Mink parvovirus virus-like particle and its preparation method and application

A mink parvovirus, virus-like technology, applied in the field of microorganisms, veterinary biopharmaceuticals, to achieve high titer, good protective effect, and good immunogenicity

Active Publication Date: 2017-07-25
CHANGCHUN SR BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, if the four amino acid sequences of polyhedrin protein are fused and expressed in mink parvovirus VP2, can the exogenous protein be expressed at a high level in Spodoptera frugiperda ovary cells (Sf9 cells), and whether the exogenous protein can be correctly packaged into virus-like particles , and the level of immunogenicity and reactogenicity, etc., there is no literature support

Method used

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  • Mink parvovirus virus-like particle and its preparation method and application
  • Mink parvovirus virus-like particle and its preparation method and application
  • Mink parvovirus virus-like particle and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Mink parvovirus virus-like particles and its preparation method

[0030] 1 material

[0031] Mink parvovirus, strain E.coli DH10Bac, Sf9 cells, and fetal bovine serum were provided by Changchun Sino Biotechnology Co., Ltd., pEASY-Blunt Simple vector was purchased from Beijing Quanshijin Biotechnology Co., Ltd., pFastBac Dual vector, liposome 2000 was purchased from Invitrogen Company, Phusion DNA polymerase, restriction endonuclease and T4 DNA ligase were purchased from NEB Company, IPTG and X-gal were purchased from Dalian Bao Biological Company, antibiotics were purchased from Sigma Company, Grace insect cell culture medium, Sf -900II SFM serum-free cell culture medium was purchased from Gibco Company, cell culture dishes and cell shaker flasks were purchased from Corning Company, and DNA extraction kits, gel recovery kits, and plasmid extraction kits were purchased from Axygen Company.

[0032] 2 methods

[0033] 2.1 Extraction of viral genome

[0034] S...

Embodiment 2

[0051] Example 2 Identification of Mink Parvovirus Virus-Like Particles and Immunization of Mink

[0052] 1 material

[0053] Fresh pig blood, rabbit serum, and parvovirus monoclonal antibody were provided by Changchun Sino Biotechnology Co., Ltd. FITC-labeled goat anti-mouse secondary antibody and HRP-labeled goat anti-mouse IgG were purchased from Sigma Company.

[0054] 2 methods

[0055] 2.1 Identification of Mink Parvovirus VLPs

[0056] 2.1.1 Hemagglutination test

[0057] Using fresh pig blood, the buffer solution is 15mM PBS with pH 6.5 for the determination of hemagglutination titer. The steps are as follows: centrifuge the freshly taken pig blood preserved in Alfred's solution at 5000rpm for 5min, wash it with PBS three times, after the centrifuged supernatant is clarified, prepare 1% erythrocytes from the centrifuged erythrocytes, and add 0.5% rabbit serum. Add 25 μL of PBS to the 96-well V-type hemagglutination plate, add 25 μL of the sample to be tested, and p...

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Abstract

The mink parvovirus virus-like particle provided by the invention optimizes the mink parvovirus VP2 gene according to the preferred codons of insect cells, and directly combines the 5' end of the optimized VP2 gene with the nucleotide sequence encoding part of the polyhedrin protein Linked, and then cloned into a transfer vector, using the baculovirus / insect cell expression system to produce mink parvovirus virus-like particles. The expression system can be used to produce mink parvovirus virus-like particles safely, efficiently and on a large scale, and the obtained virus-like particles have high titer and good immunogenicity, and both muscle immunity and oral immunization can induce mink body to produce a high level of specificity Antibodies, and can resist the attack of strong venom, provide good protection for mink, and lay the foundation for the preparation of mink viral enteritis vaccine.

Description

technical field [0001] The invention relates to the field of microorganisms, genetic engineering and veterinary biopharmaceuticals, in particular to mink parvovirus virus-like particles, a preparation method and application thereof. Background technique [0002] Mink viral enteritis is a severe infectious disease caused by mink enteritis parvovirus (Mink enteritis viurs, MEV), also known as infectious enteritis or known as panleukopenia, cats, mustelidae, mink and canids All are susceptible, and it is one of the three major viral infectious diseases that are harmful to the mink breeding industry. The disease was first discovered by Schofield in 1949 in a mink farm in Fort William, Canada. In 1952, Will extracted the pathogen and named it MEV. Subsequently, the disease spread rapidly to the United States, Denmark, Norway, Sweden, the Soviet Union, Japan and many other countries. The disease was first discovered in my country in 1974, and has since spread throughout the coun...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/866C12N7/04A61K39/23A61P31/20A61P1/00C07K16/08C12R1/93
Inventor 金宏丽夏晓红陈宪平付玉刘冰杨佳夏振强石晶
Owner CHANGCHUN SR BIOLOGICAL TECH
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