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Method for preparing recombinant novel coronavirus Spike protein

A new type of coronavirus technology, applied in the field of the preparation of recombinant novel coronavirus Spike protein, can solve the problems of limited application, it is difficult to obtain high-level recombinant expression of the full-length extracellular region, etc., to meet a large number of needs and reduce production costs. Effect

Active Publication Date: 2021-02-19
ACROBIOSYSTEMS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to its large molecular weight and its metastable trimerization state in its prefusion state, it is difficult to obtain high-level recombinant expression of its full-length extracellular region, which severely limits its application in the field of antiviral drug development and diagnosis

Method used

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  • Method for preparing recombinant novel coronavirus Spike protein
  • Method for preparing recombinant novel coronavirus Spike protein
  • Method for preparing recombinant novel coronavirus Spike protein

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1, the construction of recombinant expression vector

[0033] In this example, 10 signal peptides were screened to verify their application in the preparation of the secretion and expression of the recombinant novel coronavirus Spike protein (the amino acid sequence is shown in SEQ ID No. 1 in the sequence table).

[0034] Among them, the amino acid sequence of the signal peptide of the novel coronavirus Spike protein itself is shown in the sequence table SEQ ID No. 2. Five kinds of signal peptides known to promote high secretion and expression of recombinant proteins (hereinafter referred to as high expression signal peptides), respectively Human IL-2 signal peptide, Mouse IgG kappa signal peptide, Human tPA signal peptide, influenza virus HA signal peptide, HumanIgG The kappa signal peptide, its corresponding amino acid sequence is shown in the sequence table SEQ ID No. 3-SEQ ID No. 7. The amino acid sequences corresponding to the other four signal peptide...

Embodiment 2

[0038] Example 2. Recombinant expression vector transfected HEK293 cells and expressed novel coronavirus Spike protein

[0039] Using PEI transfection reagent, the constructed recombinant plasmids pHEK-S1~pHEK-S10 were transfected into HEK293 cells for recombinant expression of the novel coronavirus Spike protein. The day before transfection, HEK293 cells were subcultured at 1×106 / mL and cultured at 37°C. Count the cells on the day of transfection and adjust the cell density to 2×10 6 / mL, the activity rate is above 95%.

[0040] Calculate the dosage of each component in the transfection complex according to the cell density: the corresponding relationship between the plasmid dosage and the number of cells is 1×10 6 The cells correspond to 1 ug of plasmid, and the corresponding relationship between the dose of PEI and the dose of plasmid is that the quality of PEI is 3 times the quality of DNA.

[0041] Prepare the transfection complex according to the components calculated...

Embodiment 3

[0042] Embodiment 3, the separation and purification of Spike protein

[0043] The Spike recombinant protein was isolated and purified by His-tag affinity chromatography, and the effects of 10 signal peptides on improving the secretion and expression of Spike protein were compared. Collect the above cell culture supernatant by centrifugation at 1000×g for 30 min, filter the supernatant and combine with GE HisTrap excel chromatography column pre-equilibrated with PBS buffer, and then elute the target with PBS containing 30 / 100 / 500mM imidazole Protein, the elution volume of each concentration was 5 CV, and the protein eluate was collected and analyzed by SDS-PAGE.

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Abstract

The invention provides a method for preparing a recombinant novel coronavirus Spike protein. The invention firstly provides application of the following polypeptides as a signal peptide in preparationof recombinant novel coronavirus Spike protein: polypeptides composed of the amino acid sequence as shown in SEQ ID No. 10. By adopting a specific signal peptide, an expression vector containing polynucleotide for encoding the recombinant novel coronavirus Spike protein is constructed, and mammalian cells are transfected to secrete and express the recombinant novel coronavirus Spike protein, so that the secretory expression level of the Spike protein in HEK293 cells can be remarkably improved.

Description

technical field [0001] The present invention relates to a method for preparing recombinant novel coronavirus Spike protein, more specifically, relates to a method for efficiently secreting and expressing novel coronavirus Spike protein in mammalian cells by using a specific signal peptide. Background technique [0002] The new type of coronavirus (SARS-CoV-2, or COVID-19) is a new strain of coronavirus that broke out on a large scale in the world in 2020, and it belongs to the same β-coronavirus as SARS coronavirus. In order to obtain effective prevention and treatment methods for the new coronavirus as soon as possible, scientific research institutes in various countries are developing various antiviral drugs and treatment programs day and night. [0003] Spike protein is expressed on the surface of coronavirus and plays a key role in recognizing and binding to host cell surface receptors and mediating the fusion of viral envelope and cell membrane. During infection, the S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/67C07K19/00C12N5/10
CPCC07K14/005C07K2319/02C07K2319/21C07K2319/35C12N5/0686C12N15/67C12N15/85C12N2510/02C12N2770/20022C12N2770/20051C12N2800/107
Inventor 秦丽丽宋洁王芳琪
Owner ACROBIOSYSTEMS INC
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