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Construct of swine vesicular disease virus like particle using genetic engineering and the method for manufacturing thereof

A porcine vesicular disease virus and particle technology, applied to biochemical equipment and methods, cells and viruses modified by introducing foreign genetic material, etc., can solve the problems of conformation dependence and inability to form conformation structure of virus antigen site

Inactive Publication Date: 2007-10-31
大韩民国
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since porcine vesicular disease virus has a densely-packed icosahedral arrangement of 60 protomers, each protomer consists of 4 proteins (VP1, VP2, VP3 and VP) , and most of the antigenic sites are conformation dependent, however, in the case of expressing only the P1 recombinant protein in E. coli, the conformational structure of the viral antigenic site cannot be formed

Method used

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  • Construct of swine vesicular disease virus like particle using genetic engineering and the method for manufacturing thereof
  • Construct of swine vesicular disease virus like particle using genetic engineering and the method for manufacturing thereof
  • Construct of swine vesicular disease virus like particle using genetic engineering and the method for manufacturing thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Extract RNA from porcine vesicular disease virus

[0035] The porcine vesicular disease virus (UKG / 27 / 72) strain isolated in the UK in 1972 was used to inoculate IBRS-2 cells (National Veterinary Science and Quarantine Service, Korea), and then the RNeasy RNA extraction kit (Qiagen, Germany) was used to inoculate IBRS-2 cells according to the manufacturer’s Total RNA was extracted from culture supernatants according to the operating manual.

Embodiment 2

[0036] Embodiment 2: Synthesis, amplification and cloning of porcine vesicular disease virus P1 gene cDNA

[0037] For the cDNA of synthetic porcine vesicular disease virus P1 gene, the total RNA extracted in embodiment 1 and the reverse primer of SEQ ID NO: 6 are added in the test tube of RT premix kit (Bioneer, Korea), then in it according to manufacturer's The manual for synthesizing first-strand cDNA. Then the first strand of the cDNA, the forward primer DEQ ID NO: 5 and the reverse primer SEQ ID NO: 6 were added to the test tube of the PCRpremix kit (Bioneer) and heat-treated at 95° C. for 10 minutes in order to carry out Polymerase chain reaction (PCR). Then, the cDNA was amplified in a PCR amplification instrument (PE 2400; PerkinElmer), and after 30 cycles of denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 2 minutes, finally at 72°C Extend for 5 minutes.

[0038] In order to implement the above PCR reaction, the following...

Embodiment 3

[0042] Embodiment 3: Synthesis, amplification and cloning of porcine vesicular disease virus 3CD gene cDNA

[0043] When being the cDNA of synthetic porcine vesicular disease virus 3CD gene, the total RNA extracted in embodiment 1 and the reverse primer SEQ ID NO: 8 are added in the test tube of RT premix kit (Bioneer, Korea), then in it according to manufacturer's The manual for synthesizing first-strand cDNA. Then the first strand of the cDNA, the forward primer DEQ ID NO: 7 and the reverse primer SEQ ID NO: 8 were added to the test tube of the PCR premix kit (Bioneer) and heated at 95°C for 10 minutes, so that Perform polymerase chain reaction (PCR). Then, the cDNA was amplified in a PCR amplification instrument (PE 2400; Perkin Elmer), and after 30 cycles of denaturation at 94°C for 1 minute, renaturation at 55°C for 1 minute, and extension at 72°C for 2 minutes, A final extension was performed at 72°C for 5 minutes.

[0044] In order to implement the above-mentioned PC...

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Abstract

The present invention relates to a swine vesicular disease virus-like particle prepared by a gene recombination technique, as well as a preparation method thereof. More particularly, the invention relates to a method for preparing a swine vesicular disease virus-like particle, wherein the structural protein precursor (Pl) gene and 3CD protease gene of swine vesicular disease virus are inserted simultaneously into a baculovirus expression vector and are expressed in insect cells, so that the expressed 3CD protease enzyme digests the structural protein precursor into individual proteins which then form the capsid of swine vesicular disease virus by an self-assembly process.

Description

technical field [0001] The invention relates to porcine vesicular disease virus-like particles prepared by gene recombination technology and a preparation method thereof. More specifically, the present invention relates to a method for preparing porcine vesicular disease virus-like particles, wherein simultaneously the structural protein precursor (P1) gene and the 3CD protease gene in the porcine vesicular disease virus total gene are inserted into the baculovirus expression vector , and expressed in insect cells, so that the expressed 3CD protease hydrolyzes the structural protein precursor into individual proteins, and then the individual proteins undergo a self-assembly process to form a porcine vesicular disease virus capsid. Background technique [0002] Vesicular disease of swine is a highly contagious viral disease of porcine characterized by, after an incubation period of 2-7 days, on the coronary band, heels of feel and sometimes on the lips, tongue, snout (snout)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N5/16C12N15/86C12N15/09
CPCC12N7/00A61K2039/5258C12N2770/32323C12N15/63C12N15/79
Inventor 崔康锡高荣骏罗珍珠赵南仁
Owner 大韩民国
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