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mRNA-based vaccine against coronavirus and preparation method thereof

A gene and N-terminal technology, applied in the field of vaccines against coronavirus and its preparation, can solve the problems of inability to effectively take into account virus gene mutations, low proportion of antigenic determinants, and large molecular weight of expressed proteins, so as to reduce cytotoxicity and reduce Vaccine toxicity, effect of guaranteeing immune effect

Active Publication Date: 2021-12-07
成都新诺明生物科技有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The existing new crown mRNA vaccines all use the full-length sequence of the spike protein S gene, the expressed protein has a large molecular weight, and the proportion of antigenic determinants is low. The mutation of the virus gene is not conducive to the rapid upgrading of vaccines

Method used

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  • mRNA-based vaccine against coronavirus and preparation method thereof
  • mRNA-based vaccine against coronavirus and preparation method thereof
  • mRNA-based vaccine against coronavirus and preparation method thereof

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preparation example Construction

[0060] The preparation method for coronavirus vaccine based on mRNA comprises the steps:

[0061] The step of preparing mRNA-RBD+NTD+RBD preparation comprises:

[0062] Step a, synthesizing RBD+NTD+RBD gene fragments:

[0063] Query the amino acid sequence of Spike and then design the secretion signal peptide, receptor binding domain (RBD) + N-terminal domain (NTD) + receptor binding domain (RBD) + Foldon gene sequence, and optimize the codon according to the human preferred codon table For the optimized sequence, a promoter and an ApaI restriction site GGGCCC are added upstream of the optimized sequence, and a NotI restriction site GCGGCCGC is added downstream, and the designed sequence is obtained by synthesis; the gene sequence is SEQ ID No. in the sequence list. 2 shown;

[0064] Step b, construct TC-83 self-replicating vector:

[0065] Design self-replicating mRNA sequence according to the genome of alpha virus family, described self-replicating mRNA sequence comprises...

Embodiment 1

[0087] Embodiment 1 RBD+NTD+RBD gene fragment synthesis

[0088] Query Spike (SARS-CoV-2) and British variant B.1.1.7, South African variant B.1.351, Brazilian variant P.1 and Indian variant B.1.617 protein genes from NCBI, and then design secretion signal peptide, receptor binding region (RBD)+N-terminal region (NTD)+receptor binding region (RBD)+Foldon sequence gene, and then optimize according to human codons, and add ApaI restriction site and promoter upstream, and add NotⅠ downstream Restriction sites, and finally obtain the DNA sequence directly by synthesis (obtained in the form of cloning plasmid pUC57-RBD+NTD+RBD provided by the company), (the sequence is shown in SEQ ID No.2). The encoded amino acid sequence is shown in SEQ ID No.1.

[0089]

Embodiment 2

[0090] Example 2 Construction of TC-83 self-replicating vector

[0091] The self-replicating mRNA is designed based on the genome of the Venezuelan Equine Encephalomyelitis Virus (TC83 Venezuelan Equine Encephalitis Virus, VEEV) in the alphavirus family, which contains genes that encode the self-replicating components of the alphavirus, but lacks the coding for the production of infectious The structural protein of the alpha virus particle, and the constructed plasmid is used as a template to amplify, and the designed sequence is directly obtained by synthesis (the sequence is shown in SEQIDNo.3).

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Abstract

The invention relates to the field of medicine, in particular to an mRNA (messenger ribonucleic acid)-based vaccine aiming at coronavirus and a preparation method thereof. The vaccine comprises a liposome-coated mRNA-RBD+NTD+RBD vaccine; a mRNA is designed according to the genome of Venezuela equine encephalomyelitis virus TC83 in the [alpha] virus family; the mRNA-RBD+NTD+RBD contains a gene which secretes a signal peptide and a new coronal pneumonia Spike protein gene receptor binding region (RBD)+N-terminal region (NTD)+receptor binding region (RBD)+Foldon sequence; and according to the invention, the antigen of the coronavirus can be expressed by using one mRNA, so that the immunogenicity is strong, the pain of a patient can be relieved, and the immune effect can be achieved.

Description

technical field [0001] The invention relates to the field of medicine, in particular to an mRNA-based vaccine against coronavirus and a preparation method thereof. Background technique [0002] Coronaviruses are a large family of viruses that can cause severe diseases ranging from the common cold to Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), and COVID19. Common signs of infection include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. In more severe cases, infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death. Coronavirus is a zoonotic virus that spreads between animals and humans. Detailed investigation results showed that SARS-CoV was transmitted from civet cats to humans, and MERS-CoV was transmitted from dromedary camels to humans. In addition, there are several known coronaviruses that circulate in animals but have not yet infected humans. The nove...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K39/215A61P31/14
CPCC07K14/005C12N15/85A61K39/12A61P31/14C12N2770/20022C12N2770/20034C12N2800/107C12N2800/22C07K2319/02C07K2319/00A61K2039/53Y02A50/30
Inventor 朱凤才郭志军沈海浅单爱东王猛樊钒陈守春吴强张仁怀高婧
Owner 成都新诺明生物科技有限公司
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