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Method for separating and purifying whooping cough toxins and filamentous hemagglutinin

A filamentous hemagglutinin, separation and purification technology, applied in the field of separation and purification of pertussis antigenic protein, can solve the problems of easy degradation, loss of PT and FHA, low yield, etc., achieve fast purification speed, simple operation, and controllable quality Effect

Inactive Publication Date: 2015-01-28
CHENGDU OLYMVAX BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005]1. Centrifugal co-purification process: The centrifugal co-purification process adopted by most manufacturers in my country and Japan, that is, after the bacterial culture solution is harvested, it is salted out by ammonium sulfate, high-salt Solution extraction and sucrose density gradient centrifugation obtain refined antigens, and then use glutaraldehyde solution or formaldehyde solution to detoxify the antigens into toxoids; however, this method requires a huge investment in equipment, and the purity of the products produced by this process is not high enough. The rate is also low, not suitable for large-scale production;
[0006]2. Column chromatography: including the following: (1) hydroxyapatite, haptoglobin column chromatography and gel filtration; (2) blue Gel affinity chromatography and fetuin affinity chromatography; (3) blue gel affinity chromatography and hydroxyapatite chromatography; (4) perlite chromatography and hydroxyapatite column chromatography; although These processes can be used to obtain relatively pure pertussis antigens PT and FHA, but (1), (2), and (3) processes have their inevitable problems, such as: blue gum affinity chromatography, fetuin affinity Chromatography, haptoglobin affinity chromatography, the binding group of the chromatography filler is easy to degrade 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; although the process (4) can obtain PT and FHA with higher purity and yield, the culture of the bacterin has been tested. After separation and purification by perlite column chromatography, the purity of PT and FHA can only reach 62%-68%. After separation and purification by hydroxyapatite column chromatography, the purity of PT and FHA can reach about 90%. Hydroxyapatite Stone is expensive, and about 10% of PT and FHA are lost in this step

Method used

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  • Method for separating and purifying whooping cough toxins and filamentous hemagglutinin
  • Method for separating and purifying whooping cough toxins and filamentous hemagglutinin
  • Method for separating and purifying whooping cough toxins and filamentous hemagglutinin

Examples

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Embodiment 1

[0022] Example 1: A method for separating and purifying pertussis toxin and filamentous hemagglutinin, which comprises the following steps:

[0023] S1. Sample treatment: After the B. pertussis culture is completed, the temperature of the culture medium is immediately lowered to 2°C. The culture medium is centrifuged to remove bacteria and collect the supernatant. This operation is carried out at a temperature of 2°C. The supernatant was concentrated to 1 / 12 of the original volume with an ultrafiltration membrane bag with a molecular weight cut-off of 10KD, and the pH of the concentrated solution was adjusted to 6.0;

[0024] S2. One Chromatography

[0025] S21. Elution of impurity proteins: Apply the above concentrated solution to a well-balanced Capto SP ImpRes chromatography column, and elute the impurity proteins with pH 6.0, 20mM PB, 2M urea, 0.11M NaCl buffer, and the elution peak of the impurity proteins Such as figure 1 shown;

[0026] S22. Separation of pertu...

Embodiment 2

[0029] Example 2: A method for separating and purifying pertussis toxin and filamentous hemagglutinin, which comprises the following steps:

[0030] S1. Sample treatment: immediately after the B. pertussis culture, cool down the culture solution to 8°C. The culture solution is subjected to tangential flow microfiltration and pressure filtration. The pore size of the filter membrane is 0.2 μm. Carried out at a temperature of 8°C. The supernatant was concentrated to 1 / 8 of the original volume with an ultrafiltration membrane bag with a molecular weight cut-off of 10KD, and the pH of the concentrated solution was adjusted to 6.0;

[0031] S2. One Chromatography

[0032] S21. Elution of impurity proteins: Apply the above concentrated solution to a well-balanced Capto SP ImpRes chromatography column, and elute the impurity proteins with pH 6.0, 20mM PB, 2M urea, 0.11M NaCl buffer, and the elution peak of the impurity proteins Such as figure 1 shown;

[0033] S22. Separati...

Embodiment 3

[0036] Example 3: A method for separating and purifying pertussis toxin and filamentous hemagglutinin, which comprises the following steps:

[0037] S1. Sample processing: After the B. pertussis culture is completed, the temperature of the culture medium is immediately lowered to 5°C. The culture medium is centrifuged by continuous flow to remove bacteria and collect the supernatant. This operation is carried out at a temperature of 5°C. The supernatant was concentrated to 1 / 10 of the original volume with an ultrafiltration membrane bag with a molecular weight cut-off of 10KD, and the pH of the concentrated solution was adjusted to 6.0;

[0038] S2. One Chromatography

[0039] S21. Elution of impurity proteins: Apply the above concentrated solution to a well-balanced Capto SP ImpRes chromatography column, and elute the impurity proteins with pH 6.0, 20mM PB, 2M urea, 0.11M NaCl buffer, and the elution peak of the impurity proteins Such as figure 1 shown;

[0040] S22....

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Abstract

The invention discloses a method for separating and purifying whooping cough toxins and filamentous hemagglutinin. The method comprises the following steps: S1, performing sample treatment, namely ending culture of bordetella pertussis, removing the thallus, collecting the supernatant, and concentrating the supernatant by using an ultrafiltration membrane; S2, performing primary chromatography comprising the sub-steps of eluting hybrid proteins, separating the whooping cough toxins and separating and purifying the filamentous hemagglutinin; and S3, performing secondary chromatography, namely loading a coarse product of the separated whooping cough toxins onto a CaptoMMC chromatographic column, eluting the buffer solution, collecting the eluant at the OD280 elution peak, thereby obtaining the purified whooping cough toxins. With the adoption of the CaptoSPImpRes chromatographic column and the CaptoMMC chromatographic column, the supernatant of the bordetella pertussis culture solution is separated and purified, so that PT and FHA can be accurately obtained, the quality is controllable, the purity of the separated and purified whooping cough antigen proteins is over 95 percent, and the method has the advantages of simplicity in operation, high purification speed and low cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for separating and purifying pertussis antigenic protein. Background technique [0002] Whooping cough (pertussis, whooping cough) is a highly contagious respiratory disease caused by Gram-negative bacillus pertussis. Neonatal and infant patients can cause apnea and cyanosis. Pertussis is highly contagious and difficult to diagnose in the early stage of the disease, so vaccination is the most economical and effective way to prevent and control pertussis. [0003] The pathogenic substances of B. pertussis mainly include pertussis toxin (Pertussis Toxin, PT), filamentous hemagglutinin (Filamentous Hemagglutinin, FHA), pertactin (PRN), agglutinogens (AGGs), adenosine Acid cyclase toxin (Adenylate Cyclase Toxin, ACT), tracheal cytotoxin (Tracheal Cytotoxin, TCT) and heat-labile toxin (Heat-labile toxin, HLT) and other biologically active substances, these biologic...

Claims

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Application Information

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IPC IPC(8): C07K14/235C07K1/36C07K1/34C07K1/18
CPCC07K14/235
Inventor 陈道远吴强马礼耕伍长华李建霖
Owner CHENGDU OLYMVAX BIOPHARM
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