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Coxsackie virus B5 type virus-like particle as well as preparation method and application thereof

A coxsackie virus and virus-like technology, applied in the fields of genetic engineering and biomedicine, can solve the problems of long production cycle, listing virus-like particle vaccines, and difficulty in containing the outbreak of the epidemic, and achieve the effect of simple production process.

Active Publication Date: 2020-12-08
GUILIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

EV71 monovalent inactivated vaccine cannot produce cross-protection against other enteroviruses
The preparation technology of enterovirus inactivated vaccine has its advantages, but it also has great defects: 1. The production cycle is long: it takes 5-8 months from the acquisition of new subtype virus strains to the production and marketing of vaccines, and it is difficult to contain new vaccines. outbreak of the epidemic
2. High production conditions: In order to prevent man-made pollution and virus leakage and spread, the whole set of production must be carried out under strict control conditions according to regulations, and the virus inactivation must be completely and thoroughly
However, no enterovirus-associated virus-like particle vaccine is currently on the market

Method used

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  • Coxsackie virus B5 type virus-like particle as well as preparation method and application thereof
  • Coxsackie virus B5 type virus-like particle as well as preparation method and application thereof
  • Coxsackie virus B5 type virus-like particle as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] The preparation method of Coxsackievirus B5 virus-like particles in this embodiment includes the following steps:

[0074] 1, preparing the recombinant plasmid pOET5-CB5-P1-3CD.

[0075] The capsid protein P1 gene and 3CD protease gene of coxsackievirus B5 with codon optimization were synthesized respectively, and then connected to the EcoRⅠ / NotⅠ restriction site after P10 promoter and the Bamhi / Hindiii restriction site after Ph promoter of shuttle vector pOET5 of insect baculovirus, respectively, to obtain the recombinant plasmid of pOET5-CB5-P1-3CD, such as Figure 1 Shown. The main elements of the plasmid are as follows: P10 promotor is p10 promoter, polyhedrin promotor is Ph promoter, AmpR is ampicillin resistance gene and AmpR promotor is ampicillin resistance gene promoter.

[0076] In which, the nucleotide sequence of the codon optimized capsid protein P1 gene is shown in SEQ ID NO.1; The nucleotide sequence of the optimized 3CD protease gene is shown in SEQ ID NO.2

[...

Embodiment 2

[0093] This embodiment provides a coxsackievirus B5 virus-like particle vaccine. The coxsackievirus B5 virus-like particle vaccine is a liquid injection prepared by mixing 50μg / mL coxsackievirus B5 virus-like particle obtained in Example 1 with Freund's adjuvant in equal volume.

experiment example 1

[0094] Example 1: Animal immunity, determination of serum antibody and virus neutralization reaction

[0095] According to the currently used animal immunization methods of enterovirus. The animals were six-week-old BALB / c female mice without specific pathogen (SPF). The specific operation ELISA as follows: 0.3ml of coxsackievirus B5 virus-like particles (10μg total protein) emulsified with Freund's adjuvant was subcutaneously injected into each BALB / c mouse at week 0, week 2 and week 4. The same dose of coxsackievirus B5 inactivated virus was used as a positive control and PBS group as a negative control, and the mice were cut off at week 0, week 2, week 4, week 6 and week 12.

[0096]The steps of ELISA are as follows: the purified inactivated virus B5 of Coxsackie virus is diluted to 1μg / ml with coating buffer (i.e. 0.05M carbonate buffer with pH 9.6), 100μl is added to each well of 96-well plate, and the coating is carried out overnight at 4℃. Washing: The next day, washing wit...

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Abstract

The invention discloses a coxsackie virus B5 type virus-like particle as well as a preparation method and application thereof, and belongs to the technical field of gene engineering and biological medicines. The coxsackie virus B5 type virus-like particle is obtained by infecting Sf9 insect cells with recombinant baculovirus for expressing P1 capsid protein gene and 3CD protease gene of coxsackievirus B5 type, and culturing until more than 90% of the Sf9 insect cells are infected with lesions. The invention further discloses a preparation method and application of the coxsackie virus B5 typevirus-like particle. The coxsackie virus B5 type virus-like particle and a wild virus particle have similar appearance structures and sizes, the immunogenicity reaches or even exceeds the degree of the wild inactivated virus, and the coxsackie virus B5 type virus-like particle can be used for preparing a coxsackie virus B5 type virus-like particle vaccine.

Description

Technical field [0001] The invention relates to a coxsackievirus B5 virus-like particle, its preparation method and application, belonging to the technical field of genetic engineering and biomedicine. technical background [0002] Coxsackiecirus B5 (CB5) belongs to enterovirus genus of picornaviridae, and is an important member of enterovirus group B. CB5 is an icosahedral spherical particle with a diameter of 24-30nm. It consists of an unfolded capsid and a single stranded RNA of about 7400 nucleotides, and contains an open reading frame, with 5 ′ untranslated region and 3 ′ untranslated region with poly A tail on both sides. It encodes a polyprotein of about 2100 amino acids, which will be further cut to form three precursor proteins called P1, P2 and P3. P1 was split into four structural viral proteins, namely VP1, VP2, VP3 and VP4. P2 and P3 cleave to form seven kinds of nonstructural proteins, namely 2A, 2B and 2C cleaved from P2, and 3A, 3B, 3C and 3D cleaved from P3. The ...

Claims

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Application Information

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IPC IPC(8): C07K14/085C12N15/866C12N5/10A61K39/125A61P31/14
CPCA61K39/12A61K2039/5258A61K2039/54A61P31/14C07K14/005C12N15/86C12N2710/14043C12N2770/32023C12N2770/32034
Inventor 刘启亮刘洪波章宁漆琪
Owner GUILIN MEDICAL UNIVERSITY
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