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Method for preparing recombinant coxsackievirus A16 type virus-like particles

A coxsackie virus, virus-like technology, applied in the field of immunity, can solve the problems of lack of viral nucleic acid and non-infectivity, and achieve the effect of good immunogenicity, uniform shape and stable character

Active Publication Date: 2013-12-11
上海博唯生物科技有限公司 +1
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0009] The viral structural proteins of many viruses can be spontaneously assembled into virus-like particles after recombinant expression. The antigenicity of the virus-like particles is the same as that of true viruses, but they lack viral nucleic acids and are not infectious

Method used

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  • Method for preparing recombinant coxsackievirus A16 type virus-like particles
  • Method for preparing recombinant coxsackievirus A16 type virus-like particles
  • Method for preparing recombinant coxsackievirus A16 type virus-like particles

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Embodiment 1

[0053] The preparation of embodiment 1 virus particle

[0054] 1. Selection of expressed genes and optimal design of codons

[0055] The DNA sequence encoding CA16-P1 protein and CA16-3C protein refers to Chinese strain SZ / HK08-7 (GenBank: GQ279371.1), and the amino acid sequence of SZ / HK08-7 strain CA16-P1 protein is shown in SEQ ID NO:1 , the amino acid sequence of the CA16-3C protein is shown in SEQ ID NO:2.

[0056] Transform the wild-type DNA sequence encoding the CA16-P1 protein shown in SEQ ID NO:1 and the CA16-3C protein shown in SEQ ID NO:2 in the SZ / HK08-7 strain, and use the codons used in Hansenula as much as possible Higher frequency codons, while avoiding transcription factor binding regions, repetitive sequences, and RNA high-level structures that may affect expression. After codon optimization, the sequence of the recombinant gene encoding the CA16-P1 protein is shown in SEQ ID NO:3, and the sequence of the recombinant gene encoding the CA16-3C protein is sho...

Embodiment 2

[0090] Antigen performance detection of embodiment 2 virus particles

[0091] 1. VLP Vaccine Preparation

[0092] The purified CA16 virus-like particle protein is adsorbed with aluminum adjuvant to prepare the immunogenic CA16 vaccine.

[0093] 2. Determination of the immunogenicity of CA16 virus-like particles

[0094] SPF grade BALB / c mice aged 6-8 weeks were selected and divided into 4 groups with 6 mice in each group. One group of mice was immunized with buffer solution containing aluminum adjuvant (50mM PB pH7.0) (as a negative control group), and the other three groups were immunized with 1μg / mouse, 0.1μg / mouse, and 0.01μg / mouse , were immunized subcutaneously on the 0th and 14th day respectively, and were immunized twice in total, and blood was collected two weeks after the second immunization. Place the collected blood at 4°C overnight, centrifuge at 5000g for 10 minutes, and absorb the supernatant to obtain the mouse polyantiserum, store it at -20°C, and detect the...

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Abstract

The invention relates to the field of immunity technology, and particularly relates to a method for preparing recombinant coxsackievirus A16 type virus-like particles. In the method, the PI protein and 3C protein of CA16 are expressed in a double-expression plasmid through a yeast expression system. In order to realize the preparation of CA16 virus-like particles, according to the method provided by the invention, an expression vector and an expression system for CA16 virus-like particles are established, wherein the expression vector comprises two open reading frames which respectively contain a P1 protein expression sequence and a 3C protein expression sequence of CA16. According to the invention, a method for preparing recombinant coxsackievirus-like particle protein by use of co-expression of 3C protease and P1 protein is established through codon optimization; and the method can obtain virus particles with high purity, uniform form and stable characters, is used for preparing a hand-foot-and-mouth disease vaccine, and has a great market value.

Description

technical field [0001] The invention relates to the technical field of immunization, in particular to a method for preparing recombinant Coxsackievirus A16 virus-like particles. Background technique [0002] Hand, foot and mouth disease is a viral infectious disease mainly caused by enterovirus that threatens the health of children under 5 years old. The main route of infection is fecal-oral transmission. Symptoms include fever, rashes, and ulcers on hands, feet, and mouth. A small number of children may develop complications such as myocarditis, pulmonary edema, aseptic meningoencephalitis, acute flaccid paralysis, and neurogenic pulmonary edema. Children who become severely ill more quickly can lead to death. Hand, foot and mouth disease has caused explosive epidemics in many countries in the world, especially the large-scale outbreaks and significantly increased mortality in recent years. The first report of HFMD in China was in the early 1980s, and the outbreak began i...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N7/04A61K39/125A61P31/14
Inventor 张高峡
Owner 上海博唯生物科技有限公司
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