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Coronavirus fusion protein, and preparation method and application thereof

A technology of fusion protein and coronavirus, applied in antiviral immunoglobulin, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of occupying cost, achieve high detection rate, improve sensitivity, and overall use The effect of performance optimization

Active Publication Date: 2020-08-07
四川携光生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, as one of the main raw materials of the new coronavirus-specific antibody IgM / G magnetic particle chemiluminescence detection kit, the viral recombinant antigen will occupy a large amount of cost in the development of the immunoassay kit

Method used

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  • Coronavirus fusion protein, and preparation method and application thereof
  • Coronavirus fusion protein, and preparation method and application thereof
  • Coronavirus fusion protein, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Full sequence synthesis of the fusion protein gene of 2019-nCoV S protein and 2019-nCoV N protein

[0031] Artificially constructed fusion protein gene sequence, which includes N-terminal eukaryotic KOZAK sequence, bee venom signal peptide, 2019-nCoVN protein gene (GeneID: 43740575), Linker sequence (GGGGS), 2019-nCoV RBD domain gene (S protein ( GeneID: 43740568), the 306-527 amino acid sequence of the artificial fusion sequence contains BamH I and EcoR I restriction sites respectively, and finally the whole gene is synthesized by Gene Synthesis Company, and constructed into a conventional plasmid vector.

Embodiment 2

[0033] The plasmid construction of fusion protein gene in embodiment 1

[0034] The PCR fragment of the obtained protein gene S-N and the carrier pFastBac through restriction endonuclease Nco I and EcoR I double digestion TMHT A to connect. The ligation product was transformed into competent Escherichia coli DH5α, and the volume did not exceed 10% of the competent cells. Gently swirl several times to mix the contents, and put the tube in a 42°C water bath for 60 seconds for heat shock. Quickly transfer the tubes to an ice bath for 120 seconds to cool the cells, add 400 μL LB medium to each tube, and shake slowly at 37°C for 60 minutes to revive the bacteria and express the plasmid-encoded antibiotic resistance marker gene, centrifuge at low speed for 2 minutes, and remove Keep about 100 μL of medium in a centrifuge tube, resuspend the bacteria, and spread the bacteria evenly on the LB agar plate containing Amp antibiotics with a glass spreader. Place the plate upside down in...

Embodiment 3

[0036] Construction of recombinant bacmid and expression of fusion protein

[0037] According to the Bac-to-Bac instruction method (Invitrogen), the correct recombinant plasmid was transformed into Escherichia coli DH10Bac competent cells. This transformation step was similar to the chemical transformation of DH5α, but the recovery time was extended to 2-4h, and the shaker speed was increased to 200rpm. Spread 30 μL of the bacterial solution evenly on the LB agar plate containing Kan+Gent+Tet in a glass spreader, and culture the plate upside down in a constant temperature incubator at 37°C. After about 30-48 hours, blue-white colonies may appear. Pick a single positive white-white colony and insert it into 5mL Kan+Gent+Tet LB culture medium, culture at 37°C for 12-16h, take the bacterial solution for PCR identification, and the results show that the bacmid Reassembled correctly. The recombinant bacmid was transfected into insect cell sf9 with a transfection reagent, and after...

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Abstract

The invention belongs to the technical field of antigen preparation processes, and particularly relates to a coronavirus fusion protein, and a preparation method and application thereof. According tothe coronavirus fusion protein provided by the invention, the nucleotide sequence of the RBD structural domain of a 2019-nCoV S protein and the nucleotide sequence of a 2019-nCoV N protein are subjected to complete gene synthesis and then are constructed into an insect cell expression vector to prepare a recombinant plasmid containing the RBD structural domain sequence of the 2019-nCoV N protein and the RBD structural domain sequence of the 2019-nCoV S protein, escherichia coli is transformed by the recombinant plasmid to obtain a recombinant plasmid containing the gene sequence of the fusionprotein, and host cells are transfected by the recombinant plasmid to complete then fusion expression of the RBD structural domains of the 2019-nCoV N protein and the 2019-nCoV S protein. The expression process of the fusion protein is simple, meanwhile, and when the fusion protein is applied to a 2019-nCoV antibody detection kit, the sensitivity of the detection kit is remarkably improved, the specificity is also improved, the detection rate is efficiently increased, and the overall use performance is more optimized.

Description

technical field [0001] The invention belongs to the technical field of antigen preparation technology, in particular to a coronavirus fusion protein and its preparation method and application. Background technique [0002] Novel coronavirus pneumonia (CoVID-19) is a new type of coronavirus named 2019-nCoV by WHO. Coronaviruses are a large family of viruses that can cause colds as well as more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS), while 2019-nCoV is a new coronavirus that has never been found in humans before strain. 2019-nCoV is a coronavirus belonging to the genus β. It has an envelope, and its particles are round or oval, often pleomorphic, with a diameter of 60-140 nm. Its genetic characteristics are significantly different from those of SARSr-CoV and MERSr-CoV. Studies have shown that the homology with bat SARS-like coronavirus (bat-SL-CoVZC45) is more than 85%. [0003] 2019-nCoV is a type of si...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/866C12N15/70G01N33/68G01N33/569C07K16/10A61K39/215A61P31/14
CPCC07K14/005C12N15/86C12N15/70G01N33/68G01N33/56983C07K16/10A61K39/12A61P31/14C12N2770/20022C12N2710/14043G01N2333/165C12N2770/20034
Inventor 秦枫潘敬梅黄敬双张伟万文琴鲜静吴斌张小飞季纯宇颜松
Owner 四川携光生物技术有限公司
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