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Preparation method for recombinant ubiquitin blue ear disease vaccine

A blue-ear disease and ubiquitination technology, applied in the field of biotechnology genetic engineering, can solve problems such as the inability to effectively remove viruses

Active Publication Date: 2015-04-08
QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pigs immunized with these vaccines, even though they mount a good immune response, produce high levels of neutralizing antibodies, but these neutralizing antibodies are not effective in clearing the virus
This poses new challenges for vaccine development

Method used

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  • Preparation method for recombinant ubiquitin blue ear disease vaccine
  • Preparation method for recombinant ubiquitin blue ear disease vaccine
  • Preparation method for recombinant ubiquitin blue ear disease vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example Escherichia coli expression vector and the construction of expression strain

[0025]The designed polypeptide-encoding nucleotides were sent to Shanghai Handsome Biotechnology Co., Ltd. for synthesis. BamH I (5' end) and HindIII (3' end) restriction enzyme sites were designed at both ends of the nucleotide fragment. After synthesis, they were respectively cloned into the pMD18T vector, and sequence determination confirmed that the inserted gene fragment was consistent with the designed sequence (see the sequence list). The recombinant plasmids were named pMD18T-PRRSV (GP5 / 6 / 7 / Ub). The two plasmids were digested with the corresponding restriction enzymes. The Escherichia coli expression vector was the pRSETA plasmid from Invitrogen Company, and the same restriction enzymes were also used for treatment. Digestion conditions: 10 μl reaction system, adding 2 μl of plasmid, 5 activity units of restriction endonuclease (New England biolabs), 1 μl of 10× buffer was ad...

Embodiment 3

[0029] Fermentation, purification and emulsification of embodiment three engineering bacteria

[0030] The production strains were taken for fermentation, inoculated into 2ml LB liquid medium (containing 100 μg / ml ampicillin), and cultured at 37° C. with shaking at 180 rpm for 12 hours to activate the strains. Then inoculate the shake flask with an inoculation amount of 1:100, shake and culture at 37°C until OD600=3, and then inoculate it into a fermenter 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, start the tank to stir, the rotation speed is 300rpm, and sterilize the tank on-line. When the temperature of the culture solution in the tank drops to 37.0°C, calibrate the pH and dissolved oxygen (OD) zero point. The fermentation temperature is 37.0±0.1°C, the dissolved oxygen is controlled at about 40%, and the pH is controlled ...

Embodiment 4

[0033] Example 4 Safety Test of Recombinant Ubiquitinated PRRS Vaccine

[0034] Safety of Vaccines in Mice

[0035] Inject 18-22g Balb / C mice subcutaneously, 0.5ml each, and inject 5 mice in each batch of vaccine, a total of three batches, inject 15 mice, and set 2 negative controls at the same time, observe continuously for 10 days, observe Health status of mice.

[0036] Safety of Vaccines in Piglets

[0037] Select 30-day-old healthy three-element hybrid piglets, and inject recombinant ubiquitinated PRRS vaccine intramuscularly behind each ear, 5 piglets in each batch, a total of three batches, 15 piglets were injected, and 2 negative controls were set up at the same time. Head injection of physiological saline white oil emulsion 2ml, clinical observation for 10 days.

[0038] result

[0039] Safety Test of Vaccine on Mice

[0040] The results are shown in Table 1. The body temperature of one animal in the 20120208 immunization group rose slightly on the second day, an...

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Abstract

The invention relates to a preparation method and an application for a pig blue ear disease (porcine reproductive and respiratory syndrome virus, PRRSV) recombinant ubiquitin vaccine. The preparation method comprises the following steps: by taking a body fluid which contains PRRSV major structural proteins GP5, GP6 and GP7 and a cellular immune epitope as a vaccine framework structure, connecting the vaccine framework structure through a flexible linker and then connecting the vaccine framework structure with ubiquitin in series, cloning into a pRSETA vector and then transforming escherichia coli, performing processes such as fermenting, purifying and emulsifying, thereby obtaining the recombinant blue ear disease protein engineering vaccine with ideal immunogenicity. The invention further relates to a using method of the vaccine. According to the animal experiments, the toxic substance counteracting protection of the blue ear disease protein engineering vaccine is equivalent to that of an attenuated vaccine, and higher than that of an inactivated vaccine, so that T lymphocyte multiplication immune reaction on cellular level can be generated by stimulating, antibody immune reaction with virus neutralizing activity is produced on body fluid level, and the pig blue ear disease can be prevented.

Description

technical field [0001] The invention belongs to the field of biotechnology genetic engineering, and mainly relates to the preparation and application of a ubiquitinated vaccine for pig blue-ear disease. Specifically, using genetic recombination technology, the epitopes of the main structural proteins GP5, GP6, and GP7 were connected in series with porcine ubiquitin protein, cloned into a vector, transformed into a host bacterium, prepared through fermentation, purification, and emulsification processes to obtain recombinant PRRS Ubiquitination vaccine and the application of the vaccine in the prevention of major animal disease pig blue-ear disease. Background technique [0002] Porcine reproductive and respiratory syndrome is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). Immunosuppression is followed by a variety of other diseases. In recent years, porcine reproductive and respiratory syndrome virus has caused huge econo...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K39/12A61K47/48A61P31/14
Inventor 李殿明蒲勤张毓金齐春梅田春辉刘甜甜任百亮张导春党将将吴启凡
Owner QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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