CN104263687A - Animal origin free culture medium for bordetella pertussis and culture method of animal origin free culture medium - Google Patents

Animal origin free culture medium for bordetella pertussis and culture method of animal origin free culture medium Download PDF

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CN104263687A
CN104263687A CN201410515654.8A CN201410515654A CN104263687A CN 104263687 A CN104263687 A CN 104263687A CN 201410515654 A CN201410515654 A CN 201410515654A CN 104263687 A CN104263687 A CN 104263687A
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animal origin
liquid
bordetella pertussis
substratum
culture medium
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陈道远
朱冲
陈元芬
韩炼
赵丹
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Chengdu Olymvax Biopharmaceuticals Inc
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract

The invention discloses an animal origin free culture medium for bordetella pertussis and a culture method of the animal origin free culture medium. The animal origin free culture medium contains sodium glutamate, phytone, L-proline, NaCl, KH2PO4g, KCl, MgCl2.6H2O, Tris, L-cystine, niacin, vitamin C, reduced glutathione, FeSO4.7H2O, CaCl2.2H2O, beta cyclodextrin or/and agar. The culture method comprises the following steps: strain opening, secondary culture, third culture and fermentation tank culture. The animal origin free culture medium disclosed by the invention contains animal origin free components, thereby completely avoiding the risk brought to vaccine safety because an animal is carried with a pathogen, enhancing the vaccine safety and reducing the side reaction of vaccine inoculation. The method for culturing the bordetella pertussis by utilizing the animal origin free culture medium has the advantages of easiness for operation, convenience for production, easy availability of raw materials and low cost.

Description

A kind of non-animal derived substratum of bordetella pertussis and cultural method thereof
Technical field
The invention belongs to biological technical field, be specifically related to a kind of non-animal derived substratum and cultural method thereof of bordetella pertussis.
Background technology
Whooping cough is the major reason causing global Infant and child deaths.According to WHO, global Whooping cough morbidity about 1,600 ten thousand example in 2008, wherein 95% occur in developing country, about 19.5 ten thousand children are therefore dead.In China, can find out from national Epidemic Situation of Notifiable Infectious Diseases circular in recent years and fall ill about having 2000 people every year.
Whooping cough to be propagated from the infected by the spittle by bordetella pertussis (Bordetella pertussis) and is caused to susceptible individual.Recent decades, by functional quality reliable pertussis vaccine, immunization is carried out to infant, successfully prevent in infant in the world and occur serious Whooping cough case.2008, the infant in the whole world about 82% was vaccinated with 3 pins time pertussis vaccine.WHO reports, within 2008, the whole world is by inoculation pertussis vaccine, avoids about 68.7 ten thousand examples dead.
Tin-free self-polishing anti-fou ling paint be instead of the DPT vaccine of full cell in the National immunization Program of China.Traditional whole-cell vaccines are deactivated compared with the huge advantage shown after severe side effect and DTaP vaccine fast development gradually due to it.Acellular pertussis vaccine (APV) self has good immune effect and the little advantage of side reaction.The acellular vaccine that WHO recommends comprises PT, FHA (filamentous hemagglutinin), PRN, FIM2 and FIM3 five kinds of antigens, wherein, and the main component that Toxins, pertussis (PT) is APV.Desirable pertussis vaccine should form by pertussis antigen that is clear and definite by antigenic component, that have good immunogenicity and protected effect and free of toxic effects.
And pertussis vaccine manufacturer generally all adopts and Whooping cough bacterial classification is opened to bag Jiang Shi substratum (containing sheep blood component), pass gac substratum (acid hydrolyzed casein containing Niu Yuan) again, finally inoculate SS substratum (or SS substratum of improvement), improvement SS substratum may contain the acid hydrolyzed casein of Niu Yuan.All there is potential infection animal and take viruliferous risk in the animal blood composition that these add in the medium and the nitrogenous source composition of animal-origin, and complicated component, brings unsafe risk to vaccine.
Summary of the invention
The object of the invention is to the shortcoming overcoming prior art, a kind of non-animal derived substratum of bordetella pertussis is provided, this substratum is non-animal derived composition, completely avoid because animal carries the risk that pathogenic agent brings to vaccine safety, improve the security of vaccine, reduce the side reaction of vaccine inoculation;
Another object of the present invention is to provide a kind of method utilizing this culture medium culturing bordetella pertussis, the method has simple to operate, convenient for production, the advantage easy, cost is low of drawing materials.
Object of the present invention is achieved through the following technical solutions: a kind of non-animal derived substratum of bordetella pertussis, described substratum is solid medium or liquid nutrient medium, wherein, the formula of solid medium comprises following component: be the amount of 1000ml relative to solvent load, containing Sodium Glutamate: 4.1 ~ 10g, phytone: 3.0 ~ 10g, L-PROLINE: 0 ~ 0.24g, NaCl:0.75 ~ 2.5g, KH 2pO 4: 0.15 ~ 0.5g, KCl:0.06 ~ 0.2g, MgCl 26H 2o:0.03 ~ 0.2g, Tris:0.45 ~ 6.1g, CYSTINE: 1.2 ~ 4.0g, nicotinic acid: 0.12 ~ 0.4g, vitamins C: 0.12 ~ 40g, reduced glutathion: 4.5 ~ 15g, FeSO 47H 2o:0.3 ~ 1.0g, CaCl 22H 2o:0.6 ~ 2.0g, beta-cyclodextrin: 0 ~ 100g, agar: 15 ~ 20g;
Described substratum is liquid nutrient medium, and formula comprises following component: be the amount of 1000ml relative to solvent load, containing Sodium Glutamate: 4.1 ~ 10g, phytone: 3.0 ~ 10g, L-PROLINE: 0 ~ 0.24g, NaCl:0.75 ~ 2.5g, KH 2pO 4: 0.15 ~ 0.5g, KCl:0.06 ~ 0.2g, MgCl 26H 2o:0.03 ~ 0.2g, Tris:0.45 ~ 6.1g, CYSTINE: 1.2 ~ 4.0g, nicotinic acid: 0.12 ~ 0.4g, vitamins C: 0.12 ~ 40g, reduced glutathion: 4.5 ~ 15g, FeSO 47H 2o:0.3 ~ 1.0g, CaCl 22H 2o:0.6 ~ 2.0g, beta-cyclodextrin: 0 ~ 100g.
Further, described phytone is one or more combination of rice protein peptone, wheat protein peptone, soy peptone.
Apply the method for above-mentioned culture medium culturing bordetella pertussis, it comprises the following steps:
S1. bacterial classification is opened: breakdown Whooping cough working seed lots bacterial classification, is inoculated in solid medium, cultivates 48 ~ 96 hours for 35 DEG C ~ 37 DEG C;
S2. two cultures are passed: be inoculated in by the Whooping cough bacterial classification that step S1 opens in two generation shaking flask liquid nutrient mediums, initial OD 600value is 0.1 ~ 0.4,35 DEG C ~ 37 DEG C and cultivates 20 ~ 26 hours, is second-generation bacterial kind bacterium liquid;
S3. three cultures are passed: be inoculated in three generations's shaking flask liquid nutrient medium by second-generation bacterial kind bacterium liquid, initial OD 600value is 0.1 ~ 0.4,35 DEG C ~ 37 DEG C and cultivates 20 ~ 26 hours, is three generations's bacterial classification bacterium liquid;
S4. fermentor cultivation: be inoculated in fermentor liquid substratum by three generations's bacterial classification bacterium liquid, aeration-agitation is cultivated, and culture temperature is 35 DEG C ~ 37 DEG C, and incubation time is 35 ~ 45 hours, and being cultured to fermented liquid pH value is 7.2 ~ 07.4, OD 600value is 5.0 ~ 10, by tank under fermented liquid, prepares pertussis antigen by separation and purification.
Further, the air flow of aeration-agitation described in step S4 is 10 ~ 20L/min, and stirring velocity is 150 ~ 300rpm.
The present invention has the following advantages: bordetella pertussis substratum of the present invention is as non-animal derived composition, the substratum adopting plant nitrogen source, good bacterial classification recovery and amplification cultivation can be carried out when bacterial classification goes down to posterity, produce poison and also can reach industry standard, therefore substratum of the present invention completely avoid because animal carries the risk that pathogenic agent brings to vaccine safety, improve the security of vaccine, the side reaction of vaccine inoculation can be reduced; The method of this culture medium culturing bordetella pertussis that utilizes provided by the invention has simple to operate, convenient for production, the advantage easy, cost is low of drawing materials.
Embodiment
Below in conjunction with embodiment, the present invention will be further described, and protection scope of the present invention is not limited to the following stated.
Embodiment 1: a kind of non-animal derived solid medium of bordetella pertussis, the formula of solid medium comprises following component: be the amount of 1000ml relative to solvent load, containing Sodium Glutamate: 4.1g, rice protein peptone: 3.0g, NaCl:0.75g, KH 2pO 4: 0.15g, KCl:0.06g, MgCl 26H 2o:0.03g, Tris:0.45g, CYSTINE: 1.2g, nicotinic acid: 0.12g, vitamins C: 0.12g, reduced glutathion: 4.5g, FeSO 47H 2o:0.3g, CaCl 22H 2o:0.6g, agar: 15g.
Embodiment 2: a kind of non-animal derived solid medium of bordetella pertussis, the formula of solid medium comprises following component: be the amount of 1000ml relative to solvent load, containing Sodium Glutamate: 10g, wheat protein peptone: 10g, L-PROLINE: 0.24g, NaCl:2.5g, KH 2pO 4: 0.5g, KCl:0.2g, MgCl 26H 2o:0.2g, Tris:6.1g, CYSTINE: 4.0g, nicotinic acid: 0.4g, vitamins C: 40g, reduced glutathion: 15g, FeSO 47H 2o:1.0g, CaCl 22H 2o:2.0g, beta-cyclodextrin: 100g, agar: 20g.
Embodiment 3: a kind of non-animal derived solid medium of bordetella pertussis, the formula of solid medium comprises following component: be the amount of 1000ml relative to solvent load, containing Sodium Glutamate: 6g, soy peptone: 4.2g, rice protein peptone: 4g, L-PROLINE: 0.15g, NaCl:1.4g, KH 2pO 4: 0.32g, KCl:0.14g, MgCl 26H 2o:0.1g, Tris:3.82g, CYSTINE: 2.6g, nicotinic acid: 0.27g, vitamins C: 25g, reduced glutathion: 8g, FeSO 47H 2o:0.6g, CaCl 22H 2o:01.3g, beta-cyclodextrin: 56g, agar: 18g.
Embodiment 4: a kind of non-animal derived liquid nutrient medium of bordetella pertussis, the formula of liquid nutrient medium comprises following component: be the amount of 1000ml relative to solvent load, containing Sodium Glutamate: 4.1g, wheat protein peptone: 5g, rice protein peptone: 5g, NaCl:2.5g, KH 2pO 4: 0.15g, KCl:0.2g, MgCl 26H 2o:0.03g, Tris:6.1g, CYSTINE: 1.2g, nicotinic acid: 0.4g, vitamins C: 0.12g, reduced glutathion: 15g, FeSO 47H 2o:0.3g, CaCl 22H 2o:2.0g.
Embodiment 5: a kind of non-animal derived liquid nutrient medium of bordetella pertussis, the formula of liquid nutrient medium comprises following component: be the amount of 1000ml relative to solvent load, containing Sodium Glutamate: 10g, rice protein peptone: 3.0g, L-PROLINE: 0.24g, NaCl:0.75g, KH 2pO 4: 0.5g, KCl:0.06g, MgCl 26H 2o:0.2g, Tris:0.45g, CYSTINE: 4.0g, nicotinic acid: 0.12g, vitamins C: 40g, reduced glutathion: 4.5g, FeSO 47H 2o:1.0g, CaCl 22H 2o:0.6g, beta-cyclodextrin: 100g.
Embodiment 6: a kind of non-animal derived liquid nutrient medium of bordetella pertussis, the formula of liquid nutrient medium comprises following component: be the amount of 1000ml relative to solvent load, containing Sodium Glutamate: 8g, phytone: 6.2g, L-PROLINE: 0.15g, NaCl:2g, KH 2pO 4: 0.34g, KCl:0.12g, MgCl 26H 2o:0.08g, Tris:5.2g, CYSTINE: 3.6g, nicotinic acid: 0.30g, vitamins C: 34g, reduced glutathion: 12g, FeSO 47H 2o:0.7g, CaCl 22H 2o:1.3g, beta-cyclodextrin: 80g.
Embodiment 7: a kind of method of cultivating bordetella pertussis, it comprises the following steps:
S1. bacterial classification is opened: breakdown Whooping cough working seed lots bacterial classification, is inoculated in solid medium prepared by embodiment 1, cultivates 48 hours for 35 DEG C DEG C;
S2. pass two cultures: be inoculated in by the Whooping cough bacterial classification that step S1 opens in two generation shaking flask liquid nutrient mediums, liquid nutrient medium is prepared by embodiment 4, initial OD 600value is 0.1,35 DEG C and cultivates 20 hours, is second-generation bacterial kind bacterium liquid;
S3. pass three cultures: be inoculated in three generations's shaking flask liquid nutrient medium by second-generation bacterial kind bacterium liquid, liquid nutrient medium is prepared by embodiment 4, initial OD 600value is 0.1,35 DEG C and cultivates 20 hours, is three generations's bacterial classification bacterium liquid;
S4. fermentor cultivation: be inoculated in fermentor liquid substratum by three generations's bacterial classification bacterium liquid, liquid nutrient medium is prepared by embodiment 4, and aeration-agitation is cultivated, and culture temperature is 35 DEG C, and incubation time is 35 hours, and being cultured to fermented liquid pH value is 7.2, OD 600value is 5.0, by tank under fermented liquid, prepares pertussis antigen by separation and purification, and wherein, the air flow of described aeration-agitation is 10L/min, and stirring velocity is 150rpm.
Embodiment 8: a kind of method of cultivating bordetella pertussis, it comprises the following steps:
S1. bacterial classification is opened: breakdown Whooping cough working seed lots bacterial classification, is inoculated in solid medium prepared by embodiment 2, cultivates 96 hours for 37 DEG C;
S2. pass two cultures: be inoculated in by the Whooping cough bacterial classification that step S1 opens in two generation shaking flask liquid nutrient mediums, liquid nutrient medium is prepared by embodiment 5, initial OD 600value is 0.4,37 DEG C and cultivates 26 hours, is second-generation bacterial kind bacterium liquid;
S3. pass three cultures: be inoculated in three generations's shaking flask liquid nutrient medium by second-generation bacterial kind bacterium liquid, liquid nutrient medium is prepared by embodiment 5, initial OD 600value is 0.4,37 DEG C and cultivates 26 hours, is three generations's bacterial classification bacterium liquid;
S4. fermentor cultivation: be inoculated in fermentor liquid substratum by three generations's bacterial classification bacterium liquid, liquid nutrient medium is prepared by embodiment 5, and aeration-agitation is cultivated, and culture temperature is 37 DEG C, and incubation time is 45 hours, and being cultured to fermented liquid pH value is 7.4, OD 600value is 10, by tank under fermented liquid, prepares pertussis antigen by separation and purification, and wherein, the air flow of described aeration-agitation is 20L/min, and stirring velocity is 300rpm.
Embodiment 9: a kind of method of cultivating bordetella pertussis, it comprises the following steps:
S1. bacterial classification is opened: breakdown Whooping cough working seed lots bacterial classification, is inoculated in solid medium prepared by embodiment 3, cultivates 72 hours for 36 DEG C;
S2. pass two cultures: be inoculated in by the Whooping cough bacterial classification that step S1 opens in two generation shaking flask liquid nutrient mediums, liquid nutrient medium is prepared by embodiment 6, initial OD 600value is 0.3,36 DEG C and cultivates 24 hours, is second-generation bacterial kind bacterium liquid;
S3. pass three cultures: be inoculated in three generations's shaking flask liquid nutrient medium by second-generation bacterial kind bacterium liquid, liquid nutrient medium is prepared by embodiment 6, initial OD 600value is 0.2,36 DEG C and cultivates 23 hours, is three generations's bacterial classification bacterium liquid;
S4. fermentor cultivation: be inoculated in fermentor liquid substratum by three generations's bacterial classification bacterium liquid, liquid nutrient medium is prepared by embodiment 6, and aeration-agitation is cultivated, and culture temperature is 36 DEG C, and incubation time is 42 hours, and being cultured to fermented liquid pH value is 7.3, OD 600value is 8, by tank under fermented liquid, prepares pertussis antigen by separation and purification, and wherein, the air flow of described aeration-agitation is 15L/min, and stirring velocity is 220rpm.
Below by way of description of test beneficial effect of the present invention:
Substratum of the present invention and conventional medium cultivate comparative experiments
1. substratum: solid of the present invention or liquid nutrient medium, control group is conventional bag Jiang Shi substratum, gac substratum, SS substratum;
2. cultivation results: as shown in table 1:
Table 1: substratum of the present invention and conventional medium cultivate comparative result
As shown in Table 1: adopt that substratum of the present invention carries out PT content in cultivation and fermentation liquid, FHA content is significantly higher than and adopts conventional medium to carry out PT content, the FHA content cultivated.

Claims (4)

1. the non-animal derived substratum of a bordetella pertussis, it is characterized in that, described substratum is solid medium or liquid nutrient medium, wherein, the formula of solid medium comprises following component: be the amount of 1000ml relative to solvent load, containing Sodium Glutamate: 4.1 ~ 10g, phytone: 3.0 ~ 10g, L-PROLINE: 0 ~ 0.24g, NaCl:0.75 ~ 2.5g, KH 2pO 4: 0.15 ~ 0.5g, KCl:0.06 ~ 0.2g, MgCl 26H 2o:0.03 ~ 0.2g, Tris:0.45 ~ 6.1g, CYSTINE: 1.2 ~ 4.0g, nicotinic acid: 0.12 ~ 0.4g, vitamins C: 0.12 ~ 40g, reduced glutathion: 4.5 ~ 15g, FeSO 47H 2o:0.3 ~ 1.0g, CaCl 22H 2o:0.6 ~ 2.0g, beta-cyclodextrin: 0 ~ 100g, agar: 15 ~ 20g;
Described substratum is liquid nutrient medium, and formula comprises following component: be the amount of 1000ml relative to solvent load, containing Sodium Glutamate: 4.1 ~ 10g, phytone: 3.0 ~ 10g, L-PROLINE: 0 ~ 0.24g, NaCl:0.75 ~ 2.5g, KH 2pO 4: 0.15 ~ 0.5g, KCl:0.06 ~ 0.2g, MgCl 26H 2o:0.03 ~ 0.2g, Tris:0.45 ~ 6.1g, CYSTINE: 1.2 ~ 4.0g, nicotinic acid: 0.12 ~ 0.4g, vitamins C: 0.12 ~ 40g, reduced glutathion: 4.5 ~ 15g, FeSO 47H 2o:0.3 ~ 1.0g, CaCl 22H 2o:0.6 ~ 2.0g, beta-cyclodextrin: 0 ~ 100g.
2. the non-animal derived substratum of a kind of bordetella pertussis as claimed in claim 1, is characterized in that, described phytone is one or more combination of rice protein peptone, wheat protein peptone, soy peptone.
3. the method for culture medium culturing bordetella pertussis as claimed in claim 1 or 2, it is characterized in that, it comprises the following steps:
S1. bacterial classification is opened: breakdown Whooping cough working seed lots bacterial classification, is inoculated in solid medium, cultivates 48 ~ 96 hours for 35 DEG C ~ 37 DEG C;
S2. two cultures are passed: be inoculated in by the Whooping cough bacterial classification that step S1 opens in two generation shaking flask liquid nutrient mediums, initial OD 600value is 0.1 ~ 0.4,35 DEG C ~ 37 DEG C and cultivates 20 ~ 26 hours, is second-generation bacterial kind bacterium liquid;
S3. three cultures are passed: be inoculated in three generations's shaking flask liquid nutrient medium by second-generation bacterial kind bacterium liquid, initial OD 600value is 0.1 ~ 0.4,35 DEG C ~ 37 DEG C and cultivates 20 ~ 26 hours, is three generations's bacterial classification bacterium liquid;
S4. fermentor cultivation: be inoculated in fermentor liquid substratum by three generations's bacterial classification bacterium liquid, aeration-agitation is cultivated, and culture temperature is 35 DEG C ~ 37 DEG C, and incubation time is 35 ~ 45 hours, and being cultured to fermented liquid pH value is 7.2 ~ 7.4, OD 600value is 5.0 ~ 10, by tank under fermented liquid, prepares pertussis antigen by separation and purification.
4. method of cultivating bordetella pertussis as claimed in claim 3, it is characterized in that, the air flow of aeration-agitation described in step S4 is 10 ~ 20L/min, and stirring velocity is 150 ~ 300rpm.
CN201410515654.8A 2014-09-29 2014-09-29 Animal origin free culture medium for bordetella pertussis and culture method of animal origin free culture medium Pending CN104263687A (en)

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Application publication date: 20150107