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Application of SARS-COV-2 Spike protein in detection of 2019 novel coronavirus

A SARS-COV-2, 1.SARS-COV-2 technology, applied in the application field of SARS-COV-2 Spike protein in the detection of new coronary pneumonia, can solve the problems of high lethality, achieve sensitivity and specificity improvement, The effect of accurate detection

Active Publication Date: 2020-08-14
杭州泰熙生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Spike protein can be transmitted between different hosts through RBD genetic recombination or mutation, resulting in a higher lethal rate

Method used

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  • Application of SARS-COV-2 Spike protein in detection of 2019 novel coronavirus
  • Application of SARS-COV-2 Spike protein in detection of 2019 novel coronavirus
  • Application of SARS-COV-2 Spike protein in detection of 2019 novel coronavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The construction of embodiment 1 eukaryotic expression vector pcDNA3.1 (+)-SARS-COV-2 Spike (S1)

[0045] a. Codon optimization, gene synthesis and cloning vector construction

[0046] Back translation of the amino acid sequence of SARS-COV-2 Spike (S1) using mammalian cell dominant codons figure 2 , to obtain the SARS-COV-2 Spike (S1) gene sequence composed of mammalian cell dominant codons, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0047] According to the SARS-COV-2S gene (GenBank: MN908947.3), the whole gene synthesis method was adopted, and General Biosystems (Anhui) Co., Ltd. was entrusted to synthesize the whole gene, and GGTACC and TCTAGA were added to its 5' and 3' ends respectively Respectively obtain the enzyme recognition sites of endonucleases KpnI and XbaI, the sequence length is 2112bp, and the codons are replaced with the mammalian cell preference codons obtained above, the nucleotide sequence of which is shown in SEQ ID NO: 2, connect...

Embodiment 2

[0054] Example 2 HEK293 cells transfected and cultured with pcDNA3.1(+)-SARS-COV-2 Spike(S1) expression vector

[0055] a. Use liposome 2000 to transfer the plasmid into HEK293 cells (liposome 2000 was purchased from American GIBCO, Invitrogen, and operated according to the instructions of the liposome 2000 kit), DMEM medium (Dulbecco'sModified Eagle Media) plus G418 concentration Screened at 600ng / ml for 14 days, picked G418-resistant monoclonal subculture, adjusted the concentration of G418 to 300ng / ml, screened and cultured in DMEM medium, and obtained pcDNA3.1(+)-SARS-COV-2Spike(S1) positive Transfected clones.

[0056] b. Western blotting identification of recombinant proteins Collect positive clones and resuspend them with SDS-PAGE loading Buffer, boil for 10 minutes, centrifuge at 10,000rpm for 10 minutes, separate the supernatant by SDS-PAGE electrophoresis, and use a semi-dry transfer system to transfer the protein to PVDF On the membrane, block with 10g / L BSA in tur...

Embodiment 3

[0058] Example 3 Separation and purification of HEK293-SARS-COV-2 Spike (S1) protein

[0059] a, because the recombinant expression vector pcDNA3.1(+)-SARS-COV-2 Spike (S1) constructed in Example 1 has the gene sequence of the fusion tag (His Tag), so the expressed target protein also carries There is a His Tag tag. Therefore, the purification of target protein can be carried out by affinity chromatography using the agarose complexed with Ni, and the affinity chromatography column is purchased from GE Healthcare company.

[0060] Take the harvested frozen cells prepared in Example 2, lyse with pre-cooled cell lysate, resuspend in Buffer A (20mM Tris, 0.5M NaCl pH 8.0), 150W ultrasonic lysis 10 times, 5s each time, 10s interval, 4°C Centrifuge at 12000rpm / min for 20min under the conditions to collect the supernatant and prepare for loading.

[0061] The specific purification steps are as follows:

[0062] Equilibrate Ni column to OD with Buffer A 280 constant reading;

[0...

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Abstract

The invention discloses application of SARS-COV-2 Spike protein in detection of 2019 novel coronavirus. According to the invention, the HEK293 cell strain capable of stably and highly expressing the S1 protein subunit recombinant protein of the SARS-COV-2 Spike protein is obtained, and the S1 protein subunit recombinant protein of the SARS-COV-2 Spike protein is applied to the development of an immunochromatographic detection reagent. According to the invention, the SARS-COV-2 Spike protein S1 protein subunit recombinant antigen is used as a marking material and is applied to a gold-labeled immunochromatography system; the detection system adopts direct labeling capture, and compared with an IgM / IgG detection product taking prokaryotic expression SARS-COV-2 NP recombinant protein as a coating, the SARS-COV-2 Spike protein have greatly improved sensitivity and specificity.

Description

technical field [0001] The present invention relates to the field of biotechnology, more specifically, relates to the application of SARS-COV-2 Spike protein in the detection of new coronary pneumonia. Background technique [0002] Coronavirus particles are irregular in shape and about 60-220nm in diameter. Virus particles are covered with a fatty membrane (three glycoproteins on the surface of the membrane), a small envelope glycoprotein (Envelope Protein, a smaller protein that binds to the envelope) and a membrane glycoprotein (Membrane Protein, responsible for the transmembrane transport of nutrients, newborn Virus budding release and viral envelope formation). [0003] The membrane proteins of the virus mainly include the surface spike protein (Spike protein, S) and the membrane (membrane, M). Spike protein is a glycoprotein that mainly acts on cell adhesion and cell membrane fusion. In many mammals, S It is hydrolyzed into S1 and S2 by furin enzyme or other enzymes...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N33/577G01N33/569G01N33/558C12N15/85C12N15/50
CPCG01N33/587G01N33/577G01N33/56983G01N33/558C12N15/85C07K14/005G01N2333/165G01N2469/10C12N2800/107C12N2770/20022C12N2770/20051Y02A50/30
Inventor 周斌殷秀飞何鲁江
Owner 杭州泰熙生物技术有限公司
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