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Porcine Delta coronavirus virus-like particles as well as preparation method and application thereof

A coronavirus, virus-like technology, applied in the field of porcine Delta coronavirus virus-like particles and their preparation, can solve the problem that the vaccine development of porcine Delta coronavirus has not been reported, and achieve good development and application prospects, antibody titer and High safety and good immunogenicity

Active Publication Date: 2020-05-22
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the development of a vaccine for the newly discovered porcine Delta coronavirus

Method used

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  • Porcine Delta coronavirus virus-like particles as well as preparation method and application thereof
  • Porcine Delta coronavirus virus-like particles as well as preparation method and application thereof
  • Porcine Delta coronavirus virus-like particles as well as preparation method and application thereof

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preparation example Construction

[0056] The preparation method of porcine Delta coronavirus virus-like particle of the present invention comprises the steps:

[0057] (1), amplifying porcine Delta coronavirus structural protein (S, E, M, N) gene;

[0058] (2), using the structural protein gene of step (1) to construct three kinds of recombinant shuttle plasmids: pFBD-PDCoV-S, pFBD-PDCoV-N and pFBD-PDCoV-E / M;

[0059] (3), using the recombinant shuttle plasmid of step (2) to construct three kinds of recombinant bacmids: rB-PDCoV-S, rB-PDCoV-N and rB-PDCoV-E / M;

[0060] (4), 3 kinds of recombinant bacmids of step (3) are transfected into Sf9 cells respectively, obtain the recombinant baculovirus rBV-PDCoV-S, rBV expressing porcine Delta coronavirus structural protein (S, N, E / M) - PDCoV-N and rBV-PDCoV-E / M;

[0061] (5), 3 kinds of recombinant baculoviruses of step (4) are co-infected Sf9 cell, purify, obtain porcine Delta coronavirus virus-like particle PDCoV-VLP.

[0062]

[0063] The porcine Delta coron...

Embodiment 1

[0065] The design and synthesis of embodiment 1 porcine Delta coronavirus structural protein (S, E, M, N) gene

[0066] According to the whole genome sequence of porcine Delta coronavirus, the primers of this viral structural protein (S, E, M, N) gene were designed respectively, respectively in the structural protein S, M, the initiation codon of the nucleotide sequence of N gene Add an EcoRI restriction site before ATG: gaattc, add a HindIII restriction site after the stop codon: aagctt; add an XhoI restriction site before the start codon ATG of the nucleotide sequence of the structural protein E gene: ctcgag, Add a KpnI restriction site after the stop codon: ggtacc. Wherein, the nucleotide sequence of the porcine Delta coronavirus structural protein S gene is shown in SEQ ID NO.1, and the amplification forward primer PDCoV-S-PF sequence of the nucleotide sequence of the structural protein S gene is as SEQ ID NO. 5, the sequence of the reverse primer PDCoV-S-PR is shown in S...

Embodiment 2

[0067] Example 2 Construction and Identification of Recombinant Shuttle Plasmids pFBD-PDCoV-S, pFBD-PDCoV-N, pFBD-PDCoV-E / M

[0068] Use restriction endonuclease EcoRI and HindIII double enzymes to digest the purified product and pFastBac of porcine Delta coronavirus structural protein (S, M, N) gene TM The Dual plasmid was reacted at 37°C for 2 hours, separated by 1% agarose gel electrophoresis, and the Cap protein gene fragment and linearized pFastBacTMDual were recovered; 1 μL of T4DNA ligase, T4Buffer, and linearized pFastBacTMDual were added to 7 μL of S (or M or N) protein gene fragments, mix gently, add the mixture to the just-thawed Trans5α competent cells, incubate on ice for 30min, heat shock at 42°C for 90s, immediately bathe in ice for 2min, activate at 37°C for 1h and then evenly Spread on LB culture plates containing ampicillin (100 μg / mL), and incubate upside down at 37°C for 12 hours to obtain colonies of recombinant shuttle plasmids pFBD-PDCoV-S, pFBD-PDCoV-N ...

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Abstract

The invention provides porcine Delta coronavirus virus-like particles as well as a preparation method and application thereof. The method comprises the following steps: firstly, designing and amplifying a porcine Delta coronavirus structural protein gene, constructing a recombinant shuttle plasmid by using the structural protein gene, constructing a recombinant bacmid by using the recombinant shuttle plasmid, transfecting Sf9 cells with the recombinant bacmid to obtain a recombinant baculovirus expressing the porcine Delta coronavirus structural protein, infecting Sf9 cells with the recombinant baculovirus, and performing purification to obtain the porcine Delta coronavirus-like particles. The preparation method provided by the invention uses the no-serum cultured Sf9 cells for expressionpreparation, sucrose density solution ultracentrifugation is also used to obtain the PDCoV-VLP virus-like particles, the PDCoV-VLP virus-like particles have the advantages of integrity, good immunogenicity, and high titer and safety of an antibody generated by immunizing animals, and can be used for preventing and treating porcine Delta coronavirus virus diseases, so that the particles have good development and application prospects.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a porcine Delta coronavirus virus-like particle and a preparation method and application thereof. Background technique [0002] Since 2010, porcine epidemic diarrhea (porcine epidemic diarrhea, PED) characterized by high mortality of suckling piglets has broken out in my country, and the epidemic has spread to more than a dozen provinces, resulting in the death of 1 million piglets. Similarly, PED is also frequently prevalent in the United States and other places, seriously affecting the development of the world's pig industry. The research on the pathogens of PED found that the emerging porcine Delta coronavirus (Porcine deltacoronavirus, PDCoV) and porcine epidemic diarrhea virus (porcineepidemic diarrhea virus, PEDV) are important pathogens that cause the disease, and there is a phenomenon of co-infection between the two viruses . [0003] Observed und...

Claims

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Application Information

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IPC IPC(8): C12N15/50C07K14/165C12N15/866A61K39/215A61P31/14
CPCC07K14/005C12N15/86A61K39/12A61P31/14C12N2770/20023C12N2710/14043A61K2039/5258A61K2039/552C12N2770/20034Y02A50/30
Inventor 严亚贤纪立凯孙建和李莎莎
Owner SHANGHAI JIAO TONG UNIV
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