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Method for preparing proteus mirabilis-staphylococcus aureus-pseudomonas aeruginosa adsorption combined vaccine

The technology of Proteus mirabilis and Pseudomonas aeruginosa is applied in the field of preparation of Proteus mirabilis-Staphylococcus aureus-Pseudomonas aeruginosa adsorption combined vaccine, and can solve the problem that the natural structure of active components is incomplete and the recovery rate is low. , weak immunogenicity and other problems, to achieve the effect of improving immunogenicity and protection, avoiding large side effects, and covering a wide range

Inactive Publication Date: 2016-06-29
LIAONING CHENGDA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, relevant vaccine companies at home and abroad have actively carried out research and clinical trials on candidate vaccines of Staphylococcus aureus, but most of the clinical research on candidate vaccines of Staphylococcus aureus failed to achieve clinical expectations
Moreover, there are many pathogenic factors of the bacterial strain, and the content is low, and the whole bacterial vaccine and attenuated virus have certain immunogenicity, but the side effects are strong, and it is difficult to directly separate and purify the protective antigenic components from the whole bacterial body Large, complicated method, low recovery rate, unfavorable for the industrial production of vaccines, using genetic recombination technology to obtain relevant active components, the natural structure of the active components is incomplete, and the immunogenicity is not strong

Method used

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  • Method for preparing proteus mirabilis-staphylococcus aureus-pseudomonas aeruginosa adsorption combined vaccine
  • Method for preparing proteus mirabilis-staphylococcus aureus-pseudomonas aeruginosa adsorption combined vaccine
  • Method for preparing proteus mirabilis-staphylococcus aureus-pseudomonas aeruginosa adsorption combined vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Proteus mirabilis cell membrane soluble antigen stock solution.

[0033] Six strains PM13, PM28, PM43, PM62, PM92 and PM104 were cultured at high density in fermenter, and the cells were collected. Through enzymatic hydrolysis of the bacterial cell membrane, centrifugation, pre-filtration, ultrafiltration, and precipitation, the soluble fraction of the Proteus mirabilis cell membrane was separated and purified to obtain a highly protective Proteus mirabilis antigen stock solution (6 strains fermentation and purification conditions the same), the process flow is as figure 1 shown.

[0034] The purification steps are as follows.

[0035] (1) Enzymolysis: mix the bacterial cell solution obtained from high-density culture of fermenter with pH 6.0-8.0 in sterile 0.9% NaCl solution, and then mix according to 4.0-6.0×10 9 Add 1-2g trypsin to the concentration of bacteria per ml, pH7.8-8.5, digest at 50-65°C for 4.5 hours.

[0036] (2) Centrifugation: Collect the...

Embodiment 2

[0040] Example 2 Staphylococcus aureus cytoplasmic antigen stock solution.

[0041] The strain SA79 was cultured in a fermenter at a high density, and the cells were collected. Through the bead milling method, the bacteria are mechanically crushed, pre-filtered, isoelectric point precipitation 1, isoelectric point precipitation 2, enzymatic hydrolysis, and dialysis are used to separate and purify the cytoplasmic soluble components of Staphylococcus aureus to obtain highly protective gold. Staphylococcus aureus cytoplasmic antigen stock solution, the process is as follows figure 2 shown.

[0042] The purification steps are as follows.

[0043] (1) Mechanical crushing: Mix the bacteria solution obtained from high-density culture in the fermenter with a pH of 6.0-8.0 in a sterile 0.9% NaCl solution, and then pump the bacterial suspension into the crusher through a peristaltic pump for continuous crushing. The bead size range is 0.45-1.25mm, and the broken rate is not less tha...

Embodiment 3

[0050] Example 3 Staphylococcus aureus toxoid stock solution.

[0051] Staphylococcus aureus CMCC26002 was cultured in a fermenter at high density, and the bacterial suspension was collected. Separation and purification of Staphylococcus aureus toxoid components through centrifugation, detoxification, pre-filtration, ultrafiltration, acid precipitation, and centrifugation to obtain highly protective Staphylococcus aureus toxoid antigen stock solution. The process is as follows image 3 shown.

[0052] The purification steps are as follows.

[0053] (1) Centrifugation: Collect the supernatant by refrigerated high-speed centrifugation at 10,000-12,000 rpm, 4°C, for 10-15 minutes.

[0054] (2) Detoxification: add 0.4-1.0% formaldehyde solution or glutaraldehyde solution, 30-37°C, pH6.8-7.2 detoxification for 5-7 days.

[0055] (3) Pre-filtration: Use a 0.2+0.45um filter element to pre-filter the supernatant after centrifugation to obtain the pre-filtered clarified liquid.

[...

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Abstract

The invention discloses a method for preparing a proteus mirabilis-staphylococcus aureus-pseudomonas aeruginosa adsorption combined vaccine. The method includes the steps that firstly, a proteus mirabilis culture solution is subjected to in-situ digestion, high-speed centrifugation, pre-filtering, ultrafiltration and precipitation, and a proper proteus mirabilis cell membrane soluble antigen raw solution is obtained; secondly, a staphylococcus aureus bacterium suspension is subjected to centrifugation, smashing, re-centrifugation, filtering, precipitation, enzymolysis and dialysis, and a proper staphylococcus aureus cytoplast antigen raw solution is obtained; thirdly, a staphylococcus aureus and pseudomonas aeruginosa culture solution is subjected to centrifugation and pre-filtering, formalin is added for detoxification, then purification is conducted, and a proper inactivated staphylococcus aureus and pseudomonas aeruginosa toxin raw solution is obtained. The vaccine is used for preventing burn, scalding and infection caused by one or more of conditioned pathogens including proteus mirabilis, staphylococcus aureus and pseudomonas aeruginosa before and after operations in an intramuscular deep injection mode.

Description

technical field [0001] The present invention relates to a preparation method of a vaccine, in particular to a preparation method of Proteus mirabilis-Staphylococcus aureus-Pseudomonas aeruginosa adsorption combined vaccine (PSP adsorption combination vaccine), especially for prevention and treatment, Preparation method of adsorption combined vaccine for opportunistic pathogen burns, preoperative and postoperative infections, including Proteus mirabilis membrane soluble antigen, Staphylococcus aureus cytoplasmic antigen, Staphylococcus aureus toxoid, Pseudomonas aeruginosa The invention relates to a preparation method of a toxin and an aluminum hydroxide gel adsorption combined vaccine, which belongs to the field of medicine. Background technique [0002] In recent years, due to the increasingly serious use of antibiotics in China, Proteus mirabilis, Staphylococcus aureus, and Pseudomonas aeruginosa have become serious nosocomial infection pathogens, and they are also importa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/085A61K39/104A61K39/02A61P31/04
CPCA61K39/02A61K39/085A61K39/104A61K2039/70
Inventor 周荔葆杨文腰王瀛修雪亮王琦张建戴泽文
Owner LIAONING CHENGDA BIOTECH
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