Novel coronavirus trimer recombinant protein, DNA, mRNA, application and mRNA vaccine

A histone and weight technology, applied in the field of genetic engineering, can solve the problems of affecting immunogenicity, slow approval for marketing, insufficient exposure of antigenic epitopes, etc., to achieve high-efficiency expression increase, prevent structural rearrangement, and strong neutralizing antibodies effect of effect

Active Publication Date: 2021-10-22
SHENZHEN RHEGEN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] For the prevention of the new coronavirus, vaccine research and development is very important. Currently, the vaccines for the new coronavirus are mainly inactivated vaccines, but the development and safety evaluation cycle of inactivated vaccines is long, and the approval for marketing is slow, which is not enough to deal with sudden and acute viral diseases The current mRNA vaccines for the new coronavirus are all mRNA products designed for the full-length S protein sequence. After being translated into the S protein, the epitope exposure is insufficient, which affects the immunogenicity

Method used

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  • Novel coronavirus trimer recombinant protein, DNA, mRNA, application and mRNA vaccine
  • Novel coronavirus trimer recombinant protein, DNA, mRNA, application and mRNA vaccine
  • Novel coronavirus trimer recombinant protein, DNA, mRNA, application and mRNA vaccine

Examples

Experimental program
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preparation example Construction

[0041] The present invention also provides a method for preparing the above-mentioned mRNA. The above-mentioned DNA is transcribed in vitro to obtain the mRNA. The nucleotide sequence of the mRNA in the present invention includes any one of the nucleotide sequences in (a) and any one of the nucleotide sequences in (b); wherein

[0042] (a): The nucleotide sequence encoding the amino acid sequence SEQ ID No.1 is preferably one of SEQ ID No.13~SEQ ID No.15; the nucleotide sequence encoding the amino acid sequence SEQ ID No.2 is preferably SEQ One of ID No.16~SEQ IDNo.18; the nucleotide sequence encoding amino acid sequence SEQ ID No.3 is preferably one of SEQ ID No.19~SEQ IDNo.21; encoding amino acid sequence SEQ ID No The nucleotide sequence of .4 is preferably one of SEQ ID No.22~SEQ IDNo.24; the nucleotide sequence of encoding amino acid sequence SEQ ID No.5 is preferably among SEQ ID No.25~SEQ IDNo.27 kind of. (b): The nucleotide sequence encoding the amino acid sequence S...

Embodiment 1

[0055] Embodiment 1 mRNA vaccine preparation method

[0056] 1. The constructed expression plasmid is amplified as the DNA template according to the following reaction system:

[0057] Reaction volume, 50μl (reaction volume of a single tube, multiple tubes can be reacted at the same time)

[0058] Premix (2×) PCR amplification system (50 μl): PrimeSTAR Max 25 μl, Substance F (SEQ ID No.5, 10 μmol / L) 1.2 μl, Primer R (SEQ ID No.5, 10 μmol / L) 1.2 μl , DNA template (1ng / μl) 1μl and water 21.6μl.

[0059] The PCR amplification program is as follows: pre-denaturation at 98°C for 3min; denaturation at 98°C for 10s, annealing at 60°C for 5s, extension at 72°C for 2min, 34 cycles; final extension at 72°C for 10min.

[0060] After the reaction, the reaction solution was combined into a 1.5ml Tube tube. Take 10 μl for DNA agarose gel electrophoresis (1.5% agarose, 5V / min, 40min). Confirm the success of the reaction according to the size of the electrophoresis target band.

[0061] ...

Embodiment 2

[0110] Example 2 Comparative test of full-length S protein mRNA vaccine and second-generation NTD-RBD mRNA vaccine

[0111] Select 6-week-old balb / c mice, and inoculate 50ug, 100ug, and 200ug of NTD-RBD mRNA vaccine and S protein full-length mRNA vaccine respectively, and inject twice on the 1st and 14th day, respectively, and take small mice on the 35th day. Rat serum was used to detect the titer of anti-S protein-specific antibody in the serum.

[0112] 1. Coating: Dilute S1 protein (Beijing Sino Biological Science and Technology Co., Ltd., 40591-MM43) with coating buffer to a solution of 200ng / ml, add it to the microtiter plate, the volume added to each well is 100μl, each dilution Repeat for 3 wells, cover with sealing film, and coat overnight at 4°C.

[0113] 2. Plate washing: Pour out the coating liquid on the coated 96-well plate, put it on the buckle absorbent paper, and buckle the plate hard until there is no residue in the well. Prepare the eluent, dilute 50× Washi...

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Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a novel coronavirus trimer recombinant protein, DNA, mRNA, application and an mRNA vaccine. The invention provides a trimer recombinant protein of an SARS-CoV-2 virus antigen NTD-RBD, and the trimer recombinant protein can be used for synthesizing mRNA for coding a virus antigen fragment so as to realize the immunization on a novel coronavirus. According to the trimer recombinant protein disclosed by the invention, full exposure of antigen epitopes and high-efficiency expression of human cells can be realized, and immunogenicity can be improved; and the mRNA of the coding virus antigen fragment is synthesized by an in-vitro transcription method so as to immunize the novel coronavirus. The whole mRNA vaccine is short in production cycle, simple in process operation, low in production cost, long in preservation time, free of a cold chain and convenient to transport.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a new coronavirus trimeric recombinant protein, DNA, mRNA and its application and mRNA vaccine. Background technique [0002] Viruses of the genus Coronavirus widely exist in nature and have a wide range of hosts. They can infect humans and a variety of animals. They are divided into four groups: α, β, γ and δ. According to the evolutionary analysis of the existing genome sequence, SARS-CoV-2 belongs to the genus Betacoronavirus. Like other coronaviruses, its genome is about 30kb in size and is a single-stranded positive-strand RNA that contains six open reading frames (open reading frame, ORF), which encode non-structural proteins, structural proteins and some auxiliary proteins of the virus. The structural proteins of the virus consist of the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The viral genome of SARS-CoV-2 has 79.5% s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/215A61P31/14
CPCC07K14/005A61K39/12A61P31/14C12N2770/20022C07K2319/02C12N2770/20034A61K2039/53
Inventor 胡勇洪丹胡迅
Owner SHENZHEN RHEGEN BIOTECHNOLOGY CO LTD
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