PCV2, PCV3 and PCV4 triple subunit vaccine as well as preparation method and application thereof

A triple sub-vaccine technology, applied in the field of virus molecular biology technology and genetic engineering, can solve problems such as the need for verification of cross-protection ability, low gene homology, pathogenicity and host spectrum to be further studied, etc., to achieve good immunity. Protective effect, broad application prospect, good immunogenicity effect

Active Publication Date: 2022-04-29
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The positive rates of PCV3 discovered in 2016 and PCV4 discovered in 2019 are also high in domestic pigs, but their pathogenicity and host spectrum need further study
[0005] It is worth noting that the gene homology of PCV2, PCV3 and PCV4 is low, but whether the related antibodies have cross-protective ability remains to be verified
In addition, there is no research report on the triple subunit vaccine of PCV2, PCV3 and PCV4 and its preparation method

Method used

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  • PCV2, PCV3 and PCV4 triple subunit vaccine as well as preparation method and application thereof
  • PCV2, PCV3 and PCV4 triple subunit vaccine as well as preparation method and application thereof
  • PCV2, PCV3 and PCV4 triple subunit vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Construction and identification of PCV2 Cap protein, PCV3 Cap protein and PCV4 Cap protein expression plasmids.

[0039] Plasmid: prokaryotic expression vector pET-28a(+).

[0040] 1) Primer design:

[0041] According to the sequences of PCV2 (GenBank: JQ955679.1), PCV3 (GenBank: MN431641.1) and PCV4 (GenBank: MT311854.1) in GenBank to remove the nuclear localization signal (NLS), the primer sequences were designed as SEQ ID Shown in NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9.

[0042] 2Cjd-F: 5'-cgcagccatcttggccagatc-3', (SEQ ID NO.4)

[0043] 2Cjd-R: 5'-tcattaagggttaagtggggggttttaag-3', (SEQ ID NO.5)

[0044] 3Chc-F: 5'-ctagctagctatgtcagaagaaaactattcattaggaggccc, (SEQ ID NO. 6)

[0045] 3Chc-R: 5'-ccgctcgaggagaacggacttgtaacgaatccaaac-3', (SEQ ID NO.7)

[0046] 4Cjd-F: 5'-gggcagcagccatcatcatcatca-3', (SEQ ID NO.8)

[0047] 4Cjd-R: 5'-gaccttgttaactaccccaaacaggga-3'. (SEQ ID NO.9)

[0048] 2) Gene cloning and identificatio...

Embodiment 2

[0055] Example 2, Expression, purification and identification of PCV2, PCV3 and PCV4 Cap proteins

[0056] Escherichia coli transformed into pET-28a(+)-PCV2-Δ16-235Cap, pET-28a(+)-PCV3-Δ23-214Cap, pET-28a(+)-PCV4-Δ21-228Cap prepared in Example 1 Rosetta (DE3) strains, namely Rosetta-PCV2, Rosetta-PCV3 and Rosetta-PCV4.

[0057] 1) Induced expression of protein:

[0058] (1) The recombinant protein-positive strains Rosetta-PCV2, Rosetta-PCV3 and Rosetta-PCV4 prepared in Experimental Example 1 were cultured to induce expression with IPTG when the OD600 was 0.6-1.0. The induction expression conditions were: 0.8mM IPTG induction 10h, 0.8mM IPTG induction 8h, 1.4mM IPTG induction 10h.

[0059] (2) The bacterial liquid after IPTG-induced expression was collected by centrifugation at 4° C., 8000 rpm, for 30 min, and washed twice with PBS (pH=8.0).

[0060] (3) Resuspend the pellet with 20 mL of PBS (pH=8.0), and add 1% Triton X-100 and PMSF at a final concentration of 1 mM. After...

Embodiment 3

[0074] Example 3, Antibody preparation and identification of PCV2, PCV3 and PCV4 Cap proteins.

[0075] Animal: big-eared white rabbit (female).

[0076] Immunogen (recombinant protein): the recombinant proteins PCV2 Cap, PCV3 Cap and PCV4Cap purified and obtained in Example 2.

[0077] 1) Immunized animals:

[0078] Take 300 μg of each of the three target proteins obtained from expression and purification in Example 2, mix them with complete Freund’s adjuvant 1:1 (volume ratio) and fully emulsify them, then inject them subcutaneously at multiple points on the back to immunize Japanese white rabbits (female).

[0079] Blood was collected from the ear vein of each rabbit before immunization. After the separated blood was allowed to stand overnight at 4°C, it was centrifuged at 3000 rpm for 15 minutes at 4°C to separate the serum. Then the serum was divided into 1.5mL centrifuge tubes and stored at -80°C. This serum served as a negative control.

[0080] The second, third a...

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Abstract

The invention relates to a PCV2, PCV3 and PCV4 triple subunit vaccine and a preparation method thereof, the PCV2, PCV3 and PCV4 triple subunit vaccine comprises three recombinant proteins, namely a PCV2 Cap protein, a PCV3 Cap protein and a PCV4 Cap protein, the PCV2 Cap protein is obtained by expressing a gene sequence as shown in SEQ ID NO.1 through escherichia coli, the PCV3 Cap protein is obtained by expressing a gene sequence as shown in SEQ ID NO.2 through escherichia coli, and the PCV4 Cap protein is obtained by expressing a gene sequence as shown in SEQ ID NO.2 through escherichia coli. The PCV4Cap protein is obtained by expressing a gene sequence as shown in SEQ ID NO.3 through escherichia coli. The invention also discloses a preparation method of the PCV4Cap protein. The vaccine can be used for preventing and treating PCV2, PCV3 and PCV4 infection at the same time, the immunogenicity aiming at PCV2, PCV3 and PCV4 is remarkably improved, and immunized animals can be induced to generate high-titer antibodies.

Description

technical field [0001] The invention belongs to the field of virus molecular biology technology and genetic engineering, in particular to porcine circovirus type 2 (Porcine circovirus 2, PCV2), porcine circovirus type 3 (Porcine circovirus 3, PCV3) and porcine circovirus type 4 ( Porcine circovirus 4, PCV4) triple subunit vaccine and preparation method thereof, the artificially prepared Cap protein triple subunit vaccine of PCV2, PCV3 and PCV4 can be used to simultaneously prevent and treat PCV2, PCV3 and PCV4 infection. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) belongs to the genus Circoviridae in taxonomy, and is one of the smallest animal viruses found so far, with a diameter of 12-23nm. Virus particles have a diameter of 14-17nm, a 20-sided symmetrical structure, no envelope, and contain a covalently closed single-strand circular negative-strand DNA. The genome size is about 1.7kb-2kb. Based on the differences in nucleotide sequence, pat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01C12N15/34C12N15/70A61K39/12A61P31/20G01N33/569
CPCC07K14/005C12N15/70A61K39/12A61P31/20G01N33/56983C12N2750/10022C12N2750/10034A61K2039/552G01N2333/01Y02A50/30
Inventor 任林柱吉卫龙牛谷雨欧阳红生逄大欣
Owner JILIN UNIV
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