Bivalent subunit vaccine of porcine circovirus type 2b and type 2d and preparation method thereof

A subunit vaccine, recombinant baculovirus technology, applied in multivalent vaccines, chemical instruments and methods, antiviral immunoglobulins, etc. Wide range of protection, reduced stress response, excellent immune protection effect

Active Publication Date: 2020-12-22
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patent CN201710295409.4 (live attenuated chimeric porcine circovirus vaccine) discloses a chimeric porcine circovirus infectious DNA clone and a live attenuated chimeric virus, which is the capsid of PCV-2b The gene is integrated into the non-pathogenic PCV1 viral genome, and the attenuated chimeric vaccine protects pigs from PCV2b attack, but its safety is poor during production and application, and there is a potential risk of reversion in animals
In the face of the emergence of new strains, although the existing vaccines also show a certain cross-protection effect against the new strains, with the continuous mutation of the new strains, it is still necessary to accelerate the development of new PCV2 vaccines

Method used

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  • Bivalent subunit vaccine of porcine circovirus type 2b and type 2d and preparation method thereof
  • Bivalent subunit vaccine of porcine circovirus type 2b and type 2d and preparation method thereof
  • Bivalent subunit vaccine of porcine circovirus type 2b and type 2d and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Construction of recombinant baculovirus carrying porcine circovirus 2b and 2d type Cap protein coding gene

[0053] (1) Construction of recombinant transfer vectors pFastBac-PCV2d ORF2 and pFastBac-PCV2b ORF2

[0054] 1. Codon optimization and synthesis of PCV2d-ORF2 and PCV2b-ORF2

[0055] With reference to the ORF2 gene sequence (Cap protein coding gene) of PCV2d (GenBank No. JQ002671) and PCV2b (GenBank No. KM924367), in order to realize the efficient expression of Cap protein, the present invention firstly compares the above-mentioned database according to the codon preference of baculovirus The sequence in is codon-optimized. The present invention found that the expression level and purity of the obtained Cap protein cannot meet the requirements of large-scale vaccine production and preparation by adopting conventional codon optimization software or simply following the codon preference of the insect-baculovirus expression system to optimize the codon of...

Embodiment 2

[0073] Example 2 Expression and purification of target proteins PCV2d-Cap and PCV2b-Cap

[0074] 1. Expression of target proteins PCV2d Cap and PCV2b Cap

[0075] The recombinant baculovirus harvested in Example 1 was inoculated into the suspension cultured insect cell High Five TM (purchased from Invitrogen), the inoculation dose was 0.01MOI, and the cell density was 0.8*10 6 / ml, the cell volume is 400ml. After 72-96 hours, the cell culture supernatant was harvested for Western blotting to detect the expression of the target proteins PCV2d-Cap and PCV2b-Cap. The results showed that the recombinant baculovirus Ac-PCV2d Cap-1, Ac-PCV2d Cap-2 and Ac-PCV2b Cap -1. Both Ac-PCV2bCap-2 can express PCV2d-Cap and PCV2b-Cap.

[0076] 2. Purification of target protein

[0077] The purification method adopts conventional nickel column affinity chromatography. The specific operation steps are as follows: Take HighFive 72-96h after inoculation TM The cell culture supernatant was cen...

Embodiment 3

[0079] The preparation of embodiment 3 porcine circovirus 2d type and 2b type bivalent subunit vaccine

[0080] The PCV2d-Cap and PCV2d-Cap proteins purified in Example 2 were filtered after their concentration was measured, the two were mixed with equal mass, and then emulsified with sterile 201 adjuvant (purchased from SEPPIC Company) according to the mass ratio of 3:1 to prepare For the bivalent subunit vaccine, the contents of PCV2d-Cap and PCV2d-Cap antigen in each milliliter of the vaccine are both 40 μg, and stored at 4°C until use.

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Abstract

The invention relates to the field of biotechnology, in particular to bivalent subunit vaccines of type 2b and 2d porcine circoviruses and preparation methods thereof. The invention provides recombinant baculoviruses comprising one or more copies of a Cap protein encoding gene of type 2b or 2d porcine circovirus. The invention also provides the bivalent subunit vaccines of the type 2b and 2d porcine circoviruses which comprise Cap proteins of the type 2b and 2d porcine circoviruses expressed by the recombinant baculoviruses respectively. Through artificial codon optimization, high-level and high-purity expression of the Cap proteins of the type 2b and 2d porcine circovirus is achieved, and the natural structure and immunogenicity of the Cap proteins is ensured. The bivalent subunit vaccines of the type 2b and 2d porcine circoviruses have high immunogenicity and safety, and exert an excellent immunoprotective effect on the porcine circoviruses.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant baculovirus expressing Cap proteins of porcine circovirus type 2b and type 2d and a preparation method thereof, as well as preparation of porcine circovirus type 2b and type 2d by using the recombinant baculovirus A bivalent subunit vaccine approach. Background technique [0002] Porcine circovirus type 2 (PCV2) belongs to the genus Circovirus of the Circoviridae family. The PCV2 virus genome is a covalently closed single-strand circular single-stranded DNA. One of the smallest animal viruses ever discovered. Because PCV2 is a non-enveloped virus, it has strong resistance to external environmental conditions. The virus remains stable in an acidic environment with a pH of 3 and a high temperature of 56°C to 70°C. It is resistant to alcohol, chlorhexidine, and chloroform. , iodine and other fat-soluble disinfectants have strong resistance, and are more sensitive to alka...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/34C07K14/01C07K16/08A61K39/295A61K39/12A61P31/20
CPCA61K39/12A61K2039/552A61K2039/70A61P31/20C07K14/005C07K16/081C12N7/00C12N2710/14021C12N2750/10022C12N2750/10034C12N2800/22
Inventor 周明光张华伟曾小燕朱娴静郝根喜汤细彪徐高原
Owner WUHAN KEQIAN BIOLOGY CO LTD
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