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Virus-like particle as well as preparation method and application thereof

A virus-like and particle technology, applied in the field of agricultural science, animal husbandry and veterinary science, to achieve the effect of high immunogenicity and structural stability

Pending Publication Date: 2022-04-12
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the method of preparing virus-like particles

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  • Virus-like particle as well as preparation method and application thereof
  • Virus-like particle as well as preparation method and application thereof
  • Virus-like particle as well as preparation method and application thereof

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preparation example Construction

[0031] The invention provides a method for preparing virus-like particles, comprising the following steps:

[0032] 1) The viral structural protein gene VPO gene, VP1 gene and VP3 gene were respectively fused with the small ubiquitin-like modification protein gene, and the resulting fusion gene was cloned into the pET-28a vector to obtain pET / VP0-VP1 and pET / VP3;

[0033] Said virus comprises porcine vesicular disease virus and / or South African type foot-and-mouth disease virus;

[0034] 2) replacing the kanamycin resistance gene in the pET / VP3 with the ampicillin resistance gene to obtain pETa / VP3;

[0035] 3) Co-transforming the pET / VPO-VP1 described in step 1) and the pETa / VP3 described in step 3) into a prokaryotic expression system, and then recombinantly expressed to obtain three small ubiquitination-modified protease-cleaved fusion proteins;

[0036] 4) Digest the fusion proteins of the three small ubiquitinated proteases described in step 3) to remove small ubiquitina...

Embodiment 1

[0052] (1) Construction of recombinant vectors of porcine vesicular virus VP0, VP1, VP3 genes fused with small ubiquitinated modified proteins:

[0053] a. According to the published SVDV sequence (GenBank accession number: D16364.1), refer to the method of Hoover et al. (Hoover DM1, Lubkowski J. DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucl. Acids Res. (2002) 30(10):e43.) for codon optimization. The nucleotide sequence (SEQ ID NO: 2), the nucleotide sequence (SEQ ID NO: 3) of the SVDV structural protein VPO gene, the nucleotide sequence (SEQ ID NO: 3) of the VP3 gene, and the nucleotide sequence (SEQ ID NO: :4) were seamlessly fused with the small ubiquitin-modified protein gene (SEQ ID NO: 1), and respectively cloned into the pET-28a vector through Sal I / BamH I and Hind III / Xho I endonucleases to obtain pET / VP0-VP1 and pET / VP3;

[0054] (2) Expression and purification of SVDV antigen protein:

[0055] a. The constructed p...

Embodiment 2

[0064] Analysis of Immunogenicity of SVDV Virus-like Particles

[0065] Select SVDV, FMDV, 8 healthy pigs of about 20 kg who are negative for SVA antibody, and are randomly divided into 3 groups, 3 in the experimental group / group, and 2 in the control group, wherein, 1 group is the porcine vesicular virus prepared in Example 1 of the present invention 2 groups were immunized with porcine vesicular virus VP1 protein, and 3 groups were PBS control group. 100 μg of antigen was emulsified with an equal volume of 206 adjuvant and injected intramuscularly into the neck for immunization. Blood was collected before immunization and on days 7, 14, 21, and 28 after immunization, and serum was separated.

[0066] Since there is no SVDV positive serum and antigen in China, the present invention uses the IFA method to identify whether the specific antibody against VP1 is produced. The specific operation is as follows: Synthesize the SVDVVP1 capsid protein gene into the pCMV-N-Flag vector ...

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Abstract

The invention provides a virus-like particle as well as a preparation method and application thereof, and belongs to the technical field of agricultural science, animal husbandry and veterinary science. Small ubiquitin-like modified proteins are used as tag proteins to promote correct folding of virus structural proteins VP0, VP1 and VP3, meanwhile, the structural stability of the proteins is guaranteed, mass expression of the structural proteins is achieved, and after in-vitro self-assembly of the three structural proteins, a large number of virus-like particles similar to natural viruses in performance are obtained. Immunogenicity detection shows that the prepared virus-like particles have high immunogenicity and can be used as reserve vaccines to be applied to prevention and control of swine vesicular disease or foot-and-mouth disease virus transmission.

Description

technical field [0001] The invention belongs to the technical field of agricultural science, animal husbandry and veterinary medicine, and in particular relates to a virus-like particle and its preparation method and application. Background technique [0002] With the increasing frequency of international exchanges and cooperation, the world today is becoming a "global village". In this general environment, diseases such as SARS (2002) and novel coronavirus (2019) can easily develop into a global pandemic. As a large pig-raising country, my country has a heavy responsibility for the prevention and control of livestock diseases such as pigs, cattle, and sheep. In order to avoid the introduction of epidemic diseases in other epidemic areas and countries, or new outbreaks of old diseases, it is imperative to conduct research on epidemic diseases in neighboring countries and develop vaccines. Row. Foot-and-mouth disease (FMD) mainly has 7 serotypes. At present, type A and type ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/70A61K39/125A61K39/135A61K39/39A61P31/14
CPCA61K39/135A61K39/125A61K39/39A61P31/14C12N15/62C12N15/70
Inventor 郭慧琛孙世琪董虎穆素雨白满元何融泽张韵吴金恩丁耀忠冯霞尹双辉
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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