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Preparation of blue ear disease protein engineering vaccine

A technology of protein engineering and blue ear disease, applied in the field of biotechnology genetic engineering, can solve the problems of strong virulence, incomplete understanding of immune protection, poor protection effect of heterologous strains, etc., and achieve the effect of preventing infection

Inactive Publication Date: 2014-08-20
青岛宝麦德生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There are three main problems in the development of more effective PRRSV vaccines: (1) PRRSV may induce inverse regulatory signals for the immune system, there is great antigenic variability in structural proteins, and therefore, immune protection is not fully understood
Live vaccines can only protect against homologous virus challenge, but are less effective against heterologous strains (Mengeling W Let al., 2003; Meng X J, 2000)
Live vaccines only partially improve clinical symptoms, but do not prevent infection
In addition, there may be a risk of virulence reversion to live vaccines (Opriessnig T et al., 2002)
On the other hand, inactivated vaccines are less effective in preventing and controlling disease

Method used

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  • Preparation of blue ear disease protein engineering vaccine
  • Preparation of blue ear disease protein engineering vaccine
  • Preparation of blue ear disease protein engineering vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example Escherichia coli expression vector and the construction of expression strain

[0023] Send the designed polypeptide-encoding nucleotides to Shanghai Handsome Biotechnology Co., Ltd. for synthesis. EcoRI (5' end) and HindIII (3' end) restriction enzyme sites are designed at both ends of the nucleotide fragment, and the fragment is synthesized Afterwards, 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 (GP3 / 4 / 5). The two plasmids were digested with the corresponding restriction enzymes. The E. coli expression vector was the pRSETB 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× buff...

Embodiment 2

[0027] Example 2 Fermentation, purification and emulsification of engineering bacteria

[0028] 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 20%, and the pH is controlled at 7.0....

Embodiment 3

[0031] Example 3 Safety Test of Recombinant PRRS Protein Engineering Vaccine

[0032] 1 material

[0033] 1.1 Vaccines: provided by Biomed Biological R&D Center, the batch numbers are 20120611, 20120612, 20120613.

[0034] 1.2 Experimental animals: 18-22g Balb / C mice were purchased from Beijing Huafukang. 30-day-old healthy three-way hybrid piglets were provided by Guangdong Yongshun Pharmaceutical.

[0035] 2 methods

[0036] 2.1 Safety of the vaccine on mice

[0037] Inject 18-22g of 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.

[0038] 2.2 Safety of vaccines on piglets

[0039] Select 30-day-old healthy three-way hybrid piglets, inject recombinant PRRS protein engineering vaccine into each head behind the ear, 5 piglets in each batch, and inject 15 piglets in three batches, and s...

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Abstract

The invention relates to preparation and application of a pig blue ear disease (porcine reproductive and respiratory syndrome, PRRSV) protein engineering vaccine. The vaccine uses body fluid and cellular immune epitopes of the main structural proteins GP5 and minor structural proteins GP3 and GP4 of PRRSV as the vaccine frame structure, the proteins are connected by a flexible linker, then put into series connection with the Thelper epitope, cloned into a pRSETB vector, transformed into Escherichia coli, and subjected to fermentation, purification, and emulsification to obtain the recombinant PRRS protein the engineering vaccine with ideal immunogenicity. The invention also relates to the usage method of the vaccine. The animal experiment shows that the PRRS protein engineering vaccine has virus attack protecting force equal to that of an attenuated vaccine and higher than that of an inactivated vaccine, can produce rapid and effective immune response, and prevent porcine reproductive and respiratory syndrome virus infection.

Description

technical field [0001] The invention belongs to the field of genetic engineering of biotechnology, and mainly relates to the preparation and application of a protein engineering vaccine for pig PRRS. Specifically, using genetic recombination technology, the epitopes of the main structural proteins GP3, GP4, and GP5 are connected in series and Thelper epitopes are added to the framework structure, cloned into E. coli vectors, transformed into host bacteria, and prepared by fermentation, purification, and emulsification processes. The recombinant PRRS protein engineering vaccine and the application of the vaccine in preventing the major animal disease PRRS are obtained. Background technique [0002] Porcine Reproductive and Respiratory Syndrome (PRRS), also known as Blue Ear Disease (PRRS), is a new virus caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRVS) of Arteriviridae Sexually transmitted diseases are a new disease characterized by reproductive disorder...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K39/12A61P31/14
Inventor 李殿明蒲勤李毅齐春梅田春辉任百亮张导春刘甜甜顾富香
Owner 青岛宝麦德生物医药科技有限公司
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