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Recombinant porcine pseudorabies virus attenuated strain for expressing porcine circovirus Cap protein gene, and preparation method and application thereof

A technology for porcine pseudorabies and porcine circovirus, applied in the direction of biochemical equipment and methods, viruses, virus peptides, etc., can solve the problem that no one can be controlled at the same time

Active Publication Date: 2019-10-18
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no vaccine that can simultaneously control PCV2, PCV3, and PRV infection at home and abroad, and there is no vaccine that can simultaneously control the recently popular PCV2, PCV3, and new PRV infection. Therefore, in response to new clinical situations, There is an urgent need to provide a vaccine that can solve the outbreak of these diseases with one shot

Method used

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  • Recombinant porcine pseudorabies virus attenuated strain for expressing porcine circovirus Cap protein gene, and preparation method and application thereof
  • Recombinant porcine pseudorabies virus attenuated strain for expressing porcine circovirus Cap protein gene, and preparation method and application thereof
  • Recombinant porcine pseudorabies virus attenuated strain for expressing porcine circovirus Cap protein gene, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Isolation and identification of porcine circovirus type 2 and determination of Cap protein gene

[0063] 1. Source of disease material

[0064]In a pig farm immunized with commercialized PCV2 vaccine in China, there were sporadic cases of sow abortion and increased number of mummified fetuses. Affected sows exhibited symptoms of anorexia, and aborted litters contained mummified fetuses of various gestational ages, consistent with symptoms of PCV2-associated abortions. PCV2 was positive by immunohistochemistry and quantitative PCR. PCV2 was still detected from diseased pig tissues after immunization with PCV2 commercial vaccine.

[0065] 2. Isolation and cultivation of virus strains

[0066] Add the disease material to DMEM culture medium at a ratio of 1:10 (volume ratio), grind it, and prepare tissue suspension; after repeated freezing and thawing for 3 times, the tissue suspension is centrifuged at 12000r / min for 15min, and the supernatant is collected; A...

Embodiment 2

[0074] Example 2 Isolation and identification of porcine circovirus type 3 and determination of Cap protein gene

[0075] 1. Source of disease material

[0076] In a commercial pig farm in China, compared with the historical average, the mortality rate of sows increased by 9.4%, the conception rate decreased by 1.2%, and the number of mummies increased by 8.2%. Clinically, affected sows are anorexic with signs of multifocal papules, macules and superficial dermatitis. Aborted litters contained mummified fetuses of different gestational ages, consistent with symptoms of PCV2-related miscarriages. Although the overall clinical presentation and signs of abortion observed in sows were consistent with a reproductive disorder caused by porcine circovirus type 2, different tissues in all sows, including kidney, lymph nodes, lungs, skin, and stillbirth, were detected by immunohistochemistry. The detection of PCV2, PRRSV, PPV, CSFV, and Mycoplasma hyopneumoniae were all negative by c...

Embodiment 3

[0086] Example 3 Construction of porcine circovirus type 2 Cap protein gene recombinant porcine pseudorabies virus live vector

[0087] 1. Construction of recombinant vector pUC-gIA-US2B-PCV2-Cap

[0088] Porcine pseudorabies virus HN1201 strain gI, gE, 11K, 28K four gene natural deletion weakened strain HN1201-R, using HN1201-R strain viral nucleic acid as a template, using gIF and gIR primers, US2F and US2R primers to amplify gIA and The gene sequence of the homology arm of US2B was cloned into the pUC19 vector by enzyme digestion to construct the recombinant vector pUC-gIA; the pUC-gIA and US2B were digested with SalI and HindIII respectively and then ligated to obtain pUC-gIA-US2B;

[0089] Using the PCV2 genome isolated in Example 1 as a template, use PCV2-Cap-F and PCV2-Cap-R primers to amplify to obtain a PCV2Cap protein gene with a size of about 702bp, which is digested with NheI and BamHI, and combined with NheI and The pAc-GFP-C1 plasmid digested with BamHI was liga...

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Abstract

The present invention provides a recombinant porcine pseudorabies virus attenuated strain. The attenuated strain has immunogenicity of porcine pseudorabies virus antigens and immunogenicity of porcinecircovirus antigens simultaneously. A vaccine composition prepared from the recombinant porcine pseudorabies virus attenuated strain can provide complete protection for the porcine pseudorabies viruses and the porcine circoviruses simultaneously, can also completely protect the porcine circoviruses from different regional sources, and has a protection effect superior to that of corresponding subunit vaccines.

Description

technical field [0001] The invention relates to a recombinant porcine pseudorabies virus attenuated strain expressing porcine circovirus Cap protein gene, a preparation method and a vaccine composition prepared by using the recombinant virus strain, belonging to the field of veterinary biological products. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) belongs to the family Circoviridae and is the smallest vertebrate virus discovered so far. PCV is divided into three genotypes of PCV1, PCV2 and PCV3 according to pathogenicity, antigenicity and nucleotide sequence. Among them, PCV1 widely exists in pigs but has no pathogenicity to it, and its genome length is 1759bp. PCV2 is pathogenic, and its genome length is 1767bp or 1768bp. PCV3 is a porcine circovirus that was recently isolated and confirmed as the causative agent in reproductive disorders in pigs, with a genome length of 2.0 kb. [0003] Porcine pseudorabies virus (Pseudorabies virus, PRV...

Claims

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Application Information

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IPC IPC(8): C12N7/01A61K39/295A61K39/12A61K39/245A61P31/22A61P31/20
CPCC12N7/00C07K14/005A61K39/12A61P31/22A61P31/20C12N2710/16721C12N2750/10022A61K2039/70A61K2039/552C12N2750/10034C12N2710/16734
Inventor 田克恭肖燕张超林孙进忠张许科
Owner PU LIKE BIO ENG
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