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
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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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