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Preparation and use of canine parvovirus virus-like particles

A canine parvovirus and virus-like technology, which can be used in antiviral agents, medical preparations containing active ingredients, inactivation/attenuation, etc., which can solve the problem that virus-like particle vaccines are difficult to apply in clinic, and eukaryotic expression is low. , the problem of high cost

Active Publication Date: 2013-11-27
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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Problems solved by technology

The main antigenic sites are also on the VP2 protein. Therefore, VP2 has become the preferred target gene for the construction of CPV genetic engineering vaccines. At present, it has been reported that canine parvovirus can form virus-like particles in eukaryotic cells, but due to The low expression level and high cost of eukaryotic expression make it difficult to use eukaryotic expression products to prepare virus-like particle vaccines for clinical application

Method used

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  • Preparation and use of canine parvovirus virus-like particles
  • Preparation and use of canine parvovirus virus-like particles
  • Preparation and use of canine parvovirus virus-like particles

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Embodiment Construction

[0018] The present invention is illustrated below in conjunction with examples.

[0019] 1. Construction of recombinant expression vector for canine parvovirus structural protein VP2

[0020] According to the published canine parvovirus sequence (GenBabnk accession number: M: 38245), the primers for amplifying the structural protein VP2 were designed, and restriction sites were introduced into the primers. The primer sequences are as follows:

[0021] VP2F: 5'-TGTCGTCTCAAGGTATGAGTGATGGAGCAGTTC-'3 SEQ ID №2

[0022] VP2R: 5'-CGGGATCCG TTAATATAATTTTCTAGGTGCT-'3 SEQ ID №3.

[0023] Refer to the method of Yin et al. (2009) (Yin S, Sun S, Yang S, Shang Y, Cai X, Liu X. Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli. Virol J. 2010 Jul 21;7:166. doi: 10.1186 / 1743-422X-7-166.), using canine parvovirus DNA as a template, using the primer pair VP2F / VP2R to amplify the VP2 gene by polymerase chain reaction (PCR) , afte...

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Abstract

The invention discloses a canine parvovirus virus-like particle, and a preparation method and a use thereof. The canine parvovirus virus-like particles provided by the invention are formed by VP2 protein; the gene sequence of the canine parvovirus virus-like particles is SEQ ID No 1. The preparation method of the canine parvovirus virus-like particles provided by the invention comprises the following steps of: by taking the DNA (Deoxyribose Nucleic Acid) of the canine parvovirus as a template, amplifying the DNA with SEQ ID No 2 and SEQ ID No 3 as primers to obtain an amplification product, inserting the amplification product into a carrier pSMK to obtain a recombinant plasmid pSMK-VP2, transforming the pSMK-VP2 to an escherichia coli BL21-CondonPlus (DE3)-RIL line, culturing in an LB (Luria-Bertani) culture medium and expressing a recombinant protein through induction by using IPTG (Isopropyl-beta-D-Thio Galactopyranoside), and then purifying the recombinant protein and carrying out in-vitro assembly to obtain the canine parvovirus virus-like particles.

Description

technical field [0001] The invention relates to a virus-like particle of a virus, a preparation method and application of the virus particle, specifically, the present invention is a canine parvovirus virus-like particle, a preparation method and an application thereof. Background technique [0002] Canine parvovirus (Canine parvovirus, CPV) is one of the severe infectious diseases that cause canine hemorrhagic gastroenteritis and canine myocarditis. The CPV genome encodes two reading frames, of which ORF1 consists of 2007 bases, and the encoding is involved in virus replication, etc. Functionally related non-structural proteins NS1, NS2; ORF2 consists of 1755 bases, and the encoded virus mainly has structural proteins VP1 and VP2, among which VP2 protein is the main component of the virus capsid, with a content of more than 90%. Studies have shown that VP2 protein plays an important role in the immune response induced by viral infection. The main antigenic sites are also ...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/70A61K39/23A61P31/20G01N33/68G01N33/569C12R1/93
Inventor 郭慧琛孙世琪徐进魏衍全孙德惠
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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