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Novel severe acute respiratory syndrome coronavirus 2 N proteantigen variant and application thereof to detection of novel severe acute respiratory syndrome coronavirus 2 antibody

A coronavirus and protein technology, applied in the direction of viruses/phages, viruses, viral peptides, etc., can solve the problems of inability to distinguish between positive serum and healthy human serum, low false positive rate, etc., to improve specificity, reduce non-specific reactions, Improve the effect of exclusivity

Pending Publication Date: 2020-12-04
SHANGHAI NAT ENG RES CENT FORNANOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2006, a study by Maache and other scholars also pointed out that the N protein used to detect SARS antibodies could not distinguish positive serum from healthy human serum at all.
Antibody detection based on S protein has a lower false positive rate

Method used

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  • Novel severe acute respiratory syndrome coronavirus 2 N proteantigen variant and application thereof to detection of novel severe acute respiratory syndrome coronavirus 2 antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] N4 peptide synthesis

[0056] The N4 polypeptide was chemically synthesized according to the routine operation of polypeptide synthesis, and the N4 polypeptide synthesized in this example was from Shanghai Jier Biochemical Co., Ltd. The purity by HPLC was 99%. A cysteine ​​residue is added to the C-terminus of the N4 polypeptide to provide a sulfhydryl group for a coupling reaction with the amino group of BSA.

Embodiment 2

[0058] Antigen fragment N4 coupled with carrier protein BSA

[0059] BSA was purchased from SIGMA, and the N4 polypeptide was coupled to BSA using SMCC (4-(N-maleimidomethyl)cyclohexanecarboxylic acid-N-succinimidyl ester) bifunctional crosslinker. BSA was activated by SMCC cross-linking agent, the molar ratio of cross-linking agent to protein was 20, mixed evenly and incubated at room temperature for 30 minutes. After the reaction, use a desalting column to remove unreacted cross-linking agent, and dialyze for 24 hours for later use. Add the N4 polypeptide to the activated protein solution, perform coupling reaction between BSA and the synthetic polypeptide at a mass ratio of 1:1, and incubate at 4 degrees for 2 hours. The coupling product was freeze-dried after dialysis for further use.

Embodiment 3

[0061] Preparation of N4 Antigen Colloidal Gold Conjugate and Goat IgG Antigen Conjugate

[0062] Add 10 µg of N4 antigen protein to 1 mL of AuNP colloidal gold solution (pH 8.0). After incubating at room temperature for 30 min, 100 µL of 10% BSA was added to block the surface of AuNPs. After incubating at room temperature for 15 minutes, centrifuge at 8000rpm for 15 minutes, discard the supernatant, and add 1mL boric acid buffer (20mM, pH 8.0, containing 1%BSA) to resuspend. Repeat the centrifugation and resuspension steps twice, and finally resuspend in 100 µL borate buffer (20mM, pH 8.0, containing 1% BSA) for use.

[0063] Prepare the goat IgG colloidal gold conjugate in the same way, and mix it with the above-mentioned N4 antigen colloidal gold coupling solution at a ratio of 1:4 for use.

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Abstract

The invention provides a novel severe acute respiratory syndrome coronavirus 2 N proteantigen variant and an application thereof to detection of the novel severe acute respiratory syndrome coronavirus2 antibody, and relates to variants of N protein important epitope polypeptide. The variants reduce the non-specific reaction of the novel severe acute respiratory syndrome coronavirus 2 protein as acapture antigen with other coronavirus antibodies such as SARS and human influenza coronavirus antibodies. The polypeptide has coronavirus N protein epitope peptide activity, and cysteine residues are added to the C end of the polypeptide. Amino acid residue sites of the mutation are selected from: lysine at the 342rd site, lysine at the 347 site, lysine at the 387 site and lysine atthe 388 site are mutated into amino acids with relatively low homology. The sequence of the N protein 340-419 polypeptide is shown as SEQ ID NO 7. The invention further discloses an antigen composition, use and method of the variant.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a new coronavirus N protein antigen variant and its application in the detection of new coronavirus antibodies. The antigen variant can effectively capture virus antibodies while greatly reducing the concentration of the full-length N protein. The problem of false positives in virus antibody detection caused by non-specific reactions has effectively improved the specificity of new coronavirus antibody detection. Background technique [0002] The novel coronavirus (novel Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) is an RNA virus with acute respiratory infection. As of the end of July 2020, the novel coronavirus has infected about 18 million people worldwide and killed 690,000 people. [0003] The spherical new coronavirus with crown spikes on the surface is composed of a shell composed of various proteins, namely spike glycoprotein (S, Spike protein), small envelope gly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165G01N33/68G01N33/569G01N33/532G01N33/531
CPCC07K14/005G01N33/6854G01N33/56983G01N33/532G01N33/531C12N2770/20022G01N2333/165G01N2469/20
Inventor 崔大祥田静王侃刘岩磊梁辉李雪玲
Owner SHANGHAI NAT ENG RES CENT FORNANOTECH
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