Enteroviral chimeric virus-like particle vaccine and preparation method and application thereof

An enterovirus and chimeric virus technology, applied in the biological field, can solve problems such as weak immune response

Active Publication Date: 2016-04-06
河南杰瑞生物科技研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although DNA vaccine studies of EV71 have been reported, DNA vaccin

Method used

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  • Enteroviral chimeric virus-like particle vaccine and preparation method and application thereof
  • Enteroviral chimeric virus-like particle vaccine and preparation method and application thereof
  • Enteroviral chimeric virus-like particle vaccine and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0046] The construction of embodiment 1 enterovirus chimeric virus-like particle (VLPs) vaccine

[0047] 1. Synthesis of fusion fragment tHBc-SPA

[0048] The present invention obtains the fusion fragment tHBc-SPA through codon optimization and experimental screening. The fusion fragment tHBc-SPA is composed of nucleotides encoding 1-144aa (1-74aa and 82-144aa) of HBc and 208-222aa of EV71VP1 protein, 271-285aa of CA16VP1 protein and 248-263aa of EV71VP2 protein. Among them, 208-222aa of EV71VP1 protein and 271-285aa of CA16VP1 protein are linked by 9 amino acids (GGGGSGGGG) and the same 9 amino acids are introduced at both ends of the peptide to replace 75-81aa of HBc, and 248-285aa of EV71VP2 protein. 263aa is at the C-terminus of HBc1-144aa. The nucleotide sequence of the fusion fragment tHBc-SPA is shown in SEQ ID NO.2; the amino acid encoded by the fusion fragment tHBc-SPA can self-assemble into virus-like particles, and its amino acid sequence is shown in SEQ ID NO.1. ...

Embodiment 2

[0144] Example 2 Large-scale preparation and identification of enterovirus chimeric virus-like particle vaccine

[0145] 1. Large-scale preparation of enterovirus chimeric virus-like particle vaccine

[0146] 1) Large-scale induced expression of recombinant antigenic protein tHBc-SPA or tHBc

[0147] (1) Take the frozen engineered bacteria out of the -80°C refrigerator, thaw, transfer 0.1mL of the bacteria liquid to 5mL of LB medium (containing kanamycin and chloramphenicol), and rotate overnight at 37°C and 220rpm .

[0148] (2) The overnight culture was inoculated into 1 LLB medium containing kanamycin and chloramphenicol at a ratio of 1:100, and cultured with shaking at 37°C and 220rpm. To be cultured to the logarithmic growth phase (bacterial solution OD 600 value reached 0.6), IPTG was added to a final concentration of 0.2 mM, 37 ° C, 220 rpm rotating culture for 5 hours to induce protein expression.

[0149] (3) Transfer the induced culture to a 250 mL centrifuge tub...

Embodiment 3

[0173] Example 3 Functional Detection of Enterovirus Chimeric Virus-like Particles (VLPs) Vaccine

[0174] Experimental protocol

[0175] Seventy-eight female BALB / c mice aged 6-8 weeks were randomly divided into groups A, B, C, and D, with 13 mice in each group. Blood was collected from the tail of the mice before vaccination, and the serum was separated for antibody detection. The immunizations were designed according to the following: group A was injected with UV-inactivated EV71 (10 μg / monkey); group B was injected with tHBc-SPA (VLPs) at a dose of 10 μg / bird; The control group was PBS; immunized by intraperitoneal injection, each vaccine was immunized three times with an interval of two weeks; two weeks after the third immunization, blood was collected by docking the tail, and the serum was separated. After vaccination, the mice in each group were observed and weighed every day. Compared with the mice in the PBS-injected control group, the mice in the tHBc-SPA vaccine in...

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Abstract

The invention discloses an enteroviral chimeric virus-like particle vaccine and a preparation method and application thereof and belongs to the field of biotechnologies. Chimeric virus-like particles are hepatitis b virus core protein based recombinant enteroviral multi-epitope chimeric antigen protein, and the amino acid sequence of the particles is shown as SEQ ID NO.1. The nucleotide sequence of a DNA fragment encoding the virus-like particles is shown as SEQ ID NO.2. The DNA fragment shown as SEQ ID NO.2 is cloned to an escherichia coli expression vector to establish recombinant expression plasmids. The recombinant expression plasmids are converted into escherichia coli to obtain engineering bacteria. The engineering bacteria are cultured, and enteroviral chimeric virus-like particles are obtained through the operations, such as inducing expression and protein purification. The virus-like particles can induce a body to produce specific body fluid and cellular immune response, can be used for immune prevention of enterovirus EV71 and CA16 infection of people and susceptible animals and is used for preparation of the enteroviral vaccine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an enterovirus chimeric virus-like particle vaccine and its preparation method and application. Background technique [0002] Hand, foot and mouth disease (HFMD) is a viral infectious disease caused by enterovirus infection, mainly affecting infants and young children. It is characterized by fever, rashes and ulcers on the hands, feet, mouth and other parts. A small number of patients can cause severe complications such as myocarditis, pulmonary edema, and aseptic meningoencephalitis, leading to death (Lee et al., 2009; Liu et al., 2014). The main pathogens of HFMD are Enterovirus 71 (Enterovirus71, EV71) and Coxsackie A16 (CoxsackieA16, CA16). EV71 infection often causes severe central nervous system diseases and complications, while CA16 mainly causes myocarditis and pericarditis (Lee et al., 2009). In recent years, the outbreak of HFMD in my country has shown an inc...

Claims

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Application Information

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IPC IPC(8): C12N7/04A61K39/125A61P31/14G01N33/569C12N15/41C12N15/70C12R1/93
CPCA61K39/12A61K2039/523A61K2039/5258A61K2039/575C07K14/005C12N7/00C12N2770/32322C12N2770/32323C12N2770/32334C12N2770/32351G01N33/56983
Inventor 潘兹书霍春玲杨洁
Owner 河南杰瑞生物科技研究院有限公司
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