Recombinant polio type-3 VLP (virus-like particles)

A technology of polio and Pichia pastoris, applied in the field of new Pichia pastoris expression system, can solve the problems of non-infectiousness and lack of viral nucleic acid, etc., and achieve clear visible basic structural units, uniform VLP particles, and uniform product properties stable effect

Pending Publication Date: 2018-06-08
SHANGHAI ZERUN BIOTECHNOLOGY CO LTD
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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 (VLP) after recombinant expression. The antigenicity of most VLPs is the same as that of true viruses, but they lack viral nucleic acids and are not infectious

Method used

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  • Recombinant polio type-3 VLP (virus-like particles)
  • Recombinant polio type-3 VLP (virus-like particles)
  • Recombinant polio type-3 VLP (virus-like particles)

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1: Codon optimization design

[0038] There are 64 types of genetic code, but most organisms tend to use a subset of these codons. Pichia pastoris and human genes have their own preferences for codon usage. Since the genetic code is degenerate, each amino acid is encoded by more than one codon, and the codon usage frequency of the same amino acid is different in wild-type genes. The codon bias of Pichia pastoris may lead to low translation efficiency and expression level of the recombinant protein. The inventors carried out gene sequence modification based on the wild-type 3CD protein and P1 protein: all amino acid genes were used with the highest and higher frequency of use. the codon. The codon usage frequency of Pichia yeast is shown in Table 1 (see http: / / www.kazusa.or.jp / codon / ). Then on this basis, in order to avoid the GC ratio of the translated mRNA being too high, the secondary structure of the mRNA affects the efficiency of translation and some comm...

Embodiment 2

[0043] Example 2: Synthesis of Sabin 1 3CD gene and construction of 3CD expression vector

[0044] The P1 protein of poliovirus can self-assemble into a VLP structure after being cleaved by a specific 3CD enzyme, in view of the high homology of the 3CD enzyme of poliovirus. The present invention selects the 3CD enzyme of polio attenuated strain sabin 1 as the specific cutting enzyme.

[0045] In order to improve the expression level of sabin 1 3CD protein in Pichia pastoris, before the synthesis of sabin 1 3CD gene, we carried out codon preference, mRNA secondary structure, GC content and common restriction enzyme digestion on the gene of sabin 1 3CD Optimization of sites etc. Under the premise of not changing the amino acid sequence, we preferentially adopt the most frequently used codons in Pichia pastoris for all amino acids, taking into account the GC ratio of mRNA and the secondary structure of mRNA, etc., and avoid some commonly used restrictions Restriction site, desi...

Embodiment 3

[0047] Example 3: Synthesis of Polio 3 P1 gene and construction of expression vector

[0048] The P1 gene of polio type III was selected from the P1 gene of the wild-type polio 3 strain Saukett. In order to increase the expression level of the Polio 3 P1 protein in Pichia pastoris, before the synthesis of the Polio 3 P1 gene, we carried out the gene synthesis of the Polio 3P1 Optimization of codon bias, mRNA secondary structure, GC content, and commonly used restriction enzyme sites, etc. Under the premise of not changing the amino acid sequence, we preferentially adopt the most frequently used codons in Pichia pastoris for all amino acids, taking into account the GC ratio of mRNA and the secondary structure of mRNA, etc., and avoid some commonly used restrictions Restriction site, design a new DNA sequence.

[0049] The optimized DNA sequence was synthesized by Shanghai Jierui Bioengineering Co., Ltd., and connected to the vector pPICZa B through the BstBI / KpnI restriction s...

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Abstract

The invention discloses a codon-optimized gene for encoding a precursor protein of a type-3 wild polio virus coat and a gene for encoding 3CD protein of type-1 attenuated polio virus. The genes are transferred into yeast cells and can be efficiently and spontaneously assembled into VLP (virus-like particles) in the cells through coexpression. The invention further discloses a macromolecule with immunogenicity. The macromolecule is mainly prepared from the genes in the yeast cells by expressing. The invention also discloses an application and composition of the macromolecule with immunogenicity.

Description

technical field [0001] The present invention relates to a new Pichia pastoris expression system, which can be used to produce poliovirus type III virus-like particles. Background technique [0002] Poliomyelitis is an acute infectious disease caused by poliovirus, mainly transmitted through the digestive tract. The virus often invades the central nervous system, damages the motor nerve cells in the anterior horn of the spinal cord, and causes flaccid paralysis of the limbs, which is more common in children, also known as polio. Some children can recover on their own after getting sick, but most of them will have muscle atrophy and deformity of lower limbs after getting sick, resulting in permanent disability, mostly lame or even unable to stand or walk at all. There is currently no effective treatment for this disease, and it can only be prevented through vaccines. [0003] As of 2005, there were still 6 countries in the world where wild poliovirus was endemic, 3 of which ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/43C12N15/81C12N1/19C12N7/04C07K14/105C12R1/84
CPCC12N15/815C07K7/00C07K14/005C12N2770/32322C12N2770/32323C12N2800/102C12N2800/22
Inventor 仝鑫孟凡举于桂梅崔盟
Owner SHANGHAI ZERUN BIOTECHNOLOGY CO LTD
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