N33/56983A61P31/14C07K2317/20C07K2317/569G01N2333/165G01N2469/10A61K2039/505Y02A50/30
Inventor 陈锦霖潘长坤陈建明程云英
Owner MINJIANG UNIV
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