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Porcine parvovirus oral vaccine composition, preparation method and application thereof

A parvovirus and oral vaccine technology, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of low antigen titer, strong virulence, high production cost, etc., to achieve low production cost, The effect of high safety and simple operation

Pending Publication Date: 2018-06-01
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the PPV vaccines on the market at home and abroad mainly include inactivated vaccines and attenuated vaccines. Although traditional conventional vaccines have a certain effect on preventing PPV infection, they still have high production costs, low antigen titers and virulence. Backlash and other defects

Method used

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  • Porcine parvovirus oral vaccine composition, preparation method and application thereof
  • Porcine parvovirus oral vaccine composition, preparation method and application thereof
  • Porcine parvovirus oral vaccine composition, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of Porcine Parvovirus VP2 Protein Kluyveromyces marx Recombinant Expression Vector

[0044] The amino acid sequence encoding porcine parvovirus capsid protein VP2 is shown in SEQ ID No.1. According to the codon preference of Kluyveromyces marxis, the codon optimization of the gene sequence of VP2 was carried out without changing the amino acid sequence. The optimized nucleotide sequence is shown in SEQ ID No.2. Insert the optimized VP2 gene sequence into Kluyveromyces marx expression vector pUKD-N125 to construct the recombinant expression vector PUKD-N125 / VP2. The specific process is as follows:

[0045] with specific primers

[0046] VP2-N125-F (SEQ ID No. 3)

[0047] 5'-TTTTTTTGTTAGATCCGCGGATGAGCGAAAACGTGGAGC-3'

[0048] VP2-N125-R (SEQ ID No. 4)

[0049] 5'-AGCTTGCGGCCTTAACTAGTCTAGTACAACTTTCTTGGG-3'

[0050] The VP2 gene was amplified by PCR, and the amplified product was subjected to 1% agarose gel electrophoresis to recover a fragment o...

Embodiment 2

[0051] Example 2 Construction of Kluyveromyces marx genetically engineered bacteria Fim-1-pUKD-N125 / VP2

[0052] The yeast expression host strain used in the present invention is Kluyveromyces marxense Fim-1ura3Δ, and the preparation method of this strain can be constructed by the method disclosed in Example 1 of the Chinese patent publication CN105112313A, the auxotrophic strain Fim-1ura3Δ. Porcine parvovirus VP2 protein Kluyveromyces marx genetic engineering strain Fim-1-pUKD-N125 / VP2 is obtained by transforming the recombinant expression vector pUKD-N125 / VP2 into the Fim-1ura3 Δ strain, and the implementation process is as follows: Maxke Ruvermyces Fim-1ura3Δ was inoculated in a glass test tube containing 3 mL of YEPD medium, and cultured overnight on a shaker at 30°C until the OD600 was 12-15. The cells were collected and washed with LiAc-TE solution (100 mM LiAc, 10 mM Tris-HCl pH 7.5, 1 mM EDTA). Add carrier DNA, recombinant expression vector pUKD-N125 / VP2, PEG solution...

Embodiment 3

[0054] Example 3 Expression of porcine parvovirus VP2 protein in Kluyveromyces marx

[0055] Recombinant engineering bacteria Fim-1-pUKD-N125 / VP2 and control bacteria Fim-1ura3Δ were respectively inoculated in 50 ml of YD medium (1% yeast extract, 2% glucose), and cultured at 30°C and 220rpm for 66h Cells were collected by centrifugation, resuspended with 10mM Tris-HCl pH 7.5, and homogeneously disrupted by high pressure. Take 100 μl of cell lysate and add protein electrophoresis loading buffer to mix, boil water for 5 minutes, then carry out protein electrophoresis SDS-PAGE (polyacrylamide gelelectrophoresis, PAGE) and PPV-VP2 antibody immunoblotting Western Blot detection, detect porcine parvovirus PPV-VP2 protein in Max Expression levels of Kluyveromyces. The PPV-VP2 antibody used in Western Blot was polyantibody serum isolated from mice immunized with PPV-VP2 protein recombinantly expressed in Escherichia coli, and the secondary antibody was goat anti-mouse polyclonal ant...

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Abstract

The invention discloses a porcine parvovirus oral vaccine composition which includes the components a) and b), is basically composed of the a) and b), or is composed of the a) and b), wherein the component a) is viroid particles formed by self-assembling porcine parvovirus VP2 protein expressed by Kluyveromyces marxianus recombinant engineering bacteria; b) pharmaceutically or veterinarily acceptably auxiliary material for oral-taking preparations. The invention also discloses a preparation method and an application of the porcine parvovirus oral vaccine composition. The porcine parvovirus oral vaccine composition has excellent immunogenicity. An orally immunized mouse can generate protective antibodies which have high titer. The oral vaccine composition has high safety and low productioncost, can be produced to an amplified scale and has simple operation, and can be used for preventing and alleviating clinical symptoms related to PPV infection.

Description

technical field [0001] The invention relates to the fields of biotechnology and veterinary medicine, in particular to a porcine parvovirus oral vaccine composition and its preparation method and application. Background technique [0002] Porcine parvovirus (porcine parvovirus, PPV) is a very common pathogen, which can cause reproductive disorders in pigs, stillbirth and "mummy fetus" in pregnant sows, multisystem weakness syndrome in weaned piglets, etc., and sows usually have no obvious clinical symptoms. symptom. At present, the prevalence of PPV virus in my country is extremely widespread, and more than 95% of the pig farms are positive for PPV virus, thus causing huge economic losses to the pig industry. PPV belongs to the Parvoviridae family and the genus Parvovirus. The appearance of PPV is hexagonal or circular, without capsule, with a diameter of 20-23nm, icosahedral equiaxed symmetry, and the viral gene is single-stranded DNA with a molecular weight of 5.3×106Da. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/23A61P31/20A61P1/12A61P9/00A61P11/00C12N15/35C12N15/81C07K14/015A23K20/147
Inventor 吕红陈蕾余垚周峻岗
Owner FUDAN UNIV
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