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EV71virus-like particles as well as preparation method and application thereof

一种病毒样、病毒的技术，应用在生物化学设备和方法、病毒、病毒肽等方向，能够解决杆状病毒颗粒VLP分离、限制大规模的生产需求、培养条件要求较高等问题，达到原核细胞生长快、不易丢失、遗传操作简单的效果

Active Publication Date: 2013-08-21
BEIJING MINHAI BIOTECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since insect cells are mainly used to prepare VLP at present, the requirements for culture conditions are relatively high, and the purification process is complicated, which limits the large-scale production requirements; in addition, the baculovirus-insect cell expression system produces baculovirus particles and other stars that affect the vaccine effect Pollutants, baculovirus particles are difficult to separate from the prepared VLP, and measures such as inactivation treatment are required, so it has a great impact on the quality of the vaccine
The mammalian cell expression system can play a role in the processing modification and correct folding of the protein after expression, but it is not easy to control artificially and the cost is high, so the application is limited

Method used

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  • EV71virus-like particles as well as preparation method and application thereof
  • EV71virus-like particles as well as preparation method and application thereof
  • EV71virus-like particles as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] The gene sequence optimization of the P1 and 3CD protein of embodiment 1 coding EV71

[0065] According to the nucleotide sequences of the P1 and 3CD proteins of the C4a subtype strains of enterovirus type 71 popular in my country, the vector software was used to optimize the design of the P1 and 3CD gene sequences according to the most preferred codons of Hansenula spp. Expression in Hansenula cells. The nucleotide sequence of the genes encoding P1 and 3CD proteins provided by the present invention is the sequence without the yeast secretion signal peptide or the transcription termination signal recognized by the yeast. The codons of the genes encoding P1 and 3CD proteins in the present invention use the most preferred codons of Hansenula. For codon usage frequencies in Pichia angusta see http: / / www.kazusa.or.jp / codon / . In order to prevent the GC content of the translated mRNA from being too high and the secondary structure of the mRNA from affecting the translation...

Embodiment 2

[0066] The construction of embodiment 2 recombinant expression vector PMV-P1-3CD

[0067] 1. Construction process of expression vector PMV-05:

[0068] The expression vector PMV-05 of the present invention consists of 6 parts: promoter (MOX-P), terminator (MOX-T), replicon HARS, ura3 gene, ColE1 replicon, Amp resistance gene.

[0069] Using yeast genomic DNA as a template, primers MOXP-F (see SEQ ID NO.5 for sequence), MOXP-R (see SEQ ID NO.6); MOXT-F (see SEQ ID NO.7), MOXT-R respectively (see SEQ ID NO.8); HARS-F (see SEQ ID NO.9), HARS-R (see SEQ ID NO.10); Ura3-F (see SEQ ID NO.11), Ura3-R (see SEQ ID NO.12) was amplified by PCR to extract genes MOXP, MOXT, HARS, and Ura3. Using the PBR-SK plasmid (purchased from Treasure Bioengineering Dalian Co., Ltd., item number: D3050) as a template, primers Amp+ColE1-F (see SEQ ID NO.13), Amp+ColE1-R (see SEQ ID NO.14 ) for PCR amplification, and transfer the gene Amp+ColE1. The PCR reaction system was as follows: 10 μl of PCR bu...

Embodiment 3

[0078] Induced expression and detection of the EV71 bacterial strain expressed by embodiment 3 Hansenula

[0079] The recombinant expression vector PMV-P1-3CD was transformed into Hansenula ATCC26012 uracil-deficient host cell AU-0501 by electroporation (the wild-type host strain was from ATCC26012, and the ATCC26012 uracil-deficient host cell was obtained by gene knockout method) , and its deposit number is CGMCC No.7013.

[0080] The transformed clones were screened and identified by selection medium and PCR method, and the transformed clones identified as containing both P1 and 3CD genes were stably subcultured on selection medium to obtain the P1 protein and 3CD protein co-expression strain AU-PMV- P1-3CD. The expression strain was inoculated in the selection medium MDL (0.67% yeast nitrogen source medium, purchased from SIGMA Company, specification: Y1251-KG, batch number: 030M1754), 0.5% ammonium sulfate, 2% glucose), and cultured on a shaking table at 33°C After 20 ho...

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Abstract

The invention provides EV71virus-like particles as well as preparation method and application of the EV71virus-like particles. The method comprises the following steps of: connecting a P1 protein gene and 3CD protease gene of an EV71 virus with predicated mean vote (PMV) plasmid to build PMV-P1-3CD recombinant expression plasmid; turning into hansenula polymorpha AU-0501 expression bacterial strain to obtain an AU-PMV-P1-3CD recombinant expression bacterial strain; fermenting and culturing the recombinant expression bacteria strain and performing inducible expression on EV71 virus-like particle proteins by using methanol; centrifugally gathering thallus to perform homogenate crushing at high pressure; and purifying the supernate through ion-exchange column chromatography, hydrophobic chromatography and sieve chromatography to obtain the EV71virus-like particles. EV71virus-like particle vaccines provided by the invention has high immunogenicity, security, immunological characteristics and biological activity; and the EV71virus-like particles can be prepared in a large scale and purified and used for preparing vaccines for preventing EV71 infection, and has good economic value and application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an enterovirus 71 virus-like particle, a preparation method thereof, and a hand, foot and mouth disease vaccine prepared by the same. Background technique [0002] Hand, foot and mouth disease (HFMD) is one of the common infectious diseases caused by enteroviruses. Infants and young children under the age of 5 have the highest incidence. The epidemic season is from June to August every year, and the main route of transmission is respiratory and oral infection. The disease is highly contagious, has a wide transmission route, spreads quickly, and has a high epidemic intensity, which can cause a pandemic in a short period of time. Enterovirus 71 (enterovirus71, EV71) and Coxsackievirus A16 (CA16) are the main pathogens of this disease. Compared with CA16, EV71 type enterovirus is much more dangerous, more than 90% of severe cases and death cases are caused by EV71 virus. The EV71 vi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19A61K39/125A61P31/14C12N7/04C12R1/93C12R1/78
CPCA61K39/12A61K2039/5258C12N7/00C12N2770/32323C12N2770/32334C12N2770/32351A61P31/14A61K39/125A61K39/135A61K2039/55505C07K14/005
Inventor 顾美荣魏文进刘建凯宋琳琳许珊珊
Owner BEIJING MINHAI BIOTECH
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