Virus-like particle vaccine for PCV2 (porcine circovirus 2) as well as preparation method and application of virus-like particle vaccine

A porcine circovirus, virus-like technology, applied in biochemical equipment and methods, viruses, antiviral agents, etc., can solve the problems of difficult to increase virus titer and high production cost, and achieve simple operation, strong immunogenicity, suitable for effect on mass production

Inactive Publication Date: 2016-10-12
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are mainly two kinds of PCV2 commercial vaccines on the market: whole virus inactivated vaccine and subunit vaccine. Regarding the whole virus inactivated vaccine, due to the characteristics of the virus itself and the problems in the production process, there are often problems such as difficulty in increasing the virus titer. Sometimes it needs to be concentrated to meet the requirements
The subunit vaccine used in China is mainly produced by Boehringer. The PCV2 subunit vaccine produced by this company uses baculovirus to express the target protein, and the production cost is very high, which makes it difficult for many farms to use the vaccine in production

Method used

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  • Virus-like particle vaccine for PCV2 (porcine circovirus 2) as well as preparation method and application of virus-like particle vaccine
  • Virus-like particle vaccine for PCV2 (porcine circovirus 2) as well as preparation method and application of virus-like particle vaccine
  • Virus-like particle vaccine for PCV2 (porcine circovirus 2) as well as preparation method and application of virus-like particle vaccine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The construction of embodiment 1Cap protein expression vector

[0032] Acquisition of Cap protein gene

[0033] Based on the amino acid sequence of the Cap protein of the PCV2 119-GD01 strain (PCV2b gene subtype, isolated and preserved in this experiment), the amino acid sequence was translated using the common codons of E. It is a nucleotide sequence to make it suitable for expression in Escherichia coli, and then entrusts Huada Gene Company to synthesize it. Restriction sites are added to both ends of the gene synthesis. The amino acid sequence is shown in SEQID NO: 1. The synthesized gene is named rORF2. See SEQ ID NO:2 for the gene sequence.

[0034] Construction of recombinant expression vector plasmid

[0035]rORF2 was double-digested with BamHI and HindIII restriction endonucleases, and then recovered and purified. pET-43.1a was also treated in the same manner as rORF2. The treated pET-43.1a and rORF2 were ligated at 16°C for 12h, and then the ligation produc...

Embodiment 2

[0038] Expression and purification of embodiment 2 recombinant protein

[0039] Induced expression of Cap protein

[0040] Take the pET-43.1a-rORF2 strain cultured overnight and inoculate it into fresh liquid LB medium containing ampicillin at a ratio of 1:100, and culture it on a shaker at 200 rpm at 37°C for about 3 hours, so that the OD600 value reaches about 0.6. Then add IPTG with a final concentration of 0.5mmol / L, induce expression at 16°C and 150rpm for 20h. SDS-PAGE analysis was carried out after sample processing (results see figure 1 ).

[0041] Purification and de-tagging of Cap protein

[0042] Treat the induced bacteria liquid according to the following steps

[0043] (1) Centrifuge at 5000rpm for 5 minutes to collect the bacteria, and then add 10ml PBS to resuspend the bacteria according to 100ml of the bacteria solution.

[0044] (2) The resuspended bacteria are crushed by ultrasonication, crushing conditions: power 200W, working time / interval time = 4sec / ...

Embodiment 3

[0054] The Cap protein of embodiment 3 electron microscope observation expression

[0055] Take 20 ul of the 10-fold diluted purified Cap protein sample and drop it on a 200-mesh copper grid, let it stand at room temperature for 3 minutes, and gently absorb the excess liquid with filter paper. Then it was stained with 3% phosphotungstic acid for 3 minutes, and observed under a transmission electron microscope after being completely dried. Electron microscope photo results see figure 2 .

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Abstract

The invention discloses a virus-like particle vaccine for PCV2 (porcine circovirus 2) as well as a preparation method and an application of the virus-like particle vaccine, and belongs to the technical field of bio-pharmaceuticals. The virus-like particle vaccine comprises soluble protein which is obtained through expression of a PCV2 complete capsid protein gene in escherichia coli, wherein the capsid protein gene is screened from capsid protein genes of domestic PCV2 prevalent strains at present and is subjected to codon optimization, therefore, the capsid protein gene can be expressed substantively in the escherichia coli, and the expressed soluble protein can form virus-like particles. The PCV2 subunit vaccine prepared with the method has characteristics of high antigen purity, high immunogenicity and the like; besides, the vaccine preparation process is simple, the production cost is low, and quite good application prospect is realized.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a porcine circovirus type II virus-like particle vaccine and a preparation method and application thereof. Background technique [0002] Porcine circovirus 2 (PCV2) is the main pathogen of postweaning multisystemic wasting syndrome (PMWS) in weaned piglets. It mainly infects piglets aged 6-15 weeks, and the clinical manifestation is progressive emaciation. , fever, swollen lymph nodes, jaundice, dyspnea and other symptoms. In addition, PCV2 is closely related to porcine dermatitis and nephritic syndrome, porcine respiratory syndrome, type A2 congenital tremor, porcine hyperplastic and necrotizing pneumonia, and reproductive disorders. The harm of PCV2 is also that it can damage the immune function of infected pigs, thereby leading to a decline in the body's resistance, making the body prone to secondary or concurrent other infectious diseases, causing grea...

Claims

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Application Information

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IPC IPC(8): A61K39/12A61K39/39A61P31/20
CPCA61K39/12A61K39/39A61K2039/525A61K2039/575C12N2750/10034
Inventor 樊惠英苗配思刘洁高应棋谢永生
Owner SOUTH CHINA AGRI UNIV
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