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Polypeptide molecule capable of being specifically combined with nucleocapsid protein of severe acute respiratory syndrome coronavirus and preparation method

A nucleocapsid protein, coronavirus technology, applied in the fields of peptides, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of no specific binding to new coronaviruses, etc., achieve convenient screening, low preparation cost, no need Effects of the immune process

Active Publication Date: 2021-06-11
江西乐成生物医疗有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The prior art has not found reports on polypeptide molecules that can specifically bind to the new coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) nucleocapsid protein (Nucleocapsid protein, N protein)

Method used

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  • Polypeptide molecule capable of being specifically combined with nucleocapsid protein of severe acute respiratory syndrome coronavirus and preparation method
  • Polypeptide molecule capable of being specifically combined with nucleocapsid protein of severe acute respiratory syndrome coronavirus and preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0012] Example 1 Affinity panning and identification of polypeptide molecules specifically binding to the new coronavirus nucleocapsid protein

[0013] 1) The specific method for affinity panning to specifically bind polypeptide molecules to SARS-Cov-2 nucleocapsid protein is: take 0.1 mg of SARS-Cov-2 nucleocapsid protein (see genebank: MN908947.3 for the amino acid sequence), including Incubate at 4°C for 12 hours in microplate wells (150 μL / well). Take out the ELISA plate, wash 10 times with PBST (10mM PBS pH 7.4 containing 0.5% Tween-20 (v / v)), add 350μl blocking solution (5% gelatin solution) and incubate at 37°C for 2 hours, wash 10 times with PBST Then add 100 μl phage peptide library (phage display random dodecapeptide library, purchased from NEB Company, dilute the phage stock solution 10 times with PBS, about 2.0×10 11 pfu), shake at 37°C for 60 min.

[0014] 2) Wash 15 times with PBST to remove unbound phages, then add 100 μL of eluent (Gly-HCL, pH 2.2), shake gen...

Embodiment 2

[0017] Example 2 Sequencing of the polypeptide molecule coding gene and determination of its amino acid sequence

[0018] The phages identified by ELISA that specifically bind to the polypeptide molecules of the nucleocapsid protein of the new coronavirus were amplified, and the DNA sequencing templates of the phages were extracted. The brief process is as follows: carry out phage amplification, after the first step of centrifugation, transfer 800 μl of phage-containing supernatant into a new centrifuge tube. Add 200 μl PEG / NaCl to precipitate the phage. After centrifugation, resuspend the pellet in 100μl iodide buffer (10mM Tris-HCl (pH 8.0), 1mM EDTA, 4M NaI), add 250μl absolute ethanol to precipitate DNA, and wash the pellet with 70% ethanol after centrifugation (DNA sequencing template ). The pellet was finally resuspended in 20 μl sterilized water, and 2 μl was taken for agarose gel electrophoresis analysis; 5 μl of phage template was taken for DNA sequencing, and its -...

Embodiment 3

[0019] Example 3 Mass preparation of polypeptide molecules specifically binding to novel coronavirus nucleocapsid protein

[0020] 1) By means of phage amplification

[0021]The phage that specifically binds to the nucleocapsid protein of the new coronavirus was added to a 20 ml culture inoculated with Escherichia coli ER 2738, and the culture was shaken at 37 degrees and 220 rpm for 4.5 hours. Transfer the culture to another centrifuge tube, centrifuge at 10,000 rpm at 4°C for 10 minutes, transfer the upper 80% of the supernatant to a fresh tube, add 1 / 6 volume of PEG / NaCl, and let stand at 4°C for 120 minutes. Centrifuge the PEG / NaCL solution at 10,000 rpm at 4°C for 15 minutes. Discard the supernatant, centrifuge briefly and aspirate the residual supernatant. Add 1mL TBS for resuspension, which is the phage amplification solution.

[0022] 2) Preparation in the form of polypeptide-fusion protein

[0023] A. PCR amplification of exogenous coding genes of polypeptide mole...

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Abstract

The invention relates to the technical field of biology, in particular to a polypeptide molecule capable of being specifically combined with a nucleocapsid protein (N protein) of a severe acute respiratory syndrome coronavirus (SARS-CoV-2) and a preparation method of the polypeptide molecule, and relates to the technical field of biology. The amino acid sequence of the polypeptide molecule is MQPMNALGGGAY. An optimized liquid-phase affinity elutriation mode is adopted, and the polypeptide molecule capable of being specifically combined N protein of SARS-CoV-2 is obtained through rapid elutriation. Compared with a traditional antibody, the polypeptide molecule has excellent specificity and better acid-base tolerance and thermal stability. The polypeptide molecule can be obtained without an immune process, the preparation is simple, the cost is low, and the period is short. The polypeptide molecule can be massively amplified through a biological culture mode and can also be massively prepared through a chemical synthesis or genetic engineering method. A new path is provided for preparation of a SARS-CoV-2 nucleocapsid protein specific recognition element. The polypeptide molecule has good application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a polypeptide molecule capable of specifically binding to a novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) nucleocapsid protein (Nucleocapsid protein, N protein) and a preparation method. Background technique [0002] Existing studies have confirmed that novel coronavirus pneumonia (Coronavirus disease 2019, COVID-19) is caused by novel coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2). Studies have shown that the new coronavirus is a positive-sense single-stranded RNA virus with a genome length of about 30Kb, with non-coding regions at both ends, and non-structural protein coding regions and structural protein coding regions in the middle. The nonstructural protein coding region mainly includes the open reading frame ORF1a and ORF1b genes, encoding 16 nonstructural proteins. The structural protein coding region mainly encodes...

Claims

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Application Information

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IPC IPC(8): C07K7/08C12N15/11
CPCC07K7/08
Inventor 万蒙何庆华
Owner 江西乐成生物医疗有限公司
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