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Porcine circovirus type 2d and type 3 cap protein dual subunit vaccine and its preparation method and application

A subunit vaccine, porcine circovirus technology, applied in the biological field to achieve the effect of activating immune response, retaining immunogenicity and high purity

Active Publication Date: 2021-05-04
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no vaccine on the market against both PCV2d and PCV3 subtype strains

Method used

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  • Porcine circovirus type 2d and type 3 cap protein dual subunit vaccine and its preparation method and application
  • Porcine circovirus type 2d and type 3 cap protein dual subunit vaccine and its preparation method and application
  • Porcine circovirus type 2d and type 3 cap protein dual subunit vaccine and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions. Example 1 Expression and purification of porcine circovirus type 2d and type 3 Cap proteins

[0046] The present embodiment provides two kinds of recombinant baculoviruses, respectively named as Ac-PCV2d Cap and Ac-PCV3 Cap, Ac-PCV2d Cap comprises the Cap protein gene sequence (SEQ ID NO: 1) of porcine circovirus type 2d, and Ac- PCV3Cap contains the Cap protein gene sequence of porcine circovirus type 3 (SEQ ID NO: 2). The gene sequences are all derived from artificial synthesis. After codon optimization, the optimized C...

Embodiment 2

[0071] Example 2 Preparation of porcine circovirus type 2d and type 3 Cap protein dual subunit vaccine

[0072] The purified PCV2d Cap and PCV3 Cap proteins were filtered after their concentrations were measured, and the two were mixed in an equal mass ratio, and then emulsified with sterile 201 adjuvant in a mass ratio of 3:1 to prepare a dual subunit vaccine. The contents of PCV2dCap and PCV3 Cap antigens are both 40 μg, and stored at 4°C until use. At the same time, after mixing PCV2d Cap and PCV3 Cap proteins in an equal mass ratio, the vaccine was prepared according to the mass ratio of antigen and Gel01 water adjuvant 4:1, and stored at 4°C for later use.

[0073] 1. Comparative test of vaccine stability prepared with different adjuvants

[0074] The two prepared porcine circovirus type 2d and type 3 Cap protein subunit vaccines were placed at 37°C, 25°C and 4°C for observation respectively. The stability of the vaccine was regularly sampled and observed, and the test ...

Embodiment 3

[0089] Example 3 Evaluation of immune protection effect and safety evaluation of porcine circovirus type 2d and type 3 Cap protein dual subunit vaccine

[0090] Evaluation of porcine circovirus type 2d and type 3 Cap protein dual subunit vaccines in mice:

[0091] 1. Safety evaluation of porcine circovirus type 2d and type 3 Cap protein dual subunit vaccine in mice

[0092] Purchase 10 6-8-week-old female Balb / C mice and divide them into two groups, A and B, with 5 in each group. Each mouse in group A was subcutaneously injected with 0.3ml of PCV type 2d and type 3 dual vaccine (201 adjuvant) prepared in Example 2; each mouse in group B was injected with 0.3ml of dialysis buffer (20mM Tris, 200mM NaCl); After 14 days of continuous observation, the mice in the two groups were in the same state and had no abnormal reaction, which indicated that the vaccine was safe for the mice.

[0093] 2. Immunogenicity evaluation of porcine circovirus type 2d and type 3 Cap protein dual sub...

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Abstract

The invention provides a porcine circovirus type 2d and type 3 Cap protein dual subunit vaccine as well as a preparation method and application thereof. By cloning porcine circovirus type 2d and type 3 Cap protein epitope genes, the highly purified type 2d and type 3 Cap proteins were successfully expressed using the insect-baculovirus expression vector system. Utilizing the two expressed proteins, the porcine circovirus type 2d and type 3 Cap protein dual subunit vaccine was developed for the first time. The prepared dual vaccine has strong immunity and high safety and is effective against porcine circovirus type 2d and type 3 Viruses can play an ideal role in immune protection, providing effective means for the prevention and control of porcine circovirus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a porcine circovirus type 2d and type 3 Cap protein dual subunit vaccine and its preparation method and application. Background technique [0002] Porcine circovirus (Porcine Circovirus, PCV) belongs to the Circoviridae genus Circovirus, is a non-enveloped single-stranded negative-sense circular DNA virus, and is one of the smallest animal viruses. It can cause proliferative and necrotizing pneumonia (PNP), porcine respiratory disease complex (PRDC), porcine dermatitis and nephropathy syndrome (porcine dermatitis and nephropathy, PDNS) and multisystem wasting syndrome in weaned piglets (Post weaning multisystemic wasting syndrome, PMWS) and so on. Three genotypes exist, including porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3). Because PCV is a non-enveloped virus, it has strong resistance to external environmental...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/01C07K19/00C12N15/866C12N15/62A61K39/12A61P31/20
CPCA61K39/12A61K2039/552A61K2039/70A61P31/20C07K14/005C07K2319/02C07K2319/21C12N15/86C12N2710/14143C12N2750/10022C12N2750/10034C12N2800/105
Inventor 张华伟周明光朱娴静罗修鑫徐高原陈波郝根喜方玉林汤细彪陈章表金建云
Owner WUHAN KEQIAN BIOLOGY CO LTD
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