Method for preparing enterovirus virus-like particle and application thereof

A virus and baculovirus technology, which is applied in the preparation of enterovirus-like particles and its application field, can solve the problems of inability to completely eliminate and purify bottlenecks and the like

Active Publication Date: 2010-12-29
胡育诚
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

(3) Recombinant baculovirus itself has enough genetic information to infect cells and reproduce, so it does not require the existence of satellite cells or satellite viruses (helper cell lines or helper viruses). In comparison, it is more widely used in subsequent applications. For example, the production of adeno-associated virus often requires the presence of adenovirus, causing some bottlenecks in the purification of AAV
Currently commonly used traditional vaccines include deactivated vaccines and attenuated vaccines. Because the method is to deactivate the epitope of the real virus or reduce its toxicity by external force, the genetic material of the virus still exists in the vaccine, so it cannot be completely Avoid the chance of being infected after vaccination; compared with the former, the shell of the virus-like particle and the real virus can retain the epitope similar to the real virus, triggering a good immune response to resist the infection of the real virus

Method used

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  • Method for preparing enterovirus virus-like particle and application thereof
  • Method for preparing enterovirus virus-like particle and application thereof
  • Method for preparing enterovirus virus-like particle and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0065] VLP feature analysis method

[0066] Western blotting

[0067] The separated SDS-PAGE protein samples were transferred to a nitrocellulose membrane (0.45 μm, NC membrane) using a vertical transfer tank. Before transfer, cut a piece of NCmembrane of the same size as the gel, soak it in transfer buffer (transfer buffer, 2.5mM Tris, 192mM glycine, 20% v / v methanol, pH 8.3) together with the gel for five minutes, and then put the NC The membrane was placed on the colloid, and a filter paper soaked with transfer buffer was placed on the top and bottom of the membrane. The transfer current was 100V for 60 minutes. The transferred membrane was placed in blocking buffer (blocking buffer, 25mM Tris, 150mM NaCl, 5% nonfat powdered milk, pH 7.4) at room temperature for one hour or 4°C overnight. Then, the mouse anti-VP1 monoclonal antibody diluted 1500 times was used as the primary antibody to identify the VP1 protein on the membrane, and it was treated at room temperature for...

Embodiment 2

[0091] Analysis of mouse immunological experiments

[0092] Mouse immunization and serum sample collection

[0093]In order to understand the expression of the purified virus-like particles in terms of immune properties, the present invention conducts animal experiments by intraperitoneal injection. After the sample purification was completed, the total protein concentration was adjusted to 40 μg / ml and stored in a -80°C refrigerator. Female BALB / c mice were ordered by the National Animal Center and sent to Qingda Animal Room for quarantine, and then intraperitoneally injected when the mice were 6 to 8 weeks old. Samples were mixed with the same volume of adjuvant for priming injection (complete Freund's adjuvant for priming injection, Sigma, cat#F5881; incomplete Freund's adjuvant for booster injection, Sigma, cat#F5506) with a three-way valve for about 3 minutes prior to intraperitoneal injection, When the sample is mixed evenly, it is white latex. Each mouse was intrap...

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Abstract

The invention relates to preparation of enterovirus 71 type virus-like particle by using a baculovirus recombination protein expression system and application of the virion. By modifying baculovirus vector design, hydrolysis protease 3CD expression is reduced and structural protein predecessor protein P1 is enhanced to increase virus-like particle (VLP) expression. The enterovirus virus-like particle can initiate high antibody titer reaction and neutralization titer reaction of mice, thereby indicating that the VLP can be used as vaccine of the enterovirus 71 type.

Description

technical field [0001] The invention relates to the preparation of enterovirus 71 virus-like particles by using a baculovirus recombinant protein expression system, a manufacturing method and its application. Background technique [0002] Enterovirus (Enterovirus) belongs to the Piconaviridae family (Picomaviridae) in classification, is a single positive strand (positive, single strand) RNA virus, the infection route is mainly transmitted through feces or respiratory droplets, and can infect various system organs of the human body. even lead to organ failure. Traditionally, enteroviruses can be divided into polioviruses (Polioviruses, PV), Coxsackieviruses A (CAV), and Coxsackieviruses B (Coxsackieviruses 2B, CBV) using neutralizing serum tests. , Human enteric cytopathogenic orphan viruses (Enteric cytopathogenic human orphan viruses, ie Echoviruses), and 68-71 enteroviruses. Among the serotypes of enteroviruses, enterovirus 71 is particularly virulent, often accompanied ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N5/10C12P21/02A61K39/12A61P31/14C12R1/93
Inventor 胡育诚
Owner 胡育诚
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