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Mycoplasma hyopneumoniae multi-epitope mucosal vaccine

A technology of mycoplasma pneumonia and mucosal vaccines for swine, applied in the field of biotechnology genetic engineering, can solve the problems of ineffective suppression of MHP transmission, unsatisfactory, antigenic changes, etc.

Active Publication Date: 2017-07-07
QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This vaccine produced by traditional methods has encountered many problems: (1) due to the slow growth of MHP, the bacterial count titer is not very high, resulting in a long production cycle and high cost of live vaccines; , prone to antigenic changes, resulting in poor immune effect
[0010] There are two important defects in the currently used vaccines: ①The immune protection effect is not good. After vaccination, less than half of the pigs get immune protection. After the vaccine, pigs can still carry the bacteria and can spread to each other, resulting in the disease of pigs without immunity and some pigs that have been immunized, and the production performance is reduced. Experiments have confirmed that the infection rate of MHP between pigs that have been immunized and those that have not been immunized has no effect. Significant difference, indicating that the vaccines on the market did not have the expected immune protection and could not effectively inhibit the spread of MHP among pig herds
[0011] Mucosal immunity is the first line of defense of the body's immune defense, and plays a major role in defending against bacteria, viruses and other pathogens that cause intestinal and respiratory infections. However, the special physical and chemical properties of the mucosal surface weaken the strength of the mucosal immune response and even lead to immune resistance. by

Method used

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  • Mycoplasma hyopneumoniae multi-epitope mucosal vaccine
  • Mycoplasma hyopneumoniae multi-epitope mucosal vaccine
  • Mycoplasma hyopneumoniae multi-epitope mucosal vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Design Idea of ​​Multi-epitope Mucosal Vaccine Protein of Mycoplasma Porcine Pneumonia

[0053] According to the amino acid sequences of the main membrane proteins in the genome of the main epidemic strains of mycoplasma pneumoniae in China: adhesion protein (P97), lipoprotein (P65), and specific membrane protein (P46), the present invention utilizes relevant bioinformatics software to perform Th expression on it. Epitope, CTL epitope and B cell epitope analysis. The designed epitopes were concatenated and expressed in Escherichia coli, and after fermentation, purification, preparation and other processes, a multi-epitope mucosal vaccine of Mycoplasma suis pneumonia with ideal immunogenicity was obtained. The vaccine prepared by the invention can effectively prevent swine mycoplasma pneumonia.

[0054] Comprehensively analyzed the genome sequence, antigen structure, and epidemiological research progress of domestic Mycoplasma pneumonia epidemic strains in Chi...

Embodiment 2

[0055] Embodiment two Escherichia coli expression vector and the construction of expression bacterial strain

[0056] 1 Send the designed polypeptide encoding nucleotides to Shanghai Handsome Biotechnology Co., Ltd. for synthesis. BamHI (5' end) and HindIII (3' end) restriction enzyme sites are designed at both ends of the nucleotide fragment respectively. 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-CTB-Mhp (P97 / P65 / P46). Digest the plasmid with the corresponding restriction endonuclease. The expression vector of E. coli is the pRSETA plasmid from Invitrogen Company, which is also treated with the same restriction endonuclease. Digestion conditions: 10 μl reaction system, add 2 μl plasmid into the system , 5 activity units of restriction endonuclease (New England Biolabs), ...

Embodiment 3

[0060] Fermentation, purification and preparation of embodiment three engineering bacteria

[0061] 1 Fermentation Take the production strain pRSETA-CTB-Mhp(P97 / P65 / P46) / BL21(DE3, Plys), inoculate it in 2mL LB liquid medium (containing 100μg / mL ampicillin), and cultivate it with shaking at 200rpm for 12 hours at 37°C Activate the bacteria. 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 fermentation medium is a semi-synthetic medium prepared with distilled water. 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 was 37.0±0.1°C, the dissolved oxygen was controlled at about 40%, and the pH was controlled at 7.0. Afte...

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Abstract

The invention relates to preparation and application of a mycoplasma hyopneumoniae multi-epitope mucosal vaccine. A mycoplasma hyopneumoniae membrane protein, an adhesive protein P97, a lipoprotein P65, a specific membrane protein P46, a B cell epitope, a Th epitope, a CTL epitope and a cholera toxin subunit B are taken as a vaccine frame structure, a pRSETA carrier is cloned in through flexible linker connection, then Escherichia coli is transformed, and fermentation, purification and preparation technologies are carried out, so that the mycoplasma hyopneumoniae multi-epitope mucosal vaccine with ideal immunogenicity is obtained. A self-made mucosal adjuvant is used in a preparation process, so that production and using processes of the vaccine are simpler and more convenient. Animal experiments show that the mycoplasma hyopneumoniae multi-epitope mucosal vaccine not only has good safety but also can stimulate effective mucosal immunity, humoral immunity and cellular immune reactions.

Description

technical field [0001] The invention belongs to the field of biotechnology genetic engineering, and mainly relates to the preparation and application of a multi-epitope mucosal vaccine of swine mycoplasma pneumonia. Specifically, using genetic recombination technology, the cholera toxin is connected in series with the adhesion protein P97, lipoprotein P65, and specific membrane protein P46 epitopes related to Mycoplasma pneumoniae and cloned into the vector, and the host bacteria are transformed. After fermentation, purification, A recombinant multi-epitope mucosal vaccine of swine mycoplasma pneumonia and the application of the vaccine in the prevention of swine mycoplasma pneumonia were obtained through preparation and other processes. Background technique [0002] Mycoplasmal hyopneumoniae (MHP) is widely prevalent in all pig-raising countries in the world, and it is difficult to purify and eliminate pig farms once infected. The main clinical manifestations of the diseas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/02A61K39/39A61K9/12A61P31/04
CPCA61K9/12A61K39/02A61K39/39A61K2039/552A61K2039/55511A61K2039/55583A61K2039/55588C07K14/30C07K2319/00
Inventor 齐春梅许宏伟李殿明蒲勤田春辉刘甜甜任百亮张导春张晓丹吴启凡党将将
Owner QINGDAO MINGQIN BIOLOGICAL TECH CO LTD
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