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Method for preparing recombinant poliovirus-like particles

A poliovirus-like technology, which is applied in the field of related recombinant expression vectors and preparation of recombinant poliovirus-like particles, can solve the problems of lack of viral nucleic acid and non-infectivity

Inactive Publication Date: 2014-08-20
上海博唯生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 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 poliovirus-like particles
  • Method for preparing recombinant poliovirus-like particles
  • Method for preparing recombinant poliovirus-like particles

Examples

Experimental program
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Effect test

Embodiment 1

[0071] The preparation of embodiment 1 virus particle

[0072] 1. Selection of expressed genes and optimal design of codons

[0073] The DNA sequence encoding the P1 protein and 3C protein of the poliomyelitis type I Mahoney strain refers to GeneBank: V01149.1, the amino acid sequence of the P1 protein is shown in SEQ ID NO:1, and the amino acid sequence of the 3C protein is shown in SEQ ID NO:2 .

[0074] Transform the wild-type DNA sequence encoding the P1 protein shown in SEQ ID NO:1 and the 3C protein shown in SEQ ID NO:2 in the type I Mahoney strain, and the codons used in Pichia pastoris are used as codons as possible , while avoiding transcription factor binding regions, repetitive sequences, and RNA advanced structures that may affect expression. After codon optimization, the sequence of the recombinant gene encoding the P1 protein is shown in SEQ ID NO:3, and the sequence of the recombinant gene encoding the 3C protein is shown in SEQ ID NO:4.

[0075] 2. Construct...

Embodiment 2

[0093] Antigen performance detection of embodiment 2 virus particle

[0094] 1. VLP Vaccine Preparation

[0095] The purified poliovirus-like particle protein was adsorbed with aluminum adjuvant (50 μg protein: 500 μg, aluminum adjuvant), and an immunogenic poliovirus vaccine was prepared.

[0096] 2. Determination of Immunogenicity of Poliovirus-like Particles

[0097]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 (50mM PB pH7.0) containing aluminum adjuvant (as a negative control group), and the other three groups were immunized with 1 μg / mouse, 0.1 μg / mouse, and 0.01 μg / mouse (The immunization dose here refers to the quality of the virus-like particle protein amount), and the immunization was subcutaneously injected on the 0th and 14th days respectively, and a total of two immunizations were performed, and blood was collected two weeks after the second immunizatio...

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Abstract

The invention relates to the technical field of biology, especially relates to a method for preparing recombinant poliovirus-like particles, and further provides related recombinant expression vectors. The invention provides the recombinant expression vectors of the recombination poliovirus-like particles. The recombinant expression vectors contain a P1 protein expression box and a 3C protein expression box, wherein the P1 protein expression box and the 3C protein expression box are located in the same recombinant expression vector or are respectively located in two recombinant expression vectors. The virus-like particles obtained by the method are similar to a poliovirus in the structure and can be used as a vaccine antigen; and the antigen is composed of a plurality of monomers, has huge molecular weight, enables a granular repetitive structure to be observed under an electron microscope, and has stronger immunogenicity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing recombinant poliovirus-like particles, and further provides related recombinant expression vectors. Background technique [0002] Poliomyelitis (Poliomyelitis) is an acute intestinal infectious disease caused by poliovirus (Poliovirus) infection, mainly limb paralysis, mainly through fecal-oral transmission, affecting children under five years old, commonly known as polio, Most children with paralysis are left with a limp and are permanently disabled. At present, there is no specific treatment for polio, only vaccines can be used as prevention. [0003] Poliovirus belongs to the Enterovirus genus of the Picornaviridae family and contains a single-stranded positive-strand RNA gene. The protein capsid has an icosahedral stereosymmetric structure without an envelope. Poliovirus genomic RNA is about 7.5 kb long. The genome is divided into four parts: 5' non-codi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/66C12N1/19C12N7/04A61K39/125A61P31/14C12R1/93
CPCY02A50/30
Inventor 沈琼傅文彬
Owner 上海博唯生物科技有限公司