Recombinant enterovirus A71 virus-like particle and application thereof

An enterovirus, virus-like technology, applied in the field of medicine, can solve the problems of low VLP yield, unfavorable industrial production, low yield, etc., and achieve the effect of good morphology and immunogenicity

Pending Publication Date: 2022-08-02
华淞(上海)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, the yield of VLP obtained by co-expressing P1 and 3CD is low, which is not conducive to industrial production, so it is urgent to increase the yield of VLP

Method used

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  • Recombinant enterovirus A71 virus-like particle and application thereof
  • Recombinant enterovirus A71 virus-like particle and application thereof
  • Recombinant enterovirus A71 virus-like particle and application thereof

Examples

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preparation example Construction

[0036] The present invention also provides a preparation method of the recombinant enterovirus A71 virus-like particle, the preparation method comprising the following steps:

[0037] 1) culturing the cell line to express recombinant enterovirus A71 virus-like particles;

[0038] 2) The recombinant enterovirus A71 virus-like particles expressed by the cell line were isolated.

[0039] In one embodiment, the conditions for culturing the cell line are 28°C-30°C, 250-300 rpm.

[0040]In one embodiment, the cell line is obtained by transduction of the nucleic acid construct into a host cell. In one embodiment, the host cell is a Pichia cell.

[0041] In one embodiment, the preparation method of the nucleic acid construct comprises the steps:

[0042] 1) Clone the nucleotides expressing protease 3CD and the codon-optimized nucleotides expressing the enterovirus A71 P1 structural protein into different expression vectors to obtain an intermediate construct;

[0043] 2) Recombini...

Embodiment 1

[0060] Example 1 Construction of enterovirus A71 expression plasmid

[0061] In order to optimize the expression, the amino acid sequence of the enterovirus A71 structural protein P1 was optimized and synthesized according to the codon preference of Pichia pastoris, and was linked into the pPinK-HC plasmid to obtain the plasmid pPink / HC-A71 P1. The amino acid sequence of enterovirus A71 structural protein P1 is shown in SEQ ID NO: 1, and the optimized nucleotide sequence is shown in SEQ ID NO: 2.

[0062] Enterovirus A71 P1 homologous 3CD (named 3CD -O or A71 3CD -O , the amino acid sequence is shown in SEQ ID NO: 3) and P1 non-homologous 3CD (named 3CD -N or A71 3CD -N , the amino acid sequence is shown in SEQ ID NO: 4) the nucleotide sequence is synthesized and linked into the expression vector pPink-HC vector (completed by a third-party company) to obtain plasmid pPink / HC-A71 3CD -O and pPink / HC-A71 3CD -N . 3CD -O (A71 3CD -O ) and 3CD -N (A71 3CD -N ) nucleotide...

Embodiment 2

[0064] Example 2 Screening, expression and purification of enterovirus A71 high-expressing strains

[0065] Screening of high expressing strains

[0066] The final plasmid pPink / HC-A71 3CD prepared in Example 1 -O -P1 or pPink / HC-A71 3CD -N -P1 were linearized with endonuclease AflII, purified and recovered by ethanol precipitation; the linearized plasmids were introduced into different Pichia pastoris by electroporation for gene recombination, coated on PAD plates and cultured at 30°C; After 3 days, pick large white colonies in a 24-well deep-well plate and use BMMY medium for methanol-induced expression (28°C, 250rpm). A71 VLP -O of two high-expressing strains, as well as expressing A71 VLPs -N The two high expression strains of each high expression strain are consistent with the theoretical nucleotide sequence after sequencing analysis.

[0067] The rabbit anti-enterovirus A71 multi-antibody serum and the enterovirus A71-specific mouse monoclonal antibody used in the s...

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Abstract

The invention relates to the field of medicine, in particular to recombinant enterovirus A71 virus-like particles and application thereof.The recombinant enterovirus A71 virus-like particles comprise P1 structural protein and protease 3CD, and the P1 structural protein and the protease 3CD are derived from different recombinant enterovirus A71 virus strains. The invention also provides application of the recombinant enterovirus A71 virus-like particle in preparation of products for preventing hand-foot-and-mouth disease. The product for preventing the hand-foot-and-mouth disease is a pharmaceutical composition, such as a vaccine composition. The expression quantity of the A71 virus-like particles is high, the VLP obtained through purification can induce good immunogenicity, and the A71 virus-like particles can be used as candidate vaccines of the enterovirus A71 for industrial production.

Description

technical field [0001] The invention relates to the field of medicine, in particular to recombinant enterovirus A71 virus-like particles and uses thereof. Background technique [0002] Hand, foot and mouth disease is a common infectious disease of infants and young children under the age of 5. The main clinical manifestations are small herpes or small ulcers on the hands, feet and mouth. A series of complications such as myocarditis even lead to death. Hand, foot and mouth disease is caused by human enterovirus coxsackievirus A group 16, 4, 5, 7, 9, 10 and B group 2, 5 infection, as well as enterovirus 71 and echovirus 30 infection, Among them, enterovirus A71 and coxsackie virus A16 are the main pathogens causing the outbreak of hand, foot and mouth disease. At present, there is no specific treatment drug for enterovirus A71, so prevention has become an effective way to deal with hand, foot and mouth disease caused by enterovirus A71. Vaccines are the most effective way ...

Claims

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Application Information

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IPC IPC(8): C12N15/41C12N15/57C12N15/81C12N1/19A61K39/125C12R1/84
CPCC07K14/005C12N9/506C12N15/81A61K39/12A61P31/14C12R2001/84C12N2770/32322C12N2770/32323C12N2770/32333C12N2770/32334C12N2770/32351A61K2039/5258Y02A50/30
Inventor 刘庆伟王晓黎刘艳石娜张玺边金秦松程飞杨义
Owner 华淞(上海)生物医药科技有限公司
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