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COVID-19-S-RBD virus-like particles and vaccine, and preparation methods of virus-like particles and vaccine

A COVID-19-S-RBD and vaccine preparation technology, applied in the field of biomedicine, can solve the problems of high production costs, limited vaccine development, low yield of chimeric vaccines, etc., to achieve easy purification, fast immune response, and simplified screening process Effect

Active Publication Date: 2020-06-19
深圳赫兹生命科学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the yield of chimeric vaccines is relatively low, the production cost is relatively high, and in large-scale production, it is easy to cause incompatibility in product structure and components, which limits its development in vaccines

Method used

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  • COVID-19-S-RBD virus-like particles and vaccine, and preparation methods of virus-like particles and vaccine
  • COVID-19-S-RBD virus-like particles and vaccine, and preparation methods of virus-like particles and vaccine
  • COVID-19-S-RBD virus-like particles and vaccine, and preparation methods of virus-like particles and vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Provide a kind of COVID-19-S-RBD virus-like particle, described virus-like particle is by CuMV of cucumber mosaic virus TT The pET28a-CuMVTT recombinant plasmid formed after the gene is connected with the pET28a plasmid is obtained by expressing the expression strain.

[0050] Specifically, the cucumber mosaic virus CuMV TT Both the gene and the pET28a plasmid were digested with HindⅢ endonuclease and BamHI endonuclease, and then the CuMV was digested with T4 DNA ligase. TT Ligated with pET28a plasmid double digestion product to obtain pET28a-CuMV TT recombinant plasmid. The cucumber mosaic virus CuMV TT Gene to CuMV TT The viral nucleotide sequence was deduced back to the codon sequence. The expression strain adopts Escherichia coli T7 Shuffle expression strain. pET28a-CuMV was obtained by screening with 50ug / ml concentration of kanamycin TT T7 Shuffle recombinant expression strain.

[0051] The method for preparing the above-mentioned COVID-19-S-RBD virus-lik...

Embodiment 2

[0117] Provide a COVID-19-S-RBD vaccine, including the above-mentioned COVID-19-S-RBD virus-like particles, and also include pFUSE-COVID-19- The S-RBD recombinant plasmid expresses the COVID-19-S-RBD protein expressed by the cell line; the CuMV TT Virus-like particles and the COVID-19-S-RBD protein are coupled into COVID-19-S-RBD-CuMV by chemical coupling reagent SMPH TT After mixing Freund's complete adjuvant to obtain COVID-19-S-RBD vaccine.

[0118] Specifically, the expression cell line adopts the 293F expression cell line, and the pFUSE-COVID-19-S-RBD recombinant expression cell line is obtained after the pFUSE-COVID-19-S-RBD recombinant plasmid is transferred into the 293F expression cell line.

[0119] Above-mentioned COVID-19-S-RBD vaccine preparation method, comprises the following steps:

[0120] (21) Construction of recombinant plasmid: construct pFUSE-COVID-19-S-RBD recombinant plasmid after connecting COVID-19-S-RBD gene with pFUSE plasmid;

[0121] (22) Transf...

Embodiment 3

[0168] 1.1 Immunogen preparation

[0169] The above-mentioned COVID-19-S-RBD-CuMVTT was emulsified into a vaccine according to the injection amount of 50ug / mouse and Freund's complete adjuvant 1:1, and the control group had two groups injected with COVID-19-S-RBD and PBS respectively , are emulsified into vaccines with Freund's complete adjuvant 1:1.

[0170] 1.2 Immunization of mice

[0171] Six-week-old BALB / c mice were purchased from Beijing Life River Experimental Animal Technology Co., Ltd. and kept in the animal facility of Anhui Agricultural University. All animal experiments were performed in accordance with the National Animal Protection Guidelines and were approved by the Chinese Association for Laboratory Animal Science.

[0172] By subcutaneous injection of 50 μg COVID-19-S-RBD-CuMV TT or COVID-19-S-RBD (PBS as a control) to immunize six-week-old newborn BALB / c mice (3 in each group). Booster immunizations at weeks 2 and 3. Serum was collected 1 week after eac...

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Abstract

The invention discloses COVID-19-S-RBD virus-like particles and vaccine, and preparation methods of the virus-like particles and the vaccine. pET28a-CuMVTT recombinant plasmid is constructed after a CuMVTT gene is used to connect pET28a plasmid; pFUSE-COVID-19-S-RBD recombinant plasmid is constructed by a COVID-19-S-RBD gene and pFUSE plasmid; the recombinant plasmid is respectively transferred into the expression strains of escherichia coli and the expression cell lines of 293 F cells; the expression strains of escherichia coli are cultivated, biomass is separated through centrifugation, andthe virus-like particles are obtained; COVID-19-S-RBD protein is obtained by cultivating the expression cell lines of the 293 F cells; and the virus-like particles are coupled to the COVID-19-S-RBD through a chemical coupling reagent SMPH. The virus-like particles and the vaccine can be easily obtained through bacteria culture; yield can be higher than that of chimeric expression; and thus, industrial production and fast immunity can be achieved.

Description

technical field [0001] The invention relates to a COVID-19-S-RBD virus-like particle, a vaccine and a preparation method thereof, belonging to the technical field of biomedicine. Background technique [0002] Symptoms of 2019 Novel Coronavirus (COVID-19) are generally fever, fatigue, dry cough, and gradual dyspnea, and severe cases manifest as acute respiratory distress syndrome, septic shock, metabolic acidosis that is difficult to correct, and coagulation dysfunction . Human-to-human transmission of the virus has been confirmed, and the incubation period is contagious, and there is no specific treatment for the resulting disease. However, different forms of vaccines are being developed. According to news reports, for example, on February 25, 2020, Moderna, a clinical-stage biotechnology company focused on the development of mRNA drugs and vaccines, announced that it had produced the first batch of new coronaviruses for human trials. vaccine mRNA-1273. Also, on February ...

Claims

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Application Information

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IPC IPC(8): C07K14/165C12N15/70A61K39/215A61P31/14
CPCC07K14/005C12N15/70A61K39/12A61P31/14C12N2770/20023C12N2770/20034A61K2039/5258Y02A50/30
Inventor 查丽莎马丁·巴赫曼
Owner 深圳赫兹生命科学技术有限公司
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