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Antigen mimic epitope of SARS-COV-2 coronavirus and immunochromatographic test strip

A technology of SARS-COV-2 and immunochromatographic test paper, which is applied in the direction of positive-sense single-stranded RNA viruses, viruses, and viral peptides, and can solve the problems of low activity of recombinant antigens, complicated preparation, low affinity and specificity, etc.

Active Publication Date: 2021-09-14
浙江佳米生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the preparation of recombinant antigens is a relatively mature method, there are still problems such as complex preparation, low activity of expressed recombinant antigens, and low binding ability, affinity and specificity to natural novel coronavirus antibodies.

Method used

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  • Antigen mimic epitope of SARS-COV-2 coronavirus and immunochromatographic test strip
  • Antigen mimic epitope of SARS-COV-2 coronavirus and immunochromatographic test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Affinity panning and identification of SARS-COV-2 antigen mimotope

[0047] 1) Affinity panning of SARS-COV-2 antigen mimic epitopes: the specific method is: use the urine of recovered COVID-19 patients (which contains SARS-COV-2 antibodies) to coat a 96-well microtiter plate, Incubate at 37°C for 5 hours. After washing 5 times with TBST buffer, add 300 μl blocking solution and incubate at 4°C for 1 hour. After 1 hour, the blocking solution was discarded, washed 15 times with TBST buffer, and 100 μl of phage peptide library (phage display dodecapeptide library, purchased from NEB Company) was added to each well, and the phage stock solution was diluted 10 times with TBS, about 1.0×10 10 pfu), shake the reaction for 15 minutes at 22-26°C. Unbound phages were discarded, washed 10 times with TBST buffer, bound phages were eluted with 0.2M Glycine-HCl (pH 2.2), and immediately neutralized with 15 μl 1M Tris-HCl (pH 9.1). Take 10 μl of the eluted phage to measu...

Embodiment 2

[0050] Example 2. Sequencing of SARS-COV-2 antigen mimotope coding gene and determination of its amino acid sequence

[0051] The phages identified by indirect ELISA displaying the mock epitopes of the SARS-COV-2 antigen 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 the -96gIII sequencing primers were:

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Embodiment 3

[0053] A large amount of preparation of embodiment 3SARS-COV-2 antigen mimic epitope

[0054] (1) By means of phage amplification

[0055] The phage displaying the mimotope of the SARS-COV-2 antigen was added to 20 ml of culture inoculated with 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.

[0056] (2) Preparation in the form of SARS-COV-2 antigen mimic epitope-fusion protein

[0057] A. PCR amplification of exogenous coding gene of SARS-COV-2 antigen mimotope

[0058] PCR reaction system: (50μL)

[0...

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Abstract

The invention discloses an antigen mimic epitope of SARS-COV-2 with an amino acid sequence shown as SEQ ID NO.1, application of the antigen mimic epitope, and an SARS-COV-2 coronavirus detection immunochromatographic test strip. The test strip comprises a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad which are sequentially bonded on a bottom plate in a lap joint manner, wherein a colloidal gold labeled anti-human IgG antibody is sprayed on the binding pad; and a detection line for coating the antigen mimic epitope or fusion protein thereof and a quality control line for coating the human IgG antibody are arranged on the nitrocellulose membrane. A polypeptide capable of being specifically bound with the SARS-COV-2 antibody is screened from a peptide library from the SARS-COV-2 antibody in urine from a COVID-19 rehabilitative person by using a phage display peptide library technology. According to the screened SARS-COV-2 antigen mimic epitope, the novel coronavirus detection immunochromatographic test strip with high specificity and good sensitivity is prepared, and the test strip has the advantages of simplicity, convenience and rapidness in operation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an antigen mimic epitope and an immunochromatographic test strip of a novel coronavirus (SARS-COV-2). Background technique [0002] Novel coronavirus (SARS-COV-2, or 2019 novel coronavirus (2019-nCoV) is highly contagious and susceptible to novel coronavirus pneumonia (COVID-19) to humans. And SARS in 2003 The coronavirus and the 2012 MERS coronavirus belong to the genus Coronaviridae β. Compared with the 2003 "SARS" SARS CoV, 2019-nCoV has about 70% sequence similarity and 40% sequence similarity with MERS CoV. Effectively identifying patients infected with the new coronavirus is an important prerequisite and means to control the spread of the epidemic. The immunological detection method based on the immune response of antigen and antibody has the advantages of strong specificity, high sensitivity and simple operation, and does not require complicated equipment , comple...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62G01N33/68G01N33/569G01N33/558
CPCC07K14/005G01N33/68G01N33/56983G01N33/558C12N2770/20022C07K2319/24
Inventor 杨叶民
Owner 浙江佳米生物医药科技有限公司
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