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Recombinant coxsackie virus A10 VLP and application thereof

A coxsackie virus, virus-like technology, applied in the field of medicine, can solve problems affecting the uniformity of VLP

Active 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 VP0 of the prepared VLP will be degraded to different degrees to produce VP4 and VP2, which will affect the uniformity of VLP

Method used

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  • Recombinant coxsackie virus A10 VLP and application thereof
  • Recombinant coxsackie virus A10 VLP and application thereof
  • Recombinant coxsackie virus A10 VLP and application thereof

Examples

Experimental program
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preparation example Construction

[0054] The present invention also provides a method for preparing the recombinant Coxsackie virus A10 virus-like particle, which includes the following steps:

[0055] 1) culturing the cell line to express recombinant Coxsackie virus A10 virus-like particles;

[0056] 2) The recombinant Coxsackie virus A10 virus-like particles expressed by the cell line were isolated.

[0057] In one embodiment, the conditions for culturing the cell line are 28°C-30°C, 250-300 rpm.

[0058] 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.

[0059] In one embodiment, the preparation method of the nucleic acid construct comprises the steps:

[0060] 1) clone the nucleotides that express the Coxsackie virus A10 capsid protein after codon optimization into different expression vectors respectively to obtain an intermediate construct;

[0061] 2) Recombining the intermediate construct ...

Embodiment 1

[0078] Embodiment 1 Coxsackie virus A10 expression plasmid pPink / HC-A10 VPN -231 build

[0079] In order to optimize the expression, the amino acid sequence of Coxsackievirus A10 structural protein P1 was optimized and synthesized according to the codon preference of Pichia pastoris. Coxsackie virus A10 structural protein P1 amino acid sequence is shown in SEQ ID NO:1, wherein 1-69 are VP4 amino acid sequence (SEQ ID NO:3), 70-324 are VP2 amino acid sequence (SEQ ID NO:4), 325-564 are the VP3 amino acid sequence (SEQ ID NO:5) and 565-862 are the VP1 amino acid sequence (SEQ ID NO:6). The optimized nucleotide sequence is shown in SEQ ID NO:2. The nucleotide sequence of VP4 is shown in SEQ ID NO:7, ie, 1-207 in SEQ ID NO:2. The nucleotide sequence of VP2 is shown in SEQ ID NO:8, ie, 208-972 in SEQ ID NO:2. The nucleotide sequence of VP3 is shown in SEQ ID NO:9, ie, 973-1692 in SEQ ID NO:2. The nucleotide sequence of VP1 is shown in SEQ ID NO:10, ie, 1693-2586 in SEQ ID NO:2...

Embodiment 2

[0083] Example 2 Screening, expression and purification of Coxsackievirus A10 high expression strains

[0084] Screening of high expressing strains

[0085] The final plasmid pPink / HC-A10 VPN prepared in Example 1 -231 The endonuclease AflII was used for linearization, and ethanol precipitation was used for purification and recovery; the linearized plasmids were introduced into different Pichia pastoris for gene recombination by electroporation, 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 to induce expression with methanol (28°C, 250rpm). After inducing expression for 48 hours, collect the induced cells and use nutrient screening method and sandwich ELISA for expression detection. The high amount is used as the high expression strain A10 VLP -23l , the target gene sequence of the high-expressing strain was consistent with the theoretical nucleotide sequence after sequencing analysis.

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Abstract

The invention relates to the field of medicine, in particular to a recombinant coxsackie virus A10VLP and application thereof, and the recombinant coxsackie virus A10VLP is generated by a cell line with capsid proteins VP2, VP3 and VP1 for coding the coxsackie virus A10 integrated in a genome, or is generated by a cell line with capsid proteins VP4, VP2, VP3 and VP1 for coding the coxsackie virus A10 integrated in a genome. The invention also provides an application of the recombinant coxsackie virus A10VLP in preparation of a product 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. Immunogenicity research finds that the coxsackie virus A10VLP disclosed by the invention can avoid the degradation problem of VP0 and can induce good immunoreaction in a mouse body, so that the VLP can be used as a candidate vaccine of the coxsackie virus A10.

Description

technical field [0001] The invention relates to the field of medicine, in particular to recombinant Coxsackie virus A10 VLP and use 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 small number of children can cause pulmonary edema, aseptic meningoencephalitis, 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, B group 2, 5, as well as enterovirus 71 and echovirus 30 infection. Among them, enterovirus 71 and coxsackie virus A16 are the main pathogens causing the outbreak of hand, foot and mouth disease. Reports of infection by coxsackievirus A10 have become more common in recent years. There is currently no specific drug against Coxsackievirus A10, so vaccines a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/41C12N15/81C12N1/19C07K14/08A61K39/125A61P31/14C12R1/84
CPCC07K14/005C12N15/815A61K39/12A61P31/14C12N2770/32622C12N2770/32623C12N2770/32634C12N2800/102Y02A50/30
Inventor 刘庆伟王晓黎刘艳石娜张玺边金秦松赵胜涛阮丽珠
Owner 华淞(上海)生物医药科技有限公司
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