PCV2d (porcine circovirus type 2) virus-like particle vaccine and preparation method thereof

A porcine circovirus and virus-like technology, applied in the field of porcine circovirus 2d virus-like particle vaccine and its preparation, can solve the problems of immune evasion, the impact of disease prevention and control in pig farms, and the inability to effectively prevent and control the prevalence of PCV2d, etc. To achieve a good protective effect

Inactive Publication Date: 2018-08-03
JIANGSU NANNONG HI TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because PCV 2d extends a positively charged lysine at the C-terminus, which changes the original conformational epitope, the antibodies produced by the current vaccine cannot recognize it, further causing immune evasion. In 2012, the United States reported for the first time that the immune failure PCV 2d was isolated from pig farms. In order to control porcine circovirus-associated disease

Method used

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  • PCV2d (porcine circovirus type 2) virus-like particle vaccine and preparation method thereof
  • PCV2d (porcine circovirus type 2) virus-like particle vaccine and preparation method thereof
  • PCV2d (porcine circovirus type 2) virus-like particle vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] ——Expression of PCV2d Cap protein

[0085] 1) Transform the pET100-PCV 2d-SS-Cap-Pro recombinant expression plasmid into BL21(DE3) Escherichia coli (Beijing Quanshijin Company) competent cells, and spread it on LB cultured with ampicillin at a concentration of 100 μg / mL basal plate, 37 ℃ inverted culture 12 ~ 14h.

[0086] 2) Randomly pick a monoclonal colony to 10 mL of LB medium containing 100 μg / mL ampicillin antibiotic, and shake and culture at 220 r / min at 37° C. for 12 hours.

[0087] 3) According to the ratio of (V / V) 1:100, add 10 mL of bacterial solution to 1 L of α-lactose fermentation medium (0.01 mol / L) containing 100 μg / mL ampicillin antibiotic that has been autoclaved, 37 ° C 180 r Centrifuge at 8,000g for 10 minutes at 4°C, discard the supernatant, and store the E. coli pellet at -80°C for use. protein purification.

Embodiment 2

[0089] ——Purification of PCV 2d Cap protein by affinity chromatography

[0090] 1) The Escherichia coli precipitate is placed on an ice bath, and 100mL of PBS lysis buffer (Lysis buffer, its formula is: lysis buffer: NaCl 500mM, NaCl 2 HPO 4 100mM, Imidazole 30mM, KH 2 PO 4 100mM, Triton X-1001% (freshly added), pH 8.0) to lyse the centrifuged precipitate of 1L culture solution (concentrated according to the volume ratio of 1:10 times), and fully suspend the bacterial precipitate.

[0091] 2) The suspended sample is crushed and precipitated by a low-temperature ultra-high pressure continuous flow cell crusher, and the pressure is 1300bar, and the crushing is repeated 6 to 8 times. The crushed product is stained by Gram staining method, and more than 95% of E. coli are completely crushed under a microscope. The samples were then centrifuged at 16,000 g for 20 min at 4°C, and the supernatant was collected after centrifugation.

[0092] 3) Wash the Ni-NTA filler with 3 to 5...

Embodiment 3

[0101] - Assembly of PCV 2d virus-like particles

[0102] After performing SDS-PAGE analysis on the protein purified by affinity chromatography prepared in Example 2, select a high-concentration and high-purity protein and place it in a 7000D size dialysis bag and place it in a 4°C freezer in the assembly buffer of the present invention (Ammoniumcitrate 200mM, Na 3 PO 4 100mM, Tris 20mM, Arginine 20mM, pH7.0) in dialysis for 48h, the sample collected after dialysis was negatively stained with 1% phosphotungstic acid, and the virus-like particles were observed under a transmission electron microscope.

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Abstract

The invention relates to a PCV2d (porcine circovirus type 2) virus-like particle vaccine and a preparation method thereof. By means of epidemiological analysis for PCV2d strains of China, comparison of amino acid sequence information of a large quantity of collected strains as well as phylogenetic tree analysis, a Cap gene of a currently epidemic PCV2d strain in China is selected, PCV2d Cap protein is effectively expressed by an Escherichia coli prokaryotic expression system through codon sequence optimization, PCV2d virus-like particles are prepared successfully by purification and assembly in an in-vitro assembly and dialysis buffer solution, and form, size and concentration of the virus-like particles are not affected when the virus-like particles are placed for 6 months in a storage buffer solution at 4 DEG C and subzero 20 DEG C; the prepared PCV2d virus-like particle vaccine immunizes 21-day-old piglets, and a PCV2d challenge test proves that the vaccine has a good protecting effect for the piglets.

Description

technical field [0001] The content of this special invention includes the preparation method of porcine circovirus type 2d (PCV2d) virus like particles (virus like particles, VLPs), the in vitro assembly buffer (buffer) successfully dialyzed into VLPs, the reagent formula of VLPs long-term storage buffer and PCV2d The invention relates to an evaluation system of VLPs assembly efficiency, and the prepared virus-like particle vaccine is used to prevent porcine circovirus type 2d disease infection, belonging to the field of veterinary biological products. Background technique [0002] In 1991, Porcine circovirus type 2 (PCV 2) was first isolated from pigs in Canada. Currently, the genotypes of PCV2 are divided into: PCV 2a, PCV 2b, PCV 2c, PCV 2d (mPCV 2b) , PCV2c is currently only reported in Denmark. Initially, PCV2a was the main genotype of the epidemic, until 2003, the genotype of the main epidemic strain of PCV2 changed from PCV 2a to PCV 2b. In 2010, it was first report...

Claims

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Application Information

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IPC IPC(8): A61K39/12A61P31/20C12N15/70C12N15/34
CPCA61K39/12A61K2039/523A61K2039/53A61K2039/552A61P31/20C07K14/005C12N15/70C12N2750/10022C12N2750/10034C12N2800/22
Inventor 杨毅王东亮王乃东邹亚文雷昕诺湛洋余婉婷董彦鹏
Owner JIANGSU NANNONG HI TECH
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