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Pertussis vaccine preparation and combined vaccine thereof

A combined vaccine and pertussis technology, which can be used in drug combinations, medical preparations with inactive ingredients, and medical preparations containing active ingredients, etc., and can solve problems such as insufficient direct evidence.

Inactive Publication Date: 2015-09-16
BEIJING ZHIFEI LVZHU BIOPHARM +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other acellular pertussis vaccine components are thought to increase pertussis vaccine protection, but direct evidence is insufficient

Method used

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  • Pertussis vaccine preparation and combined vaccine thereof
  • Pertussis vaccine preparation and combined vaccine thereof
  • Pertussis vaccine preparation and combined vaccine thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Fermentation culture of pertussis bacteria

[0038] Open the strain of Bacillus pertussis, inoculate it on the modified bag-ginger medium (Bordet-Gengou), cultivate it at 35-37°C for no more than 72 hours, and transfer it to S-S medium (Stainer-Scholte) for no more than 48 hours hour to prepare the seed solution. Inoculate the seed solution in a fermenter containing liquid medium, the initial seed concentration OD 600 0.1-0.4, cultured at 35-37°C to bacterial concentration OD 600 Stop fermentation at 5.0-10.0, take samples for Gram staining microscopic examination and pure bacteria experiment. Add thimerosal to the non-polluting culture to sterilize it, and separate the bacteria and culture solution by a continuous flow centrifuge for future use.

Embodiment 2

[0040] Pertussis toxin extraction and detoxification

[0041] Example 1 The culture solution harvested by centrifugation was concentrated by ultrafiltration and replaced with a buffer solution (pH7.6) of 0.2M PB and 0.5M NaCl. After filtration and clarification, the sample was loaded on Butyl Sepharose (Butyl) hydrophobic chromatography After the column was fully washed with buffer, it was eluted with 0.2M PB (pH 6.2), and the eluate was collected. After diluting the eluate with water for injection, load it onto a Trisacryl Blue column with 0.05M PB (pH 6.2), wash it thoroughly, and elute linearly with 0.1M PB (pH 7.6) with a NaCl concentration of 0–500mM to collect the target peak . After the eluate was concentrated by ultrafiltration, it was loaded on a Sephacryl S-200 chromatographic column, and the elution peak before Kd 0.1 was collected to obtain the target protein pertussis toxin.

[0042] Add glutaraldehyde to the pertussis toxin to a final concentration of 0.5±0.1%,...

Embodiment 3

[0044] Lipooligosaccharide extraction and conjugate preparation

[0045] Extract lipooligosaccharides by conventional hot phenol method, collect them by ultracentrifugation, dissolve them into water for injection / 5% sodium nitrite / 30% acetic acid solution mixed at the same ratio at a concentration of 2-3mg / ml, incubate at 25°C for 4 hours, 200,000g After ultracentrifugation for 2 hours, the supernatant was collected, concentrated and purified by Bio-Gel P-2 chromatography to obtain detoxified lipooligosaccharides.

[0046] Mix and dissolve detoxified lipooligosaccharide and tetanus toxoid with 0.5M dipotassium hydrogen phosphate solution (pH 9.0), wherein the dissolved concentration of detoxified lipooligosaccharide is 10 mg / ml, and the dissolved concentration of tetanus toxoid is 2.5 mg / ml, after adding sodium borocyanide equivalent to sugar, airtight and react at 37°C for 7 days, the reaction solution was purified by Sephadex G-100 to obtain lipooligosaccharide conjugates. ...

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Abstract

The invention relates to a pertussis vaccine preparation and combined vaccine thereof. Pertussis lipooligosaccharide is obtained through bacterial culture purification or chemical synthesis, wherein the pertussis lipooligosaccharide obtained through purification is hydrolyzed to remove the endotoxin activity, the pertussis lipooligosaccharide obtained through chemical synthesis comprises a pertussis lipooligosaccharide tail end trisaccharide structure, and the lipooligosaccharide and carrier protein are coupled to prepare conjugate, namely the pertussis lipooligosaccharide conjugate with immunogenicity. The pertussis toxin is detoxicated pertussis toxin obtained through chemical detoxication or genetic engineering detoxication, and the weight proportion of the pertussis lipooligosaccharide conjugate to the pertussis toxin is 5-20 [mu]g:15-30 [mu]g.

Description

1. Technical field [0001] The invention relates to a novel pertussis component vaccine preparation and a combined vaccine containing the preparation, especially the pertussis component contains lipooligosaccharide component, which exists in a conjugated state and is used for the prevention of specific infectious diseases. 2. Background technology [0002] Pertussis is an acute respiratory infectious disease caused by Bordetella. The typical symptoms are sudden paroxysmal cough with an inspiratory epilogue, sometimes accompanied by vomiting. It can last for several months and mainly infects children. Adults who are not immune can also become infected. B. pertussis has humans as the only natural host. Although there is good vaccination coverage, there are still 50 million cases and about 300,000 deaths per year. 90% of the cases occur in developing countries. [0003] There are currently 2 pertussis vaccines, the whole-cell pertussis vaccine (wP) and the acellular pertussis v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/10A61K39/29A61K47/48A61P31/04A61P11/14A61K39/13A61K39/102
CPCY02A50/30
Inventor 杜琳
Owner BEIJING ZHIFEI LVZHU BIOPHARM
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