Separation and purification method for pertussis antigen

A technology for separation and purification of whooping cough, which is applied in the direction of bacterial antigen components, chemical instruments and methods, antibody medical components, etc. It can solve the problems that are not conducive to the production and income generation of vaccine companies, harsh elution conditions, and easy to degrade, and achieve rapid separation. Purification method, high purity of antigen, easy-to-obtain effect

Inactive Publication Date: 2015-12-23
BEIJING MINHAI BIOTECH
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

But these methods in the prior art all have respective deficiencies, for example: blue gum affinity chromatography, fetuin affinity chromatography, haptoglobin affinity chromatography, the bonding group of chromatographic filler is easy degraded and affect the quality of the vaccine
In addition, the eluent contains toxic substances or the elution conditions are too harsh, which will directly affect the yield and quality of the antigen
Furthermore, because the medium used for purification is very expensive, the production cost is increased, which is not conducive to the production and income generation of vaccine companies

Method used

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  • Separation and purification method for pertussis antigen
  • Separation and purification method for pertussis antigen
  • Separation and purification method for pertussis antigen

Examples

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Effect test

Embodiment 1 100

[0036] Example 1 Pertussis Antigen Isolation and Purification Method

[0037] Select domestic diatomite (Beijing Chuangqingyuan Filter Material Co., Ltd.), sieve to remove smaller and larger particles, and obtain a particle range of 60-200 μm. Use water for injection to wash the diatomite to remove floating and suspended matter , packed into a chromatographic column (Zhongsheng Huamei (Beijing) Technology Co., Ltd., column diameter 100mm), the packing height was 200mm, and the chromatographic column was washed with 5 times of 10mmol / LPB (pH8.0).

[0038] Open the freeze-dried strain bottle of Bordetella pertussis CS strain, purchased from China National Institutes for Food and Drug Control, transfer the strain to the modified ginger medium, culture at 35.5°C for 72 hours, collect and transfer to activated carbon half Cultivate on comprehensive medium at 35.5°C for 48 hours, then collect and transfer to activated carbon semi-comprehensive medium for 48 hours to collect strains....

Embodiment 2

[0043] Embodiment 2 diatomaceous earth specific adsorption PT and FHA

[0044] After 10 ml of the fermented broth concentrated in Example 1 was adjusted to 4-6 ms / cm with water for injection, it was thoroughly mixed with diatomaceous earth and allowed to stand at 4° C. for 10 minutes. Pour off the supernatant, wash the diatomaceous earth precipitate three times with 10ml10mmol / LPB (pH8.0), shake well each time, let it stand for 10 minutes, pour off the supernatant, after cleaning, add 10ml of 50mmol / LPB (pH 8.0) containing 0.5M NaCl was used for elution. The elution method was to shake the diatomaceous earth with buffer solution and then stand at 4°C for 10 minutes, and collect the supernatant. The supernatant was detected by electrophoresis, and the main components were PT and FHA. The results show that diatomite has specific adsorption on FHA and PT, and the elution of FHA and PT is related to the salt concentration.

Embodiment 3

[0045] Example 3 Purified PT and FHA purity detection

[0046] The purified PT and FHA were subjected to SDS-PAGE electrophoresis, and then the protein purity was determined by gel scanning.

[0047] The test method is to carry out SDS-PAGE electrophoresis with the 12% separation gel of the purified PT and PT standard substance with a concentration (the electrophoresis results are shown in Figure 4 ), the purified FHA and the FHA standard substance were carried out by SDS-PAGE electrophoresis with a concentration of 8% separating gel (see the electrophoresis results in Figure 5 ), after electrophoresis, the gel was scanned and analyzed to calculate the purity of the protein. Result shows that the PT of purification and PT standard substance, the FHA of purification and FHA standard substance have the same electrophoretic pattern, illustrate that PT and PT standard substance obtained by the method of embodiment 1, FHA and FHA standard substance have consistency, and purity a...

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Abstract

The invention provides a separation and purification method for a pertussis antigen. The separation and purification method comprises the steps of with diatomite and hydroxylapatite as column chromatography materials, after concentrating, regulating and conducting a bordetella pertussis fermentation solution, enriching the pertussis antigen by using the diatomite, and then, purifying by using the hydroxylapatite to obtain pertussis toxin (PT) and filamentous hemagglutinin (FHA). Proven through SDS-PAGE electrophoresis detection, the purities of both a PT protein and an FHA protein are higher than 95%. The method provided by the invention is simple in operation and capable of obtaining a high-purity antigen, not only greatly reducing the production cost, but also remarkably improving the quality of a product and increasing the yield of the product so as to be the simple and rapid separation and purification method for the pertussis antigen; in addition, the economic benefit is high, and the market application prospect is wide.

Description

technical field [0001] The invention relates to the technical field of biological product preparation, in particular to a method for separating and purifying pertussis antigens. Background technique [0002] Whooping cough has a high morbidity and mortality rate among children under the age of four. Early countries used whole-cell pertussis vaccines for prevention, but whole-cell pertussis vaccines may cause side effects such as fever, vomiting, and redness at the injection site. Individuals may also develop more serious brain neurosis and even die. Therefore, some countries, such as: the United Kingdom, Sweden and Japan have stopped vaccination of whole-cell pertussis vaccine. But then came the whooping cough epidemic. Therefore, in the 1980s, Japan first prepared an acellular pertussis vaccine instead of a whole-cell vaccine, which extracted effective antigenic proteins of Bacillus pertussis, such as filamentous hemagglutinin (FHA) and pertussis toxin (PT), and then pass...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/235C07K1/16A61K39/02A61P31/04A61P11/14
CPCC07K14/235A61K39/099
Inventor 张俊李国顺徐兴军刘建凯
Owner BEIJING MINHAI BIOTECH
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