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Porcine circovirus bivalent genetic engineering vaccine

A porcine circovirus, amino acid technology, applied in the field of biotechnology genetic engineering, to achieve good safety, high immunogenicity, and the effect of preventing infection

Active Publication Date: 2018-12-21
QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Currently there is no domestic or international registered PCV-3 vaccine and PCV-1 and PCV-3 bivalent vaccine

Method used

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  • Porcine circovirus bivalent genetic engineering vaccine
  • Porcine circovirus bivalent genetic engineering vaccine
  • Porcine circovirus bivalent genetic engineering vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 The source of the fusion protein gene

[0021] The present invention comprehensively analyzes the gene sequence, antigen structure and epidemiological research progress of the main epidemic strains of porcine circovirus type 1 and type 3 at home and abroad, according to the amino acid sequence of its structural protein Cap protein, uses relevant bioinformatics software to analyze its relative Water, antigenicity, plasticity, surface accessibility and secondary structure are analyzed to predict possible B-cell epitopes and T-cell epitopes, and relevant reports are combined to determine PCV-1 and PCV-3 Cap proteins subunit. The vaccine skeleton structure is formed by flexible Linker connection and then connected in series with IL-5. The overall structure of the vaccine is:

[0022] PCV1 Cap - PCV3 Cap - IL-5 - Tag

Embodiment 2

[0023] Example 2 Construction of Escherichia coli expression vector and expression strain

[0024] The designed polypeptide encoding nucleotides in Example 1 were sent to Shanghai Handsome Biotechnology Co., Ltd. for synthesis. BamHI (5' end) and HindIII (3' end) restriction enzyme sites were designed at both ends of the nucleotide fragment respectively. The synthesized fragment was cloned into the pMD18T vector, and sequence determination confirmed that the inserted gene fragment was consistent with the involved sequence (see the sequence list). The recombinant plasmid was named pMD18T-PCV-IL-5, and the plasmid was digested with the corresponding restriction endonuclease. The Escherichia coli expression vector was pRSETB plasmid from Invitrogen Company, and the same restriction endonuclease was also used. Digestion conditions: 10 μL reaction system, 2 μL plasmid, 5 activity units of restriction endonuclease (New England biolabs), 1 μL 10× buffer, complete with deionized water...

Embodiment 3

[0028] Example 3 Fermentation, purification and emulsification of engineering bacteria

[0029]Fermentation Inoculate the production strains into 2 mL of LB liquid medium containing 100 μL / mL ampicillin, and culture at 37°C for 12 hours with shaking at 180 rpm to activate the strains. Put the activated strains into shake flasks at an inoculum size of 1:100, shake and culture at 37°C until OD600=3, and then inoculate them into fermenters at a ratio of 10%. The medium used for fermentation is a semi-synthetic medium prepared with distilled water and does not contain any antibiotics. Calibrate the dissolved oxygen and pH electrodes, turn on the tank to stir at 300 rpm, and sterilize the tank on-line. When the temperature of the culture solution in the tank drops to 37°C, calibrate the pH and dissolved oxygen (OD) zero point. The fermentation temperature is 37.0°C±0.1°C, the dissolved oxygen is controlled at about 20%, and the pH is controlled at 7.0. After inoculation, when the ...

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Abstract

The invention relates to preparation of a porcine circovirus bivalent genetic engineering vaccine. The vaccine is prepared from a PCV1 structural protein Cap protein subunit, a PCV3 structural proteinCap protein, a porcine interleukin-5, and a purification tag. The preparation process of the vaccine is stable and suitable for large-scale production. Animal experiments show that the porcine circovirus bivalent genetic engineering vaccine provided by the invention is good in safety, can induce a stronger immune response in an animal body, and effectively prevents the infection of a porcine circovirus type 1 and a porcine circovirus type 3.

Description

technical field [0001] The invention belongs to the field of biotechnology genetic engineering and relates to a fusion protein used for preventing porcine circovirus type 1 and porcine circovirus type 3. Specifically, using gene recombination technology, the subunit of porcine circovirus type 1 Cap protein, porcine circovirus type 3 Cap protein and molecular adjuvant IL-5 were connected in series, and cloned into the vector, transformed into host bacteria, fermented and purified A porcine circovirus bivalent genetically engineered vaccine and the application of the vaccine in preventing porcine circovirus type 1 and porcine circovirus type 3 are obtained through processes such as emulsification and emulsification. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) is a circular single-stranded DNA virus belonging to the family Circoviridae. The genome size of PCV is about 1.7kb, and the Cap protein is the only structural protein of PCV, which is rela...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/295A61K39/12A61P31/20
CPCA61K39/12A61K39/295A61K2039/552A61P31/20C07K14/005C12N2750/10022C12N2750/10034
Inventor 李殿明蒲勤张晓丹田春辉齐春梅刘甜甜任百亮张导春吴启凡党将将
Owner QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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