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High-prolificacy porcine circovirus type-2 strain and application thereof

A porcine circovirus and strain technology, applied in the field of virology, can solve the problems of poor reproduction ability of PCV2 strains and high vaccine cost, and achieve good immune protection effect

Active Publication Date: 2012-11-21
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The invention provides a new strain of porcine circovirus type 2 with high reproductive ability (high titer), which solves the problems of poor reproductive ability of existing PCV2 strains and high cost of application in developing vaccines

Method used

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  • High-prolificacy porcine circovirus type-2 strain and application thereof
  • High-prolificacy porcine circovirus type-2 strain and application thereof
  • High-prolificacy porcine circovirus type-2 strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction and Identification of Infectious Clones of Nucleotide Site 1376 Deletion Mutant Strain

[0033] 1.1 Viruses, cells and plasmids

[0034] PCV2-HZ0201 strain (Genbank NO.AY188355), Escherichia coli strain TG1, carrier pMD18-T, PCV negative PK-15 cell line, PCV2 / Cap protein monoclonal antibody, FITC goat anti-mouse IgG, all are test materials, no specificity sexual demands.

[0035] Wherein, the preparation of PCV2 / Cap protein monoclonal antibody and FITC goat anti-mouse IgG can refer to literature (Shang SB et al.Fine mapping of antigenic epitopes on capsid proteins of porcine circovirus and antigenic phenotype of porcine circovirus Type 2.Molecular Immunology.2009.46 : 327-334), and the rest are commercially available products.

[0036] 1.2 Primer design and synthesis

[0037]According to the PCV2-HZ0201 strain sequence provided in GenBank, a continuous single-nucleotide deletion was designed for 1376-1379 bp. Using the Prime5.0 biological soft...

Embodiment 2

[0057] In vitro replication ability of embodiment 2 single-site nucleotide deletion strain

[0058] 2.1 Test method

[0059] Through the detection of indirect immunofluorescence (IFA) on the primary and passaged viruses, the successfully rescued mutant viruses were obtained. Infect PK-15 cells with mutant virus P0, culture at 37°C for 72 hours, place at -70°C, freeze and thaw three times repeatedly, and collect the virus liquid after filtering and sterilizing with a filter. PK-15 cells were suspended and inoculated at a ratio of 1:10 for subculture of the virus. Continuously cultured in PK-15 cells for 8 generations until the virulence was stable.

[0060] 2.2 Determination of Reproductive Ability Infect PK-15 cells with successfully rescued PCV2-ZJ / H virus at a ratio of 1:10 for subculture. Viruses were assayed for replication capacity. The TCID of the cultured virus was measured at each generation 50 .

[0061] The virus solution of PCV2-ZJ / H was diluted with MEM respe...

Embodiment 3

[0063] The inactivated vaccine that embodiment 3 strains of the present invention prepares is to the protective power test of mouse

[0064] The immunogenicity of PCV2-ZJ / H strain was determined using clean-grade Balb / C mice.

[0065] 3.1 prepare inactivated vaccine with strain of the present invention

[0066] Make the potency greater than 10 7.0 TCID 50 The PCV2-ZJ / H strain virus solution per ml was inactivated at 4°C for 48 hours with a final concentration of 0.05% β-propiolactone, then in a water bath at 37°C for 2 hours, and the inactivated venom was properly diluted with MEM culture solution, and then Mix and emulsify the venom with ISA206VG adjuvant (Sepic, France) at a ratio of 3:1 (v / v) to prepare an oil-in-water PCV2-ZJ / H strain inactivated vaccine.

[0067] 3.2 Experimental animals and experimental program

[0068] Eight-week-old clean-grade BALB / C mice with negative PCV2 antigen and antibody tests were randomly divided into groups, 10 in each group. After the ...

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Abstract

The invention discloses a high-prolificacy porcine circovirus type-2 strain and application thereof. The porcine circovirus type-2 strain is named as PCV2-ZJ / H strain, the preservation number of the strain is CGMCC No.6391, the gene sequence of the strain is shown as SEQ ID NO:1, and nucleotide at a 1376 locus in the nucleotide sequence of a genome of the strain is absent. The porcine circovirus type-2 strain has high prolificacy, the virus titer (TCID50) in PK-15 cells reaches 107.6 / mL and is more than 10 times higher than that (106.3 / mL) of a parent virus, and a vaccine prepared with the strain has high immune protection capacity.

Description

technical field [0001] The invention belongs to the field of virology, and in particular relates to a novel strain of porcine circovirus type 2 with high reproductive capacity obtained through transformation of virus molecular genetics technology and its application in the preparation of inactivated vaccines. Background technique [0002] According to the pathogenicity, antigenicity and nucleotide sequence of porcine circovirus (Porcine circovirus, PCV), it can be divided into two genotypes, namely porcine circovirus type 1 (PCV1) and porcine circovirus 2 type (PCV2). [0003] PCV2 is a new virus that can cause porcine circovirus virus-associated disease (PCVAD) in the past ten years. The pathogenicity of PCV2 to pigs is characterized by damage to the immune system, causing the infected pigs to be in a state of severe immunosuppression, clinically manifested as emaciation, lymphadenopathy and other lesions, mixed infection with various pathogens can also lead to multisystem...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/34C12N15/63A61K39/12A61P31/20C12R1/93
Inventor 周继勇金玉兰
Owner ZHEJIANG UNIV
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