Method for preparing novel coronavirus nucleocapsid protein by using HEK293 cells

A nucleocapsid protein, coronavirus technology, applied in biochemical equipment and methods, viruses, viral peptides, etc., can solve the problem of reducing the accuracy of immunodiagnosis and antibody preparation, protein folding errors, post-translational modification, different structures and modifications, etc. problems, to achieve the effect of high repeatability, high accuracy and broad prospects of the method

Active Publication Date: 2021-02-26
ACROBIOSYSTEMS INC
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Problems solved by technology

However, because the E. coli prokaryotic expression system has many defects such as protein folding errors and post-translational modifications compared with the eukaryotic expression system, the N protein prepared by E. coli expression has the same structure as the N protein expressed by human cells after the new coronavirus infection. and modified different risks, thereby reducing the accuracy of immunodiagnosis and antibody preparation

Method used

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  • Method for preparing novel coronavirus nucleocapsid protein by using HEK293 cells
  • Method for preparing novel coronavirus nucleocapsid protein by using HEK293 cells
  • Method for preparing novel coronavirus nucleocapsid protein by using HEK293 cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of recombinant expression vector

[0047] Construction of recombinant pHEK-N1 expression vector: Shanghai Bioengineering Co., Ltd. synthesized a polynucleotide (SEQ ID NO: 5) encoding an amino acid sequence as shown in SEQ ID NO: 4, and added a restriction enzyme BamH at the 5' end Ⅰ enzyme cutting site, Kozak sequence; the 3' end adds a stop codon TAA and a restriction endonuclease Xho Ⅰ enzyme cutting site. The synthesized product was double-digested with restriction endonucleases BamH Ⅰ and Xho Ⅰ, then subjected to agarose gel electrophoresis, and the target fragment was recovered by cutting the gel. The target fragments were respectively digested with pcDNA3.1(+) (Invitrogen Company, catalog number: V790-20) ( figure 1 ) to construct a recombinant eukaryotic expression vector named pHEK-N1. The schematic diagram of recombinant plasmid construction is shown in figure 2 . Transform Escherichia coli E.coli DH5α with pHEK-N1, culture at 37°C ...

Embodiment 2

[0048] Example 2 Recombinant expression vector transfected HEK293 cells and expressed the new coronavirus nucleocapsid protein

[0049] The constructed recombinant plasmid pHEK-N1 was transfected into HEK293 cells with PEI transfection reagent for recombinant expression of the N protein of the new coronavirus. The day before transfection, HEK293 cells were divided into 1 × 10 6 / mL subcultured 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 over 95%.

[0050] 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.

[0051] Prepare the transfection complex according to the components calculated ab...

Embodiment 3

[0052] Example 3 Separation and purification of the new coronavirus nucleocapsid protein

[0053] Use the His-tag affinity chromatography column to separate and purify the recombinant protein, specifically: collect the above cell culture supernatant by centrifugation at 1000g for 30 min, filter the supernatant and combine with GE HisTrap excel chromatography column pre-balanced with PBS buffer , and then gradient elution was performed with PBS buffer containing 0, 30, 100, 250 and 500 mM imidazole in sequence, and the elution volume of each concentration was 10 CV, and the protein eluate was collected and analyzed by SDS-PAGE ( image 3 ). According to the results of SDS-PAGE, the purity of the N protein eluted under the conditions of 100, 250 and 500 mM imidazole is as high as 95%, and the part of the protein is mixed and then dialyzed. The dialysate was dialyzed overnight, and the dialysate used was: PBS buffer (pH 7.4). The dialyzed samples were tested by SDS-PAGE. The re...

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Abstract

The invention provides a method for preparing novel coronavirus nucleocapsid protein by using HEK293 cells. The method comprises the following steps: 1) constructing a recombinant expression vector ofthe novel coronavirus nucleocapsid protein (N protein); 2) transfecting HEK293 cells by using the recombinant expression vector; and 3) culturing the cells in vitro, and separating and purifying theN protein from the culture supernatant. A large amount of new coronavirus N protein can be obtained in a short time by utilizing an HEK293 expression system, and N protein with the purity of 98% or above can be obtained through a one-step affinity chromatography method. Compared with escherichia coli, the N protein prepared by adopting the HEK293 expression system has great advantages in the aspects of antibody binding activity and novel coronavirus antibody colloidal gold detection, and the protein space conformation of the N protein prepared by adopting the HEK293 expression system is closeto the protein expression conformation of a virus N gene in a host body; the invention has higher accuracy of immunodiagnosis and antibody preparation, and has a wide prospect in preparation of diagnostic reagents and vaccines.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing a novel coronavirus nucleocapsid protein using HEK293 cells. Background technique [0002] Novel 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 genus of beta coronavirus as SARS coronavirus. Common signs after a person is infected with the new coronavirus include respiratory symptoms, fever, cough, shortness of breath, and dyspnea. In more severe cases, infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death, posing a serious threat to human health. [0003] The new coronavirus nucleocapsid protein (N protein) is one of the most important structural proteins in the new coronavirus capsid, accounting for the largest proportion of the virus' structural proteins. The COVID-19-N protein and viral genome RNA intertwine t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C07K14/165G01N33/569G01N33/58A61K39/215A61P31/14
CPCA61K39/12A61P31/14C07K14/005C12N15/85C12N2770/20022C12N2770/20034C12N2770/20051C12N2800/107G01N33/56983G01N33/585G01N2333/165G01N2469/20
Inventor 秦丽丽王晓菲张林张晓慧
Owner ACROBIOSYSTEMS INC
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