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Corona Virus Disease 2019 (COVID-19) DNA vaccine and preparation method and application thereof

A technology of DNA vaccines and coronaviruses, applied in the field of vaccines, can solve problems such as the long process of vaccine development, achieve the effect of maintaining functionality and antigenicity, and increasing expression levels

Inactive Publication Date: 2020-08-25
吉林省中科生物工程股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Relatively speaking, the development process of such vaccines is lengthy

Method used

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  • Corona Virus Disease 2019 (COVID-19) DNA vaccine and preparation method and application thereof
  • Corona Virus Disease 2019 (COVID-19) DNA vaccine and preparation method and application thereof
  • Corona Virus Disease 2019 (COVID-19) DNA vaccine and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of plasmid pVAX1-RBD△N

[0039] (1) Acquisition of RBD△N gene

[0040] ①Template preparation

[0041] The commercialized novel coronavirus pneumonia virus S gene full-length plasmid pUC-57-S produced by Shanghai Shenggong Company was used as a template for PCR amplification.

[0042] ②Primer design

[0043]Using the primer design software, refer to the relevant sequences registered in Genbank, and design primers as shown in Table 1, that is, to amplify the RBD△N gene from pUC-57-S.

[0044] Table 1 Design primer sequence

[0045] Primer name sequence upstream primer GGGAGACCCAAGCTGGCTAGCGCCACCATGATTACAAACTTGTGCCCTTTTG downstream primer GGTTTAAACGGGCCCCTTAGATTAACCTGTGCCTGTTAAACCATT

[0046] ③PCR reaction

[0047] Use NEB company Q5 high-fidelity DNA polymerase to amplify RBD△N gene, add 5×Q5Buffer 10 μL, dNTP Mixture (2.5mM) 8 μL, upstream primer 1 μL, downstream primer 1 μL, plasmid 1 μL, Q5 enzyme 0.5 μL accordi...

Embodiment 2

[0051] Example 2 Identification of New Coronavirus DNA Recombinants

[0052] (1) Enzyme digestion and identification of recombinant DNA of the new coronavirus

[0053] The ligation product was transformed into Escherichia coli competent cells, 200 μL was spread on LB agar plate, and cultured overnight at 37°C. Pick a single colony and inoculate it in 2 mL of LB culture medium containing corresponding antibiotics, and prepare a small amount of plasmid DNA. The recombinant plasmid was digested with restriction endonucleases Nhe I and Xba I, and analyzed by agarose gel electrophoresis. Select those whose digestion results are the same as expected for further identification, and those whose digestion results are exactly the same as expected are positive recombinant plasmids, such as figure 2 shown.

[0054] (2) New coronavirus DNA recombinant transfected cells

[0055] ① Cell count

[0056] Take 0.5mL of cell suspension, add 0.5mL of trypan blue solution, shake fully, place ...

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Abstract

The invention discloses a Corona Virus Disease 2019 (COVID-19) DNA vaccine and a preparation method and application thereof. According to the DNA vaccine, glycosylated asparaginate on an N-1 site of aconventional Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Receptor Binding Domain (RBD) sequence is deleted, an expression level can be improved, and functionality and antigenicity ofthe DNA vaccine are maintained. RBD triangle N can trigger a high-titer neutralizing antibody for pseudoviruses and live viruses. Meanwhile, an encoding protein uses pVAX1 as vector skeleton molecules, and front and rear base sequences of a gene initial codon ATG are designed and regulated so as to conform to the Kozak rule; and a recombinant of the DNA vaccine transfects 293 cells, and RT-PCR and Western Blot detection shows that a target protein is successfully expressed and an expression product can be identified by standard serum.

Description

technical field [0001] The present invention relates to the technical field of vaccines, in particular to a novel coronavirus pneumonia DNA vaccine and its preparation method and application. Background technique [0002] Novel coronavirus disease 2019 (COVID-19), referred to as "new crown pneumonia", is an acute respiratory infectious disease caused by a new type of coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2). The novel coronavirus pneumonia still poses a serious threat to the life and health of all mankind, and has also had a huge impact on the development of the global economy. [0003] Novel coronavirus pneumonia can be transmitted through droplets and close contact without protection. There is no report of air transmission, but there may be the possibility of air transmission due to aerosols produced by medical operations. Latitude and temperature had little to no association with the spread of COVID-19, and humidity was also weakly associ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/215A61P31/14A61P11/00C12N15/85C12N15/50
CPCA61K9/0019A61K39/12A61K2039/53A61K2039/54A61P11/00A61P31/14C07K14/005C12N15/85C12N2770/20022C12N2770/20034C12N2800/107
Inventor 梁磊宿晓云刘天戟董欣
Owner 吉林省中科生物工程股份有限公司
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