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Large-scale preparation method of recombinant staphylococcus aureus vaccine

A staphylococcus, golden yellow technology, applied in the field of protein stock solution, can solve the problems of rapid spread of drug resistance, difficult to control, high incidence of nosocomial infection of Staphylococcus aureus, and incapable of large-scale preparation, etc., and achieves a simple preparation method and high quality Good, realize the effect of industrialized scale production

Pending Publication Date: 2019-10-18
CHENGDU OLYMVAX BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the present invention is that the incidence of nosocomial infection of Staphylococcus aureus is relatively high at present, and its drug resistance spreads quickly and is difficult to be controlled. The existing recombinant Staphylococcus aureus vaccine cannot be prepared on a large scale, and the preparation efficiency is relatively Low, the purpose is to provide a large-scale preparation method of recombinant Staphylococcus aureus vaccine, and solve the problem of large-scale preparation of recombinant Staphylococcus aureus vaccine

Method used

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  • Large-scale preparation method of recombinant staphylococcus aureus vaccine
  • Large-scale preparation method of recombinant staphylococcus aureus vaccine
  • Large-scale preparation method of recombinant staphylococcus aureus vaccine

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Experimental program
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Embodiment 1

[0044] The large-scale preparation method of recombinant Staphylococcus aureus vaccine of the present invention comprises the following steps:

[0045] (1) mSEB protein working seed batch strains were opened and inoculated in a Erlenmeyer flask. The inoculum size is strain: culture medium = 1:100 (ml:ml), and each Erlenmeyer flask is filled with 25±2ml LB liquid medium. Place it in a constant temperature shaking incubator at 36-38°C, and cultivate it at 180-300rpm for 6-10 hours, and it is the first-generation production strain.

[0046] (2) The first-generation production strain is inoculated in the seed tank for second-generation culture. The seed tank is filled with 30L of LB liquid medium, the inoculation amount is the first-generation strain: culture medium = 1:2000 (ml:ml), the parameters of the control seed tank are temperature: 36~38°C, 50~500rpm, air flow: 30~ 70L / min, pH: 7.2-7.5 and cultured for 6-10 hours, it is the second-generation production strain. During th...

Embodiment 2

[0063] (1) HI protein working seed batch strains were opened and inoculated in a Erlenmeyer flask. The inoculum size is strain: culture medium = 1:100 (ml:ml), and each Erlenmeyer flask is filled with 25±2ml LB liquid medium. Place it in a constant temperature shaking incubator at 36-38°C, and cultivate it at 180-300rpm for 6-10 hours, and it is the first-generation production strain.

[0064](2) The first-generation production strain is inoculated in the seed tank for second-generation culture. The seed tank is filled with 30L of LB liquid medium, the inoculation amount is the first-generation strain: culture medium = 1:2000 (ml:ml), the parameters of the control seed tank are temperature: 36~38°C, 50~500rpm, air flow: 30~ 70L / min, pH: 7.2-7.5 and cultured for 6-10 hours, it is the second-generation production strain. During the cultivation process, samples were taken every hour to detect the OD600 value. When the OD600 value was greater than 1.5, the seed tank cultivation ...

Embodiment 3

[0080] (1) MntC protein working seed batch strains were opened and inoculated in a Erlenmeyer flask. The inoculum size is strain: culture medium = 1:100 (ml:ml), and each Erlenmeyer flask is filled with 25±2ml LB liquid medium. Place it in a constant temperature shaking incubator at 36-38°C, and cultivate it at 180-300rpm for 6-10 hours, and it is the first-generation production strain.

[0081] (2) The first-generation production strain is inoculated in the seed tank for second-generation culture. The seed tank is filled with 30L of LB liquid medium, the inoculation amount is the first-generation strain: culture medium = 1:2000 (ml:ml), the parameters of the control seed tank are temperature: 36~38°C, 50~500rpm, air flow: 30~ 70L / min, pH: 7.2-7.5 and cultured for 6-10 hours, it is the second-generation production strain. During the cultivation process, samples were taken every hour to detect the OD600 value. When the OD600 value was greater than 1.5, the seed tank cultivati...

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Abstract

The invention discloses a large-scale preparation method of a recombinant staphylococcus aureus vaccine. The method comprises the following steps: opening a protein working seed lot, inoculating the protein working seed lot strain in a conical flask, inoculating first-generation production strain in a seeding tank, performing continuous second-generation cultivation, inoculating the second-generation production strain in a fermentation tank, and performing continuous third-generation cultivation; centrifugally removing supernate from a bacterial solution through a continuous flow after fermentation is completed; and performing bacterium body dissolving and bacterium disruption; performing combination, washing the mixture after combination is completed, performing digestion, purifying the protein digestion solution by adopting a chromatographic column and performing desalination, endotoxin removing, sterile filtration and storage. The preparation method is simple and can be used for preparing recombinant staphylococcus aureus vaccine in a large scale.

Description

technical field [0001] The invention relates to the field of protein stock solution, in particular to a large-scale preparation method of recombinant Staphylococcus aureus vaccine. Background technique [0002] Staphylococcus aureus is an important pathogen that causes nosocomial infection and community infection. It develops multi-drug resistance and becomes a difficult point in clinical treatment. It is urgent to develop a vaccine to control its infection and epidemic. [0003] Staphylococcus aureus, also known as "sarcophagus", is a representative of Gram-positive bacteria and is an important pathogen that causes hospital infections and community infections. Infection is characterized by acute and purulent, localized purulent infection of skin and soft tissue, which will not heal after a long time; whole body can lead to severe pneumonia, sepsis, endocarditis, septic arthritis, osteomyelitis, etc. Infection and complications, the mortality rate is as high as 20%. At the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P21/02C07K14/31A61K39/085A61P31/04
CPCC12N1/20C12P21/02C07K14/31A61K39/085A61P31/04
Inventor 樊绍文邹全明张卫军陈克平章金勇曾浩敬海明顾江朱冲刘唯樊钒
Owner CHENGDU OLYMVAX BIOPHARM
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