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Expression vector of novel coronavirus vaccine, construction method and application of expression vector and vaccine

An expression vector, virus vaccine technology, applied in the direction of virus/phage, application, vaccine, etc., can solve the problems of fast mutation speed of coronavirus RNA virus, limited protection effect, complicated fire extinguishing vaccine process, etc., to induce humoral immunity and cellular immunity, The effect of strong immune protection

Pending Publication Date: 2021-07-30
浙江格源致臻生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex process of preparing fire-fighting vaccines and the high requirements for the production environment
In addition, more importantly, due to the risk of infection of inactivated vaccines, and the rapid mutation of the coronavirus itself, its protective effect is limited, and it can only play a preventive role, and has no effect on asymptomatic infections.

Method used

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  • Expression vector of novel coronavirus vaccine, construction method and application of expression vector and vaccine
  • Expression vector of novel coronavirus vaccine, construction method and application of expression vector and vaccine
  • Expression vector of novel coronavirus vaccine, construction method and application of expression vector and vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] as attached figure 1As shown, this embodiment provides a method for constructing an expression vector of a new coronavirus vaccine, which includes the following steps:

[0034] S1. Link the nucleotide sequence expressing the new coronavirus S protein and the NP protein with a 2A peptide to synthesize a fusion gene; wherein, the nucleotide sequence expressing the S protein is: , specifically as shown in the sequence table SEQ ID NO:4 The nucleotide sequence of the expressed NP protein is: ATGCGGGTCACGGCACCCCGAACCCTGATCCTGCTGCTCTCGGGGGCCCTGGCCCTGACCGAGACCTGGGCCGGCTCCATGTCTGATAATGGACCCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAA CCAGAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCCAAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGACCTTAAATTCCCTCGAGGACAAGGCGTTCCAATTAACACCAATAGCAGTCCAGATGACCAAATTGGCTACTACCGAAGAGCTACCAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGA...

Embodiment 2

[0040] This embodiment provides a method for constructing an expression vector of a new coronavirus vaccine, which includes the following steps:

[0041] S1, linking the nucleotide sequence (as shown in SEQ ID NO: 5) of expressing the new coronavirus S protein (as shown in the sequence table SEQ ID NO: 4) and the NP protein using a 2A peptide to synthesize a fusion gene; Wherein, the amino acid sequence of the 2A peptide is: GSGEGRGSLLTCGDVEENPGP, as shown in SEQ ID NO: 2 in the sequence table.

[0042] S2. Contain XbaI and SalI restriction sites at both ends of the fusion gene, and load them into the pcDNA3.1+ plasmid to obtain a recombinant plasmid.

[0043] S3. Using XbaI and SalI endonucleases to perform double enzyme digestion on the above-mentioned recombinant plasmid, and cut the gel to recover the target gene fragment.

[0044] S4. The original pcDNA3.1+ plasmid is double digested with XbaI and SalI endonucleases, and the vector fragment is recovered by cutting the ge...

Embodiment 3

[0053] This example is used to compare the antibody-inducing ability of the expression vectors obtained in the above-mentioned Example 1 and Comparative Example 1, which specifically includes the following steps:

[0054] 1. Preparation of RNA vaccine by in vitro transcription of expression vector:

[0055] (1) Use Xhol enzyme plasmid linearization;

[0056] (2) Use a DNA purification kit to recover linearized DNA;

[0057] (3) Use an in vitro transcription kit to transcribe RNA in vitro;

[0058] (4) Use the RNA purification kit to purify and recover the RNA to obtain the S protein and NP protein RNA vaccines prepared by the expression vectors obtained in Example 1, and the S protein RNA vaccines prepared by the expression vectors obtained in Comparative Example 1.

[0059] 2. The acquisition of mouse DC:

[0060](1) 6-10 week mice were sacrificed by cervical dislocation, and all femurs and tibias were surgically removed, and the muscular tissues around the bones were remo...

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Abstract

The invention is applicable to the technical field of biology, and provides an expression vector of a new coronavirus vaccine, a construction method and application of the expression vector and the vaccine, and the construction method of the expression vector comprises the following steps: connecting nucleotide sequences for expressing S protein and NP protein of a new coronavirus by using 2A peptide to synthesize a fusion gene; two ends of the fusion gene respectively comprise two restriction enzyme cutting sites, and the fusion gene is loaded to a plasmid to obtain a recombinant plasmid; carrying out double enzyme digestion on the recombinant plasmid, and carrying out gel cutting to recover a target gene segment; carrying out double enzyme digestion on original plasmids, and carrying out gel cutting to recover carrier fragments; and connecting the target gene fragment with the vector fragment to obtain the expression vector. According to the embodiment of the invention, the coronavirus S protein receptor binding region and the NP protein are expressed at the same time, so that cells infected by the expression vector not only can induce antibody reaction, but also can induce T cell reaction, humoral immunity and cellular immunity are effectively induced, and stronger immune protection is provided for subjects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an expression vector of a novel coronavirus vaccine, its construction method, application and vaccine. Background technique [0002] The biggest problem facing global public health at present is emerging and re-emerging infectious diseases. Coronaviruses are such a class of infectious viruses that can cause serious diseases. Coronaviruses are widely found in humans, mammals, and birds. They are enveloped RNA viruses that can cause respiratory, intestinal, liver, and nervous system diseases. . The deadly zoonotic viruses among the current strains of coronaviruses are severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome virus (MERS-CoV). The explosive and severe infectious disease caused by coronavirus has the characteristics of strong infectivity, long incubation period, variable symptoms, and fatality, which has caused a heavy bl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/50C12N15/66C07K14/165A61K39/215A61P31/14
CPCA61K39/12A61K2039/53A61P31/14C07K14/005C12N15/66C12N15/85C12N2770/20022C12N2770/20034
Inventor 苏小平李锐李伟迎张齐
Owner 浙江格源致臻生物医药科技有限公司
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