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Novel coxsackie virus group A 6 recombinant subunit protein vaccine and preparation method thereof

A coxsackie virus and protein vaccine technology, applied in the field of medical biology, can solve problems such as high cost and limited production capacity, and achieve high production capacity, reduced production cost, and low cost

Active Publication Date: 2020-04-14
ZHEJIANG PUKANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because this preparation method involves the cultivation of stromal cells for production, the cost is relatively high, and the production capacity is also greatly limited.

Method used

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  • Novel coxsackie virus group A 6 recombinant subunit protein vaccine and preparation method thereof
  • Novel coxsackie virus group A 6 recombinant subunit protein vaccine and preparation method thereof
  • Novel coxsackie virus group A 6 recombinant subunit protein vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Expression of Coxsackievirus Group A Type 6 Recombinant Subunit Protein CRM197-CVA6VP1

[0025] (1) Utilizing the codon merger principle and the codon usage frequency of Escherichia coli, using the amino acid sequence described in SEQ ID NO.1 as a template, designing the nucleotide sequence SEQ ID NO.2 expressing the CRM197-CVA6VP1 recombinant protein, The synthesized gene was inserted into pET28a Escherichia coli expression plasmid through two restriction sites of XhoI and NcoI to obtain a recombinant expression plasmid. The resulting recombinant expression plasmid was transferred into E. coli BL21 (DE3) competent cells by 42-degree heat activation method, and monoclonal screening was carried out through LB solid medium plates to obtain recombinant E. coli monoclonal colonies expressing the target protein, and pick the recombinant Escherichia coli monoclonal colonies were cultured overnight at 37°C in 5 ml of LB liquid medium to obtain recombinant E. coli see...

Embodiment 2

[0031] Example 2 Preparation of Coxsackievirus Group A Type 6 Recombinant Subunit Protein Vaccine

[0032](1) Utilizing the codon merger principle and the codon usage frequency of Escherichia coli, using the amino acid sequence described in SEQ ID NO.1 as a template, designing the nucleotide sequence SEQ ID NO.2 expressing the CRM197-CVA6VP1 recombinant protein, And at the 5' end of the nucleotide sequence, a histidine tag atgcatcacc atcatcaccac was introduced, and the gene sequence SEQ ID NO.3 was synthesized by artificial gene synthesis technology to facilitate subsequent purification, and the synthesized gene sequence was synthesized by XhoI and NcoI restriction sites. The gene was inserted into pET28a Escherichia coli expression plasmid to obtain a recombinant expression plasmid. The resulting recombinant expression was transformed into E. coli BL21 (DE3) competent cells by 42-degree heat activation method, and monoclonal screening was performed on LB solid medium plates t...

Embodiment 3

[0044] Example 3 Coxsackievirus Group A Type 6 Recombinant Subunit Protein Vaccine Induces the Body to Produce Neutralizing Antibodies

[0045] (1) Using the CVA6VP1 protein sequence as a template, design the nucleotide sequence SEQ ID NO.4 for expressing the CVA6VP1 protein. A separate CVA6VP1 recombinant protein vaccine was prepared according to the preparation method of Example 2;

[0046] (2) Take 30 4-week-old long-clawed gerbils and divide them into three groups: a test group, a separate CVA6VP1 recombinant protein group and a control group, 10 in each group, and the test group is immunized with Coxsackie virus A group type 6 recombinant subunit protein Vaccine, the immunization dose is 20ug / rat; the CVA6VP1 recombinant protein group alone is immunized with the CVA6VP1 recombinant protein vaccine alone, the immunization dose is 20ug / rat; the control group is injected with the same amount of phase-changing solution plus aluminum adjuvant. A booster immunization was given...

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Abstract

The invention provides a novel coxsackie virus group A 6 recombinant subunit protein vaccine and a preparation method thereof. The invention relates to the novel coxsackie virus group A 6 recombinantsubunit protein vaccine which is artificially designed. The vaccine comprises a recombinant protein formed by fusion design of a diphtheria toxin non-toxic mutant CRM197 protein with an immunologic adjuvant effect and a CV-A6 antigen structural protein. The recombinant protein is subjected to escherichia coli expression, chromatographic column separation, purification and compatibility, and finally the recombinant subunit protein vaccine with good immunogenicity is prepared. The vaccine can effectively activate an organism to generate a neutralizing antibody aiming at the coxsackie virus groupA 6, and can effectively prevent the infection of the coxsackie virus group A 6 on an organism. The vaccine disclosed by the invention realizes efficient expression of the target protein in an escherichia coli expression system by carrying out password liberation on an expression gene, so that the preparation efficiency is greatly improved, the production cost is reduced, and a foundation is laidfor popularization and immunization of large-scale crowds.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, in particular to a method for preparing a novel Coxsackievirus group A type 6 recombinant subunit protein vaccine. Background technique [0002] Hand, foot and mouth disease (HFMD) is a common infectious disease caused by a variety of enteroviruses. Most patients only manifested as fever or herpes and rashes in the mouth, hands, and feet. Complications leading to death. Hand, foot and mouth disease can infect children of different age groups, mainly children under 5 years old, especially children under 3 years old, accounting for 85%-95% of the number of cases. In recent years, hand, foot and mouth disease has broken out and become popular in many parts of the world. Hand, foot and mouth disease has been reported in most provinces in my country, and a few provinces have reported outbreaks and reported deaths, and the number of cases is increasing year by year. Therefore, in 2008, my countr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/125A61K39/39A61P31/14C12N15/70C12N15/62C07K19/00
CPCA61K39/12A61K39/39A61P31/14C12N15/70C07K14/005A61K2039/55505C12N2800/22C12N2770/32022C12N2770/32034C07K2319/55Y02A50/30
Inventor 高孟沈钱通杨宏宏庄昉成毛子安
Owner ZHEJIANG PUKANG BIOTECH
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