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Production method of acellular pertussis vaccine

A production method and pertussis technology, applied in the direction of antibacterial drugs, bacterial antigen components, recombinant DNA technology, etc., can solve the problems of material resources, extended production time, bottleneck of fermentation process optimization, and danger of experimenters, so as to reduce production costs , Simplify the production process and improve the yield

Active Publication Date: 2012-11-28
CANSINO BIOLOGICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] 3. A large number of experiments to evaluate the degree of Pt toxicity recovery in each batch of preparations is very time-consuming;
[0007] 4. Exposure to toxic reagents during the detoxification process is dangerous for experimenters
[0009] Therefore, the components FHA, Pt, and Prn in the acellular pertussis vaccine in the prior art are produced by fermentation with wild-type Bacillus pertussis strains, which cannot make the expression of each antigen reach the optimal level under the mastered technical ability , will cause a bottleneck in the optimization of the fermentation process; in addition, the simultaneous expression of FHA, Pt and Prn in the same wild-type B. pertussis strain will cause mutual interference during the purification process. These unfavorable factors will undoubtedly increase the manpower and material resources of production. extension of time

Method used

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  • Production method of acellular pertussis vaccine
  • Production method of acellular pertussis vaccine
  • Production method of acellular pertussis vaccine

Examples

Experimental program
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Effect test

Embodiment 1

[0053] The Fha pertussis genetically engineered strain of pt gene knockout is obtained by the following method:

[0054] (1) Obtaining pBS-ptx_up-KANA-ptx_down (SEQ ID.No.1) linear DNA

[0055] The linear DNA obtained after digestion with the single restriction site ScaI on the plasmid pBSptx_up-KANAptx_down was electrotransformed.

[0056] (2) Electrotransformation and homologous recombination

[0057] The linear DNA above 2ug was electroporated at a voltage of 1500V / 1mm to 200ul competent cells of B. pertussis CS strain (see reference 1 for the preparation method) and spread on a resistance plate containing 25ug / ml kanamycin for resistance screening , Bacterial colonies grown on a culture plate containing 25ug / ml kanamycin were screened for Fha pertussis genetically engineered strains with knockout of the pt gene. The kanamycin resistance gene can also be replaced with a tetracycline resistance gene or a chloramphenicol resistance gene.

Embodiment 2

[0059] The Pt pertussis genetically engineered strain of fha gene knockout is obtained by the following method:

[0060] (1) Obtaining linear DNA of pBS-fha_up-TET-ptx_down (SEQ ID.No.2) plasmid

[0061] The linear DNA obtained after digestion with the single restriction site ScaI on the plasmid pBS-fha_up-TET-ptx_down was electrotransformed.

[0062] (2) Electrotransformation and homologous recombination

[0063] The linear DNA above 2ug was electroporated at a voltage of 1500V / 1mm to 200ul competent cells of CS strain of B. Colonies grown on a tetracycline / ml culture plate were screened for fha knockout tetracycline-resistant Tet R &△FHA. pertussis strains. The tetracycline resistance gene can also be replaced with a kanamycin resistance gene or a chloramphenicol resistance gene.

[0064] (3) Obtaining pBS-fha_up-Pt-KANA-ptx_down (SEQ ID.No.3) linear DNA

[0065] The linear DNA obtained after digestion with the restriction site DraI on the plasmid pBS-ptx_up-KANA-ptx_d...

Embodiment 3

[0069] The Prn pertussis genetically engineered strain of fha gene knockout is obtained by the following method:

[0070] (1) Obtaining linear DNA of pBS-fha_up-TET-ptx_down (SEQ ID.No.2) plasmid

[0071] The linear DNA obtained after digestion with the single restriction site ScaI on the plasmid pBS-fha_up-TET-ptx_down was electrotransformed.

[0072] (2) Electrotransformation and homologous recombination

[0073] The linear DNA above 2ug was electroporated at a voltage of 1500V / 1mm to 200ul competent cells of CS strain of B. Colonies grown on a tetracycline / ml culture plate were screened for fha knockout tetracycline-resistant Tet R &△FHA. pertussis strains. The tetracycline resistance gene can also be replaced with a kanamycin resistance gene or a chloramphenicol resistance gene.

[0074] (3) Obtaining pBS-fha_up-Prn-KANA-ptx_down (SEQ ID.No.4) linear DNA

[0075] The linear DNA obtained after digestion with the restriction site DraI on the plasmid pBS-fha_up-Prn-KANA...

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Abstract

The invention discloses a production method of an acellular pertussis vaccine. The production method comprises the following steps of: fermenting and purifying a Fha bordetella pertussis genetic engineering strain in which pt gene is knocked out to obtain an antigen component fha; fermenting and purifying the Pt bordetella pertussis genetic engineering strain in which fha gene is knocked out to obtain an antigen component pt; fermenting and purifying Prn bordetella pertussis genetic engineering strain in which fha gene is knocked out to obtain an antigen component prn; and preparing the acellular pertussis vaccine from the antigen components fha, pt and prn. According to the method, high-efficiency expression of a single target antigen can be realized, so that the growing speed of the bordetella pertussis genetic engineering strain is remarkably accelerated, and the production time and production cost can be reduced.

Description

technical field [0001] The invention relates to the field of research and development of vaccine production strains, in particular to a production method of an acellular pertussis vaccine. Background technique [0002] Pertussis is an acute respiratory infectious disease caused by Bacillus pertussis. Its clinical features are paroxysmal spasmodic cough accompanied by deep and long "chicken crowing"-like inspiratory roar. The pertussis bacillus, Bordetella pertussis, mainly affects children, and half of the cases are infants under one year old. [0003] The pathogenic factor of Bacillus pertussis is mainly pertussis toxin, pertussis toxin, PT, which is a protein complex composed of five different subunits s1-s5 (encoding genes are ptxA, ptxB, ptxD, ptxE, ptxC, and its toxicity comes from s1 The ADP-ribosyltransferase activity of the subunit. In the acellular pertussis vaccine that has been adopted, in addition to PT, it also contains other antigenic molecules such as filame...

Claims

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Application Information

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IPC IPC(8): A61K39/10A61P31/04C12N15/09
Inventor 朱涛段磊宇学锋邵忠琦毛慧华邱东旭
Owner CANSINO BIOLOGICS INC
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