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CMV virus-like particle for producing VLP recombinant vaccine and preparation method thereof

A recombinant vaccine, virus-like technology, applied in the fields of virology, molecular biology, immunology and medicine, which can solve the problems of inability to achieve high-efficiency modification at sites, low modification efficiency of chemical linkers, and inhomogeneity.

Pending Publication Date: 2021-04-30
深圳赫兹生命科学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the efficiency of chemical linker modification is still low and uneven, and it cannot be modified with high efficiency. This solution can achieve fixed-point condensation between the target antigen and CMV through the fixed-point catalysis of sorteaseA enzyme. The catalytic efficiency is high and the process is simple. The fixed-point modification of CMV can be achieved by engineering

Method used

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  • CMV virus-like particle for producing VLP recombinant vaccine and preparation method thereof
  • CMV virus-like particle for producing VLP recombinant vaccine and preparation method thereof

Examples

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Embodiment 1

[0028] Example 1 of the present invention provides a CMV virus-like particle for the production of a VLP recombinant vaccine. The CMV virus-like particle clones the C-terminal G4SLPETG-modified plant virus cucumber mosaic virus CMV capsid gene into a prokaryotic expression vector to obtain a recombinant expression vector , the recombinant expression vector was transfected into Escherichia coli BL21(DE3), and obtained through expression of the recombinant Escherichia coli BL21(DE3); the amino acid sequence of the CMV modified with G4SLPETG at the C-terminus is SEQ ID NO.1.

Embodiment 2

[0030] see figure 1 and figure 2 Embodiment 2 of the present invention provides a method for preparing CMV virus-like particles for producing VLP recombinant vaccines, comprising the following steps:

[0031] (1) Construction of the recombinant plasmid: the CMV-G4SLPETG gene was directly synthesized by BGI into the pET28a vector NdeI restriction site and XhoI restriction site to obtain the recombinant plasmid his-TEV-CMV-G4SLPETG-pET28a;

[0032] (2) Transfer the recombinant plasmid into the expression strain: transfer the his-TEV-CMV-G4SLPETG-pET28a recombinant plasmid into the Escherichia coli BL21 (DE3) expression strain to obtain the recombinant expression strain his-TEV-CMV-G4SLPETG-pET28a-BL21;

[0033] (3) Bacterial culture and purification of his-TEV-CMV-G4SLPETG virus-like particles: culture the recombinant expression strain his-TEV-CMV-G4SLPETG-pET28a-BL21, and induce expression with IPTG to obtain his-TEV-CMV-G4SLPETG virus-like particles.

[0034] (4) Preparation ...

Embodiment 3

[0046] Example 3 of the present invention provides a VLP recombinant vaccine, which is prepared by mixing the CMV virus-like particles of Example 1 and the target antigen at a molar ratio of 1:1, and then catalyzed by Sortease A, which simplifies the production of the vaccine.

[0047] Specifically, the CMV-G4SLPETG virus-like particle in Example 1 and the target antigen modified by three glycines at the N-terminus were prepared in PBS (10 mM) to make 1 mg / ml respectively, mixed at a ratio of 1:1, and his-Sotase A5 enzyme was added, According to the proportion of 100ug catalyzed 2mg mixed protein, shake and catalyze at 37°C for 5h, then pass the catalytic solution through the nickel column, the penetration solution is CMV-G4SLPETGGG-target antigen virus-like particles, and his-Sotase A recombinase will hang on the nickel column , using a 3K concentration tube to concentrate the penetrating fluid, ie, CMV-G4SLPETGGG-target antigen virus-like particles, to obtain a VLP recombinan...

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Abstract

The invention discloses a CMV virus-like particle for producing a VLP recombinant vaccine and a preparation method thereof, the CMV virus-like particle is obtained by cloning a C-terminal G4SLPETG modified plant virus cucumber mosaic virus CMV capsid gene into a prokaryotic expression vector to obtain a recombinant expression vector, transfecting escherichia coli BL21 (DE3) with the recombinant expression vector, and expressing by the recombinant escherichia coli BL21 (DE3); the amino acid sequence of the C-terminal G4SLPETG modified CMV is shown as SEQ ID NO. 1. Tests prove that the recombinant strain constructed by the invention is stable in exogenous protein expression. The recombinant protein expressed by the invention is used for preparing virus-like particles for antigen coupling, the coupling efficiency is high, the antigen purity is high, the safety is good, no pathogenicity is caused to animals such as mice and the like, and the safety evaluation is easy to pass.

Description

technical field [0001] The present invention relates to the fields of molecular biology, virology, immunology and medicine. The invention includes a CMV virus-like particle for producing VLP recombinant vaccine, VLP recombinant vaccine and a preparation method for CMV virus-like particle for producing VLP recombinant vaccine. Background technique [0002] Plant viruses and their derived virus-like particles (VLPs) have attracted much attention, mainly due to the ability of plants to provide unique post-translational modifications and cost-effectiveness, and are an economical and rapid alternative platform for the production of VLP vaccines with high Security, production speed and scalability. [0003] Currently, although progress has been made in the development of VLP-based vaccines, further unique VLP systems are still needed. In particular, anergy occurs in the context of generally poor antibody responses in the elderly and in situations where vaccines induce variable a...

Claims

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Application Information

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IPC IPC(8): C07K14/08C12N15/70C12R1/19
CPCC07K14/005C12N15/70C12N2770/14023C12N2770/00034C12N2770/00051
Inventor 查丽莎周宇杭郑琪
Owner 深圳赫兹生命科学技术有限公司
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