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Porcine circovirus 3 Cap protein, nucleic acid, virus-like particles, vaccine, and preparation method and application of porcine circovirus 3 Cap protein

A porcine circovirus, virus-like technology, applied in the field of molecular biology, can solve the problems of low VLP expression, complex production process, endotoxin contamination, etc., to improve yeast expression efficiency, no endotoxin contamination, and simple production process. Effect

Active Publication Date: 2020-06-09
天康制药股份有限公司
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  • Abstract
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Problems solved by technology

However, the PCVcap protein expressed by Escherichia coli has low solubility and is polluted by endotoxin, and the endotoxin removal process in the later stage is complex and expensive; the VLP expressed by the insect baculovirus system has a good immune effect, but the insect baculovirus produced VLP expression is low, production cost is expensive, the production process is complicated, and it is difficult to produce large-scale production as an animal vaccine
As an efficient protein expression system, the yeast expression system has many advantages, but due to the influence of the PCV3 cap protein's own nuclear localization sequence, it is easy to form insoluble protein aggregation when directly expressing the protein, so the soluble expression of the PCV3 cap protein in the yeast system challenging

Method used

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  • Porcine circovirus 3 Cap protein, nucleic acid, virus-like particles, vaccine, and preparation method and application of porcine circovirus 3 Cap protein
  • Porcine circovirus 3 Cap protein, nucleic acid, virus-like particles, vaccine, and preparation method and application of porcine circovirus 3 Cap protein
  • Porcine circovirus 3 Cap protein, nucleic acid, virus-like particles, vaccine, and preparation method and application of porcine circovirus 3 Cap protein

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preparation example Construction

[0038] In some preferred embodiments, the present invention also provides a method for preparing the above-mentioned porcine circovirus type 3 Cap protein. The recombinant plasmid is transferred into a yeast strain, and after being expressed by the yeast strain, the porcine circovirus type 3 Cap protein is obtained.

[0039]Using the yeast expression system to express porcine circovirus type 3 Cap protein, compared with the existing baculovirus system expressing PCV3VLP, the production cost is low, the process is simple, the expression level is high, and it is easy to scale up to production; compared with the existing Escherichia coli expression system In comparison, it can express high-quality soluble target protein without endotoxin contamination. Therefore, the preparation method of porcine circovirus type 3 Cap protein provided by the present invention has the advantages of low production cost, simple production process, a large amount of target protein can be obtained thr...

Embodiment 1

[0064] Expression of embodiment 1PCV 3cap protein and assembly of virus-like particle (VLP)

[0065] Through epidemiological investigation and PCV3 virus evolution analysis, the cap protein sequence (SEQ ID NO.1) of a well-conserved and widespread strain was selected.

[0066] (1) About 50 amino acids at the N-terminal of the primary sequence of the PCV3 cap protein are its nuclear localization sequence. In this example, the NLS of the PCV3 cap protein is mutated according to the prediction website (cNLS mapper), and the mutated sequence is predicted by NLS The sequence (SEQ ID NO.2) used was obtained when the predicted site no longer had the characteristics of a nuclear localization sequence. Then carry out codon optimization to its sequence, add base GCCACC (to form KOZAK sequence together with initiation codon ATG) at the 5' end of the nucleic acid sequence after codon optimization, obtain the nucleic acid sequence (SEQ ID NO. 3). This step aims to continuously enhance it...

Embodiment 2

[0082] The preparation of embodiment 2 PCV 3 virus-like particle vaccines

[0083] Using the method described in this application, the purified PCV 3cap protein was emulsified with SEPPIC 206 adjuvant at a volume ratio of 1:1 (see the instructions of SEPPIC 206 for the emulsification method). The content of the emulsified PCV3 virus-like particle (VLP) protein antigen is 50 μg / mL. According to the requirements of the appendix of "Chinese Veterinary Pharmacopoeia" (current version), after the sterility test, viscosity measurement, and stability measurement are qualified, it is placed at 4°C for later use.

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Abstract

The invention provides a porcine circovirus 3 Cap protein, nucleic acid, virus-like particles, vaccine, and a preparation method and application of the porcine circovirus 3 Cap protein, and relates tothe technical field of molecular biology. The porcine circovirus 3 Cap protein provided by the invention has an amino acid sequence shown as SEQ ID NO.2, can be expressed in a soluble manner in a yeast expression system, and effectively improves yeast expression efficiency of the porcine circovirus 3 Cap protein. In addition, the porcine circovirus 3 Cap protein provided by the invention can be self-assembled into the virus-like particles, the vaccine prepared by using the virus-like particles has the characteristics of cell immunity and humoral immunity, and immunologic related experiments show that the immune effect is good. The method for expressing the porcine circovirus type 3 Cap protein by utilizing the yeast expression system has the advantages that production cost is low, the production process is simple, and a large amount of target proteins can be obtained through high-density fermentation, has no endotoxin pollution, and can be used for large-scale production.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a porcine circovirus type 3 Cap protein, a nucleic acid, a virus-like particle, a vaccine, a preparation method and an application. Background technique [0002] Porcine circovirus (PCV) is a single-stranded circular DNA virus with a gene length of about 1.7kb. It is one of the smallest animal DNA viruses. It mainly causes multisystem wasting syndrome, pneumonia, dermatitis, nephrotic syndrome, and reproductive impairment in piglets. , Bring heavy losses to the breeding of live pigs. PCV3 is a new type of circovirus, and there is no commercial vaccine against PCV3 in China. The homology of PCV3 and PCV2 cap protein genes is very low, only 30% identity, which also implies that the existing PCV2 vaccines cannot protect pigs from PCV3. Therefore, the research and development of PCV3 corresponding vaccine is very important. [0003] Recent studies have shown that PCV cap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01C12N15/34C12N15/81A61K39/12A61P31/20C12R1/84
CPCC07K14/005C12N15/815A61K39/12A61P31/20C12N2750/10022C12N2750/10023C12N2750/10034C12N2800/22A61K2039/5258A61K2039/552
Inventor 贺笋郭苗苗张国庆闫鹏先肖升东李延涛王清华曾钰潘毅平
Owner 天康制药股份有限公司
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