Fusion protein for novel coronavirus variant strain Delta, nasal spray vaccine as well as preparation method and application thereof

A fusion protein and virus mutation technology, applied in the field of virus vaccines, can solve the problems of enhancing respiratory diseases, vaccines have poor immunogenicity, and safety cannot be guaranteed

Pending Publication Date: 2021-11-09
NANHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Vaccines with a whole virus structure cannot guarantee their safety, and may cause ineffective antibodies in the vaccinated subjects
The production of ineffective antibodies often does not have a beneficial effect on the response to viral infections and may even lead to side effects such as enhanced dependent infection and enhanced respiratory diseases
However, single-protein vaccines may not have good immunogenicity, or face the risk of failure in the face of other mutant strains of the same type

Method used

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  • Fusion protein for novel coronavirus variant strain Delta, nasal spray vaccine as well as preparation method and application thereof
  • Fusion protein for novel coronavirus variant strain Delta, nasal spray vaccine as well as preparation method and application thereof
  • Fusion protein for novel coronavirus variant strain Delta, nasal spray vaccine as well as preparation method and application thereof

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preparation example Construction

[0038] In the present invention, the preparation method of the nasal spray vaccine preferably comprises the following steps:

[0039] The recombinantly expressed fusion protein is mixed with an adjuvant to obtain colostrum, which is then mixed with PVA, washed with double distilled water, dissolved in Tris buffer, and the solvent is removed to obtain a nasal spray vaccine.

[0040] In the present invention, the adjuvant is preferably PLGA. The adjuvant is preferably mixed with the fusion protein in the form of a 50 mg / ml solution of PLGA in dichloromethane. The mass ratio of the fusion protein to PLGA is 1:12.5 according to U-type. The solvent of the fusion protein is preferably PBS solution.

[0041] The invention provides the application of the fusion protein in preparing a vaccine against the new coronavirus mutant strain Delta. The vaccine is preferably a nasal spray vaccine. The preparation method of the vaccine is the same as that described above, and will not be rep...

Embodiment 1

[0045] The preparation method of S-M-N protein vaccine

[0046] 1 Find the amino acids of S, M, and N of Delta virus in the Uniprot protein database, connect the 5' sequence of the S protein with the 3' sequence of the M protein, and the 5' sequence of the M protein and the 3' sequence of the N protein with a flexible linker. The linker is composed of glycine G and serine S (GGGGS) 3 .

[0047] 2 Digest the 5' and 3' ends of the S-M-N protein coding gene with Xhol I, connect the pET-28a plasmid with T4 ligase, transform into competent cells, and obtain the S-M-N gene expression plasmid, and obtain the stable expression by single clone screening Cell line for recombinant S-M-N protein.

[0048] 3. Expand the culture of the above cell lines, carry out secretion expression and purification, and obtain the purified recombinant Delta S-M-N protein.

[0049] 3.1 Inoculate the cell line of recombinant S-M-N protein in the bacteriolysis broth medium (LB) of 50 μ g / ml kanamycin, 37 ...

Embodiment 2

[0066] Encapsulation efficiency determination and characterization analysis observation of S-M-N vaccine

[0067] 1 Nanoparticle drug loading and encapsulation efficiency

[0068] Determination of S-M-N protein concentration using the BCA protein quantification assay

[0069] Take by weighing 10mg of the above-mentioned prepared PLGA-S-M-N nanopowder in 2ml of 0.05mol / L NaOH and 1% SDS aqueous solution, detect the amount of protein contained in the supernatant after centrifugation, calculate the drug loading by subtracting free protein from the total protein (see Formula I) and encapsulation efficiency (see formula II) are 8.2%, 63.2%, respectively.

[0070] Drug loading (LC)=(total protein-free protein) / total nanoparticles×100% formula I;

[0071] Wherein, the total protein is the total protein with a fixed mass added, and the free protein is the quality of the S-M-N protein; the total nanoparticle is the quality of the nano-encapsulated protein;

[0072] Encapsulation Eff...

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Abstract

The invention provides a fusion protein for a novel coronavirus variant strain Delta, a nasal spray vaccine as well as a preparation method and application thereof, and belongs to the technical field of virus vaccines. The fusion protein for the novel coronavirus variant strain Delta is formed by fusing spike protein S, transmembrane protein M and nucleocapsid protein N; and coding genes of the protein S, the protein M and the protein N are sequentially shown as SEQ ID NO: 1-3. The nasal spray vaccine prepared by mixing the fusion protein and an adjuvant has good immunogenicity, safety and biological activity, and can induce an organism to generate an effective antibody, so that the growth of the novel coronavirus variant strain Delta is inhibited, and the Delta infection can be effectively prevented; by adopting the form of the nasal spray vaccine, injection is not needed, the operation is simple and convenient, and adverse reactions after use of an injection vaccine are avoided; and the preparation method of the vaccine is simple, and the vaccine is easy to purify and high in safety, and can be quickly applied to clinical tests.

Description

technical field [0001] The invention belongs to the technical field of virus vaccines, and in particular relates to a fusion protein, a nasal spray vaccine and a preparation method and application thereof for the mutant strain Delta of the new coronavirus. Background technique [0002] SARS-CoV-2 (or 2019-nCoV) is the causative virus of new coronary pneumonia. The Delta virus strain is a mutated strain of the new coronavirus, which has the characteristics of significantly enhanced transmission ability, shortened incubation period and intergenerational interval, and can lead to increased disease severity. At present, there is no specific drug for the treatment of new coronary pneumonia caused by Delta variant infection. Faced with the difficulty of finding a cure, developing a vaccine has become a top priority. [0003] The existing vaccine production includes traditional vaccines (attenuated live vaccines, inactivated vaccines), subunit vaccines, virus vector vaccines, nuc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K39/215A61K39/385A61P31/14
CPCC07K14/005A61K39/12A61K39/385A61P31/14C07K2319/00C12N2770/20022C12N2770/20034A61K2039/55511A61K2039/543A61K2039/6031
Inventor 左建宏谢卓熠黄佳璐
Owner NANHUA UNIV
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