CN104263678A - Corynebacterium diphtheriae culture medium and method for preparing diphtheria toxoid by applying same - Google Patents
Corynebacterium diphtheriae culture medium and method for preparing diphtheria toxoid by applying same Download PDFInfo
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Abstract
The invention discloses a corynebacterium diphtheriae culture medium. The corynebacterium diphtheriae culture medium consists of a basic culture medium, an I liquid and an II liquid, wherein the basic culture medium consists of the materials of HyPep5630, Amisoy, Na2HPO4.12H2O, lactic acid, yeast powder and calcium chloride; the I liquid is L-cystine solution; the II liquid consists of the materials of magnesium sulfate, beta-alanine, niacin, pimelic acid, copper sulfate pentahydrate, zinc sulfate heptahydrate and manganese chloride tetrahydrate. The method for preparing diphtheria toxoid by applying the corynebacterium diphtheriae culture medium is culture enabling, two-generation culturing, three-generation culturing, four-generation culturing and fermentation tank culturing. The culture medium is the animal-free culture medium; good culture recovery and multiplication culture are carried out during culture passage; and the toxin production can also reach the requirement of the existing pharmacopoeia criterion. The method for preparing the diphtheria toxoid by using the culture medium has the advantages of being simple to operate, convenient to produce, easy in taking of materials and low in cost.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of diphtheria corynebacterium substratum and apply its method preparing diphtheria toxoid.
Background technology
Diphtheria is that dyspnoea can be caused even to suffocate, and be one of childhood disease of most terrorise in history, the outburst of this disease has destructiveness always by the Acute respiratory infectious disease that can produce ectotoxic diphtheria corynebacterium and cause.2006 the World Health Organization (WHO) be reported in and use in a large number before diphtheria toxoid the eighties in 20th century, about have 1,000,000 cases every year in developing country, 50,000 ~ 60,000 examples are dead.Even if in recent years, in endemic area, the report case fatality rate of diphtheria is still more than 10%.
People is unique natural host of diphtheria corynebacterium.Diphtheria is propagated by means of only the spittle and close contact, and therefore keep the vaccine height rate of vaccination of children and adult very important, this point obtains confirmation from the outburst of the many local diphtheria in the world, and the diphtheria especially occurring in USSR (Union of Soviet Socialist Republics) the nineties in 20th century is very popular.
The composition of diphtheria vaccine is diphtheria toxoid, and namely treated diphtheria toxin can induce the toxinicide with provide protection.Expand after the Immune Programming (EPI) starts from WHO in 1974, during 1980 ~ 2000 years, the general report case load of diphtheria reduces amplitude and is not less than 90%, and the provide protection of vaccine is fairly obvious.After conventional diphtheria vaccine fundamental immunity program completes, average guard time is about 10 years.For prevention adult diphtheria, be necessary the adult being in some epidemiology environment every 10 years booster immunizations 1 time, to keep immunizing power.
The Production Flow Chart that diphtheria toxoid is commonly used is opened in solid LuShi blood serum medium with diphtheria corynebacterium bacterial classification, reaches liquid improvement Lin Shi (Huang Shi) substratum and produce diphtheria toxin after recovery amplification, or employing CY substratum.But will use the horse serum without deactivation in the preparation of solid LuShi blood serum medium, horse serum is a kind of foreign preteins to human body, may form allergen, cause the generation of the propagation of pathogenic agent and rejection, cause human body uncomfortable; Lin Shi substratum main component trysinization beef obtains, and CY substratum major nitrogen source is that hydrochloric acid hydrolysis casein food grade obtains, and beef and casein food grade are all ox source property, and pancreatin is ox source or pig source.These compositions all have certain potential risk.And confirm that mad cow disease, the 1996 annual reports mankind newly make a variation after creutzfeldt-Jacob disease (vCJD) first from Britain in 1986, although there is no evidence in the world shows that the biological products such as vaccine are the paathogenic factors of variability creutzfeldt-Jacob disease at present, only theoretic by vaccine broadcasting vCJD to the danger of the mankind, but for safety, as prevention, National Drug Administration of China calendar year 2001 starts just medicine, biological products to be produced to the ox source property material used and given stringent regulations.The animal source such as horse serum and beef substratum is not used to avoid generation and the transmission of disease of abnormal response.
Summary of the invention
The object of the invention is to the shortcoming overcoming prior art, a kind of diphtheria corynebacterium substratum is provided, this substratum is non-animal derived composition, the substratum adopting plant nitrogen source, can carry out good bacterial classification recovery and amplification cultivation, produce poison and also can reach existing standards of pharmacopoeia requirement when bacterial classification goes down to posterity;
Another object of the present invention is to provide a kind of method utilizing this medium preparing diphtheria toxoid, 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 diphtheria corynebacterium substratum, and described substratum is made up of basic medium, No. I liquid and No. II liquid, and weight ratio is 100:0.3 ~ 0.6:0.3 ~ 0.6;
Described basic medium is made up of the raw material of following concentration: HyPep5630:10 ~ 20g/L, Amisoy:10 ~ 20g/L, Na
2hPO
412H
2o:1 ~ 5g/L, lactic acid: 2 ~ 3g/L, yeast powder: 0.1 ~ 0.5g/L, calcium chloride: 5 ~ 15g/L;
Described No. I liquid is CYSTINE solution, and concentration is 150 ~ 250g/L;
Described No. II liquid is made up of the raw material of following concentration: magnesium sulfate: 100 ~ 120g/L, Beta-alanine: 0.5 ~ 1.5g/L, nicotinic acid: 0.5 ~ 1.5g/L, pimelic acid: 0.04 ~ 0.15g/L, cupric sulfate pentahydrate: 0.3 ~ 0.8g/L, Zinc Sulphate Heptahydrate: 0.2 ~ 0.8g/L, tetrahydrate manganese chloride: 0.1 ~ 0.2g/L.
As preferably, described substratum is made up of basic medium, No. I liquid and No. II liquid, and weight ratio is 100:0.3 ~ 0.6:0.3 ~ 0.6;
Described basic medium is made up of the raw material of following concentration: HyPep5630:13 ~ 18g/L, Amisoy:13 ~ 18g/L, Na
2hPO
412H
2o:1.5 ~ 3.5g/L, lactic acid: 2.3 ~ 2.7g/L, yeast powder: 0.25 ~ 0.35g/L, calcium chloride: 6 ~ 10g/L;
Described No. I liquid is CYSTINE solution, and concentration is 180 ~ 230g/L;
Described No. II liquid is made up of the raw material of following concentration: magnesium sulfate: 105 ~ 112g/L, Beta-alanine: 0.8 ~ 1.2g/L, nicotinic acid: 0.8 ~ 1.2g/L, pimelic acid: 0.05 ~ 0.09g/L, cupric sulfate pentahydrate: 0.4 ~ 0.6g/L, Zinc Sulphate Heptahydrate: 0.3 ~ 0.6g/L, tetrahydrate manganese chloride: 0.13 ~ 0.16g/L.
Further, described substratum is made up of basic medium, No. I liquid and No. II liquid, and weight ratio is 100:0.3 ~ 0.6:0.3 ~ 0.6;
Described basic medium is made up of the raw material of following concentration: HyPep5630:16.5g/L, Amisoy:16.5g/L, Na
2hPO
412H
2o:2.8g/L, lactic acid: 2.5g/L, yeast powder: 0.3g/L, calcium chloride: 8g/L;
Described No. I liquid is CYSTINE solution, and concentration is 200g/L;
Described No. II liquid is made up of the raw material of following concentration: magnesium sulfate: 109.88g/L, Beta-alanine: 1.15g/L, nicotinic acid: 1.15g/L, pimelic acid: 0.075g/L, cupric sulfate pentahydrate: 0.5g/L, Zinc Sulphate Heptahydrate: 0.458g/L, tetrahydrate manganese chloride: 0.15g/L.
Utilize the method for above-mentioned medium preparing diphtheria toxoid, it comprises the following steps
S1. bacterial classification is opened: open diphtheria bacterial classification, be inoculated on diphtheria corynebacterium substratum, 30 ~ 38 DEG C of quiescent culture 48 ~ 72h, liquid medium face length goes out mycoderm layer, is generation diphtheria corynebacterium bacterial classification;
S2. pass two cultures: by generation diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 30 ~ 38 DEG C of quiescent culture 10 ~ 14h, liquid medium face length goes out mycoderm layer, be two generation diphtheria corynebacterium bacterial classification;
S3. pass three cultures: by two generation diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 30 ~ 38 DEG C of quiescent culture 14 ~ 20h, liquid medium face length goes out mycoderm layer, is three generations's diphtheria corynebacterium bacterial classification;
S4. pass four cultures: by three generations's diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 30 ~ 38 DEG C of quiescent culture 42 ~ 48h, liquid medium face length goes out mycoderm layer, be four generation diphtheria corynebacterium bacterial classification;
S5. fermentor cultivation: by the diphtheria corynebacterium substratum in four generation diphtheria corynebacterium strain inoculation fermentor tanks, at the temperature of 30 ~ 38 DEG C, 42 ~ 48h is cultivated in aeration-agitation, wherein, air flow is 10 ~ 20L/min, stirring velocity is 300 ~ 400rpm, being 7.5 ~ 8.2 by adding the pH of maltose maintain liquid in fermentor cultivation, through fermentor cultivation, separation and purification, obtaining diphtheria toxoid.
The present invention has the following advantages: diphtheria 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 existing standards of pharmacopoeia requirement, therefore substratum of the present invention can substitute containing the LuShi blood serum medium of horse serum with by improvement Lin Shi substratum prepared by beef source material, avoids the potential hazard to vaccine recipient; The method of this medium preparing diphtheria toxoid that utilizes provided by the invention has simple to operate, convenient for production, the advantage easy, cost is low of drawing materials.
Accompanying drawing explanation
Fig. 1 is that different culture media produces diphtheria toxin SDS-PAGE comparison diagram.
Embodiment
Below in conjunction with drawings and Examples, the present invention will be further described, and protection scope of the present invention is not limited to the following stated.
Embodiment 1:
1. prepare diphtheria corynebacterium substratum
Described substratum is made up of basic medium, No. I liquid and No. II liquid, and weight ratio is 100:0.3:0.3;
Described basic medium is made up of the raw material of following concentration: HyPep5630:10g/L, Amisoy:10g/L, Na
2hPO
412H
2o:1g/L, lactic acid: 2g/L, yeast powder: 0.1g/L, calcium chloride: 5g/L;
Described No. I liquid is CYSTINE solution, and concentration is 150g/L;
Described No. II liquid is made up of the raw material of following concentration: magnesium sulfate: 100g/L, Beta-alanine: 0.5g/L, nicotinic acid: 0.5g/L, pimelic acid: 0.04g/L, cupric sulfate pentahydrate: 0.3g/L, Zinc Sulphate Heptahydrate: 0.2g/L, tetrahydrate manganese chloride: 0.1g/L.
2. prepare the method for diphtheria toxoid, it comprises the following steps
S1. bacterial classification is opened: open diphtheria bacterial classification, be inoculated on diphtheria corynebacterium substratum, 30 DEG C of quiescent culture 48h, liquid medium face length goes out mycoderm layer, is generation diphtheria corynebacterium bacterial classification;
S2. pass two cultures: by generation diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 30 DEG C of quiescent culture 10h, liquid medium face length goes out mycoderm layer, be two generation diphtheria corynebacterium bacterial classification;
S3. pass three cultures: by two generation diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 30 DEG C of quiescent culture 14h, liquid medium face length goes out mycoderm layer, is three generations's diphtheria corynebacterium bacterial classification;
S4. pass four cultures: by three generations's diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 30 DEG C of quiescent culture 40h, liquid medium face length goes out mycoderm layer, be four generation diphtheria corynebacterium bacterial classification;
S5. fermentor cultivation: by the diphtheria corynebacterium substratum in four generation diphtheria corynebacterium strain inoculation fermentor tanks, at the temperature of 30 DEG C, 48h is cultivated in aeration-agitation, wherein, air flow is 10L/min, stirring velocity is 300rpm, be 7.5 ~ 8.2 by adding the pH of maltose maintain liquid in fermentation culture, after fermentor cultivation, add the formaldehyde sterilization of concentration 0.2% (v/v), the results cotton-shaped unit of diphtheria toxoid is not less than 150Lf/ml; Adopt centrifugal segregation diphtheria thalline, add final concentration 25% ammonium sulfate, 1% gac, 0.5% sodium bicarbonate, fully stirs, and places 12 hours for 2 ~ 8 DEG C; Collected by centrifugation supernatant, continues to add ammonium sulfate to final concentration 45%; Centrifugal collecting precipitation, dissolve with 0.5% sodium bicarbonate, 10kd molecular weight cut-off carries out the ultrafiltration being not less than 10 times of volumes, removes ammonium sulfate, adds 0.2% (v/v) formaldehyde and Methionin, detoxification 28 days at the temperature of 37 DEG C, i.e. obtained diphtheria toxoid.
Embodiment 2:
1. prepare diphtheria corynebacterium substratum
Described substratum is made up of basic medium, No. I liquid and No. II liquid, and weight ratio is 100:0.6:0.6;
Described basic medium is made up of the raw material of following concentration: HyPep5630:20g/L, Amisoy:20g/L, Na
2hPO
412H
2o:5g/L, lactic acid: 3g/L, yeast powder: 0.5g/L, calcium chloride: 15g/L;
Described No. I liquid is CYSTINE solution, and concentration is 250g/L;
Described No. II liquid is made up of the raw material of following concentration: magnesium sulfate: 120g/L, Beta-alanine: 1.5g/L, nicotinic acid: 1.5g/L, pimelic acid: 0.15g/L, cupric sulfate pentahydrate: 0.8g/L, Zinc Sulphate Heptahydrate: 0.8g/L, tetrahydrate manganese chloride: 0.2g/L.
2. prepare the method for diphtheria toxoid, it comprises the following steps
S1. bacterial classification is opened: open diphtheria bacterial classification, be inoculated on diphtheria corynebacterium substratum, 38 DEG C of quiescent culture 72h, liquid medium face length goes out mycoderm layer, is generation diphtheria corynebacterium bacterial classification;
S2. pass two cultures: by generation diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 38 DEG C of quiescent culture 14h, liquid medium face length goes out mycoderm layer, be two generation diphtheria corynebacterium bacterial classification;
S3. pass three cultures: by two generation diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 38 DEG C of quiescent culture 20h, liquid medium face length goes out mycoderm layer, is three generations's diphtheria corynebacterium bacterial classification;
S4. pass four cultures: by three generations's diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 38 DEG C of quiescent culture 48h, liquid medium face length goes out mycoderm layer, be four generation diphtheria corynebacterium bacterial classification;
S5. fermentor cultivation: by the diphtheria corynebacterium substratum in four generation diphtheria corynebacterium strain inoculation fermentor tanks, at the temperature of 38 DEG C, 48h is cultivated in aeration-agitation, wherein, air flow is 20L/min, stirring velocity is 400rpm, be 7.5 ~ 8.2 by adding the pH of maltose maintain liquid in fermentation culture, after fermentor cultivation, add the formaldehyde sterilization of concentration 0.2% (v/v), the results cotton-shaped unit of diphtheria toxoid is not less than 150Lf/ml; Adopt centrifugal segregation diphtheria thalline, add final concentration 25% ammonium sulfate, 1% gac, 0.5% sodium bicarbonate, fully stirs, and places 12 hours for 2 ~ 8 DEG C; Collected by centrifugation supernatant, continues to add ammonium sulfate to final concentration 45%; Centrifugal collecting precipitation, dissolve with 0.5% sodium bicarbonate, 10kd molecular weight cut-off carries out the ultrafiltration being not less than 10 times of volumes, removes ammonium sulfate, adds 0.2% (v/v) formaldehyde and Methionin, detoxification 28 days at the temperature of 37 DEG C, i.e. obtained diphtheria toxoid.
Embodiment 3:
1. prepare diphtheria corynebacterium substratum
Described substratum is made up of basic medium, No. I liquid and No. II liquid, and weight ratio is 100:0.4:0.5;
Described basic medium is made up of the raw material of following concentration: HyPep5630:13g/L, Amisoy:13g/L, Na
2hPO
412H
2o:1.5g/L, lactic acid: 2.3g/L, yeast powder: 0.25g/L, calcium chloride: 6g/L;
Described No. I liquid is CYSTINE solution, and concentration is 180g/L;
Described No. II liquid is made up of the raw material of following concentration: magnesium sulfate: 105g/L, Beta-alanine: 0.8g/L, nicotinic acid: 0.8g/L, pimelic acid: 0.05g/L, cupric sulfate pentahydrate: 0.4g/L, Zinc Sulphate Heptahydrate: 0.3g/L, tetrahydrate manganese chloride: 0.13g/L.
2. prepare the method for diphtheria toxoid, it comprises the following steps
S1. bacterial classification is opened: open diphtheria bacterial classification, be inoculated on diphtheria corynebacterium substratum, 32 DEG C of quiescent culture 55h, liquid medium face length goes out mycoderm layer, is generation diphtheria corynebacterium bacterial classification;
S2. pass two cultures: by generation diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 32 DEG C of quiescent culture 12h, liquid medium face length goes out mycoderm layer, be two generation diphtheria corynebacterium bacterial classification;
S3. pass three cultures: by two generation diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 32 DEG C of quiescent culture 16h, liquid medium face length goes out mycoderm layer, is three generations's diphtheria corynebacterium bacterial classification;
S4. pass four cultures: by three generations's diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 32 DEG C of quiescent culture 42h, liquid medium face length goes out mycoderm layer, be four generation diphtheria corynebacterium bacterial classification;
S5. fermentor cultivation: by the diphtheria corynebacterium substratum in four generation diphtheria corynebacterium strain inoculation fermentor tanks, at the temperature of 32 DEG C, 46h is cultivated in aeration-agitation, wherein, air flow is 13L/min, stirring velocity is 320rpm, be 7.5 ~ 8.2 by adding the pH of maltose maintain liquid in fermentation culture, after fermentor cultivation, add the formaldehyde sterilization of concentration 0.2% (v/v), the results cotton-shaped unit of diphtheria toxoid is not less than 150Lf/ml; Adopt centrifugal segregation diphtheria thalline, add final concentration 25% ammonium sulfate, 1% gac, 0.5% sodium bicarbonate, fully stirs, and places 12 hours for 2 ~ 8 DEG C; Collected by centrifugation supernatant, continues to add ammonium sulfate to final concentration 45%; Centrifugal collecting precipitation, dissolve with 0.5% sodium bicarbonate, 10kd molecular weight cut-off carries out the ultrafiltration being not less than 10 times of volumes, removes ammonium sulfate, adds 0.2% (v/v) formaldehyde and Methionin, detoxification 28 days at the temperature of 37 DEG C, i.e. obtained diphtheria toxoid.
Embodiment 4:
1. prepare diphtheria corynebacterium substratum
Described substratum is made up of basic medium, No. I liquid and No. II liquid, and weight ratio is 100:0.5:0.4;
Described basic medium is made up of the raw material of following concentration: HyPep5630:18g/L, Amisoy:18g/L, Na
2hPO
412H
2o:3.5g/L, lactic acid: 2.7g/L, yeast powder: 0.35g/L, calcium chloride: 10g/L;
Described No. I liquid is CYSTINE solution, and concentration is 230g/L;
Described No. II liquid is made up of the raw material of following concentration: magnesium sulfate: 112g/L, Beta-alanine: 1.2g/L, nicotinic acid: 1.2g/L, pimelic acid: 0.09g/L, cupric sulfate pentahydrate: 0.6g/L, Zinc Sulphate Heptahydrate: 0.6g/L, tetrahydrate manganese chloride: 0.16g/L.
2. prepare the method for diphtheria toxoid, it comprises the following steps
S1. bacterial classification is opened: open diphtheria bacterial classification, be inoculated on diphtheria corynebacterium substratum, 35 DEG C of quiescent culture 58h, liquid medium face length goes out mycoderm layer, is generation diphtheria corynebacterium bacterial classification;
S2. pass two cultures: by generation diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 34 DEG C of quiescent culture 13h, liquid medium face length goes out mycoderm layer, be two generation diphtheria corynebacterium bacterial classification;
S3. pass three cultures: by two generation diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 34 DEG C of quiescent culture 14 ~ 20h, liquid medium face length goes out mycoderm layer, is three generations's diphtheria corynebacterium bacterial classification;
S4. pass four cultures: by three generations's diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 34 DEG C of quiescent culture 45h, liquid medium face length goes out mycoderm layer, be four generation diphtheria corynebacterium bacterial classification;
S5. fermentor cultivation: by the diphtheria corynebacterium substratum in four generation diphtheria corynebacterium strain inoculation fermentor tanks, at the temperature of 34 DEG C, 45h is cultivated in aeration-agitation, wherein, air flow is 15L/min, stirring velocity is 350rpm, be 7.5 ~ 8.2 by adding the pH of maltose maintain liquid in fermentation culture, after fermentor cultivation, add the formaldehyde sterilization of concentration 0.2% (v/v), the results cotton-shaped unit of diphtheria toxoid is not less than 150Lf/ml; Adopt centrifugal segregation diphtheria thalline, add final concentration 25% ammonium sulfate, 1% gac, 0.5% sodium bicarbonate, fully stirs, and places 12 hours for 2 ~ 8 DEG C; Collected by centrifugation supernatant, continues to add ammonium sulfate to final concentration 45%; Centrifugal collecting precipitation, dissolve with 0.5% sodium bicarbonate, 10kd molecular weight cut-off carries out the ultrafiltration being not less than 10 times of volumes, removes ammonium sulfate, adds 0.2% (v/v) formaldehyde and Methionin, detoxification 28 days at the temperature of 37 DEG C, i.e. obtained diphtheria toxoid.
Embodiment 5:
1. prepare diphtheria corynebacterium substratum
Described substratum is made up of basic medium, No. I liquid and No. II liquid, and weight ratio is 100:0.4:0.4;
Described basic medium is made up of the raw material of following concentration: HyPep5630:16.5g/L, Amisoy:16.5g/L, Na
2hPO
412H
2o:2.8g/L, lactic acid: 2.5g/L, yeast powder: 0.3g/L, calcium chloride: 8g/L;
Described No. I liquid is CYSTINE solution, and concentration is 200g/L;
Described No. II liquid is made up of the raw material of following concentration: magnesium sulfate: 109.88g/L, Beta-alanine: 1.15g/L, nicotinic acid: 1.15g/L, pimelic acid: 0.075g/L, cupric sulfate pentahydrate: 0.5g/L, Zinc Sulphate Heptahydrate: 0.458g/L, tetrahydrate manganese chloride: 0.15g/L.
2. utilize the method for above-mentioned medium preparing diphtheria toxoid, it comprises the following steps
S1. bacterial classification is opened: open diphtheria bacterial classification, be inoculated on diphtheria corynebacterium substratum, 36 DEG C of quiescent culture 68h, liquid medium face length goes out mycoderm layer, is generation diphtheria corynebacterium bacterial classification;
S2. pass two cultures: by generation diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 36 DEG C of quiescent culture 13h, liquid medium face length goes out mycoderm layer, be two generation diphtheria corynebacterium bacterial classification;
S3. pass three cultures: by two generation diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 36 DEG C of quiescent culture 18h, liquid medium face length goes out mycoderm layer, is three generations's diphtheria corynebacterium bacterial classification;
S4. pass four cultures: by three generations's diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 36 DEG C of quiescent culture 46h, liquid medium face length goes out mycoderm layer, be four generation diphtheria corynebacterium bacterial classification;
S5. fermentor cultivation: by the diphtheria corynebacterium substratum in four generation diphtheria corynebacterium strain inoculation fermentor tanks, at the temperature of 36 DEG C, 42h is cultivated in aeration-agitation, wherein, air flow is 18L/min, stirring velocity is 385rpm, be 7.5 ~ 8.2 by adding the pH of maltose maintain liquid in fermentation culture, after fermentor cultivation, add the formaldehyde sterilization of concentration 0.2% (v/v), the results cotton-shaped unit of diphtheria toxoid is not less than 150Lf/ml; Adopt centrifugal segregation diphtheria thalline, add final concentration 25% ammonium sulfate, 1% gac, 0.5% sodium bicarbonate, fully stirs, and places 12 hours for 2 ~ 8 DEG C; Collected by centrifugation supernatant, continues to add ammonium sulfate to final concentration 45%; Centrifugal collecting precipitation, dissolve with 0.5% sodium bicarbonate, 10kd molecular weight cut-off carries out the ultrafiltration being not less than 10 times of volumes, removes ammonium sulfate, adds 0.2% (v/v) formaldehyde and Methionin, detoxification 28 days at the temperature of 37 DEG C, i.e. obtained diphtheria toxoid.
Below by way of test, beneficial effect of the present invention is described:
One, the Selection experiment of substratum
1. test strain
Adopt diphtheria corynebacterium PW8 strain (CMCC38007);
2. test medium
As shown in table 1, table 2:
Table 1. culture medium prescription
Table 2. culture media nitrogen source
| Test number | Nitrogenous source | Producer | Source |
| 1 | N-Z-AMINEAS | KERRY | Acid hydrolyzed casein (Niu Yuan) |
| 2 | HyPep5603 | KERRY | Little rice gluten, wheat protein |
| 3 | Amisoy | KERRY | Soybean protein |
| 4 | HyPep4601N | KERRY | Wheat protein |
| 5 | HyPep5603+Amisoy | KERRY | Millet, wheat, soybean protein |
| 6 | Selected soya peptone | BD | Soybean protein |
| 7 | ProteosePeptoneNo.2 (showing No. 2, peptone) | BD | Beef |
| 8 | ProteosePeptoneNo.3 (showing No. 3, peptone) | BD | Beef |
| 9 | Lin Shi Digestive system | Self-control | Beef |
The preparation method of Lin Shi Digestive system:
Be 1000 grams of calculating with beef, calculate material amounts according to following ratio:
Beef: 1000 grams, water for injection: 3000 milliliters, pancreatin: 3 unit of activity/gram meat, glacial acetic acid: the water yield 2.5% (V/V).
Be mixed in heated and boiled in saucepan by 1/4 of hamburger and full dose water for injection, add water for injection (20 DEG C) to total amount; Adjustment temperature 50 ~ 51 DEG C, with 20% sodium hydroxide solution adjustment pH8.2; Full dose pancreas enzyme powder is divided into 7 parts, starts to add 2/7, after 30 minutes, add 2/7, then digest and add 2/7 in 30 minutes, then add 1/7 after digesting 30 minutes, digest 2.5 hours altogether; PH8.2 is kept, temperature 50 ~ 51 DEG C in digestive process; Digestion end adds people's Glacial acetic acid and stirs, heated and boiled; After boiling, extract solution in filter pocket, filter with canvas, collection filtrate is for subsequent use;
3. experimental result: it is in the substratum of 1 ~ 9 that diphtheria corynebacterium PW8 strain is inoculated in test number, and measure the cotton-shaped unit of diphtheria toxin after static gas wave refrigerator 120h, test-results is as shown in table 3:
Table 3. experimental result
| Test number | Nitrogenous source | Cotton-shaped unit (Lf/ml) after static gas wave refrigerator 120h |
| 1 | N-Z-AMINEAS | 70 |
| 2 | HyPep5603 | 70 |
| 3 | Amisoy | 30 |
| 4 | HyPep4601N | 20 |
| 5 | HyPep5603+Amisoy | 90 |
| 6 | Selected soya peptone | 20 |
| 7 | Show peptone No. 2 | 100 |
| 8 | Show peptone No. 3 | 140 |
| 9 | Lin Shi Digestive system | 120 |
As shown in Table 3: select HyPep5603+Amisoy to produce toxic effect fruit as the test group of the diphtheria toxin producing medium of the animal origin-free of major nitrogen source and animal-origin close, illustrate that HyPep5603+Amisoy can substitute the diphtheria toxin producing medium that major nitrogen source is animal-origin.
Two, culture medium culturing result of the present invention
1. detected object: in microscopy embodiment 1 ~ 5 and adopt the generation bacterial classification of Loeffler's serum medium, improvement Lin Shi substratum, second-generation bacterial kind, three generations's bacterial classification, four generation bacterial classification and fermentor tank in thalline and measure the cotton-shaped unit of diphtheria toxin;
2. test-results:
Table 4. microscopy result and each stage produce malicious situation
Experimental result shows to adopt culture medium culturing of the present invention to produce poison and Lv Shi serum, improve Lin Shi substratum go down to posterity produce malicious basically identical, each generation bacterial classification microscopy form is consistent, and its culture efficiency is no significant difference compared with current cellar culture (having animal source substratum).
Three, electrophoresis detection
1. detected object: embodiment 4 (non-animal derived substratum) and the Irreversible toxoiding adopting improvement Lin Shi substratum (having animal source substratum) to produce, carries out polyacrylamide gel electrophoresis.
2. experimental result: as shown in Figure 1.
As shown in Figure 1: the diphtheria toxin that the diphtheria toxin that the present invention produces is produced with improvement Lin Shi substratum is basically identical, the molecular size range of two kinds of toxin is all substantially in same level line between 44.3kDa ~ 66.4kDa, all only has this obvious master tape, molecular size range meets the 58kDa ~ 62kDa about existing bibliographical information, and toxin purity is also higher.
Above-mentioned experimental result shows: adopt culture medium culturing of the present invention to produce poison and Lv Shi serum, improve Lin Shi substratum go down to posterity produce malicious basically identical, each generation bacterial classification microscopy form is consistent, and the Irreversible toxoiding molecular size range that different culture media is produced is consistent, purity is higher, and Production of Toxin efficiency of the present invention produces no significant difference compared with (having animal source substratum) with at present conventional.
Claims (4)
1. a diphtheria corynebacterium substratum, is characterized in that, described substratum is made up of basic medium, No. I liquid and No. II liquid, and weight ratio is 100:0.3 ~ 0.6:0.3 ~ 0.6;
Described basic medium is made up of the raw material of following concentration: HyPep 5630:10 ~ 20g/L, Amisoy:10 ~ 20g/L, Na
2hPO
412H
2o:1 ~ 5g/L, lactic acid: 2 ~ 3g/L, yeast powder: 0.1 ~ 0.5g/L, calcium chloride: 5 ~ 15g/L;
Described No. I liquid is CYSTINE solution, and concentration is 150 ~ 250g/L;
Described No. II liquid is made up of the raw material of following concentration: magnesium sulfate: 100 ~ 120g/L, Beta-alanine: 0.5 ~ 1.5 g/L, nicotinic acid: 0.5 ~ 1.5g/L, pimelic acid: 0.04 ~ 0.15g/L, cupric sulfate pentahydrate: 0.3 ~ 0.8g/L, Zinc Sulphate Heptahydrate: 0.2 ~ 0.8g/L, tetrahydrate manganese chloride: 0.1 ~ 0.2g/L.
2. a kind of diphtheria corynebacterium substratum as claimed in claim 1, is characterized in that, described substratum is made up of basic medium, No. I liquid and No. II liquid, and weight ratio is 100:0.3 ~ 0.6:0.3 ~ 0.6;
Described basic medium is made up of the raw material of following concentration: HyPep 5630:13 ~ 18g/L, Amisoy:13 ~ 18g/L, Na
2hPO
412H
2o:1.5 ~ 3.5g/L, lactic acid: 2.3 ~ 2.7g/L, yeast powder: 0.25 ~ 0.35g/L, calcium chloride: 6 ~ 10g/L;
Described No. I liquid is CYSTINE solution, and concentration is 180 ~ 230g/L;
Described No. II liquid is made up of the raw material of following concentration: magnesium sulfate: 105 ~ 112g/L, Beta-alanine: 0.8 ~ 1.2 g/L, nicotinic acid: 0.8 ~ 1.2g/L, pimelic acid: 0.05 ~ 0.09 g/L, cupric sulfate pentahydrate: 0.4 ~ 0.6g/L, Zinc Sulphate Heptahydrate: 0.3 ~ 0.6g/L, tetrahydrate manganese chloride: 0.13 ~ 0.16g/L.
3. a kind of diphtheria corynebacterium substratum as claimed in claim 1, is characterized in that, described substratum is made up of basic medium, No. I liquid and No. II liquid, and weight ratio is 100:0.3 ~ 0.6:0.3 ~ 0.6;
Described basic medium is made up of the raw material of following concentration: HyPep 5630:16.5g/L, Amisoy:16.5g/L, Na
2hPO
412H
2o:2.8g/L, lactic acid: 2.5g/L, yeast powder: 0.3g/L, calcium chloride: 8g/L;
Described No. I liquid is CYSTINE solution, and concentration is 200g/L;
Described No. II liquid is made up of the raw material of following concentration: magnesium sulfate: 109.88g/L, Beta-alanine: 1.15g/L, nicotinic acid: 1.15g/L, pimelic acid: 0.075g/L, cupric sulfate pentahydrate: 0.5 g/L, Zinc Sulphate Heptahydrate: 0.458g/L, tetrahydrate manganese chloride: 0.15g/L.
4. utilize the method for the medium preparing diphtheria toxoid in claim 1-3 described in any one, it is characterized in that, it comprises the following steps
S1. bacterial classification is opened: open diphtheria bacterial classification, be inoculated on diphtheria corynebacterium substratum, 30 ~ 38 DEG C of quiescent culture 48 ~ 72h, liquid medium face length goes out mycoderm layer, is generation diphtheria corynebacterium bacterial classification;
S2. pass two cultures: by generation diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 30 ~ 38 DEG C of quiescent culture 10 ~ 14h, liquid medium face length goes out mycoderm layer, be two generation diphtheria corynebacterium bacterial classification;
S3. pass three cultures: by two generation diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 30 ~ 38 DEG C of quiescent culture 14 ~ 20h, liquid medium face length goes out mycoderm layer, is three generations's diphtheria corynebacterium bacterial classification;
S4. pass four cultures: by three generations's diphtheria corynebacterium strain inoculation on diphtheria corynebacterium substratum, 30 ~ 38 DEG C of quiescent culture 42 ~ 48h, liquid medium face length goes out mycoderm layer, be four generation diphtheria corynebacterium bacterial classification;
S5. fermentor cultivation: by the diphtheria corynebacterium substratum in four generation diphtheria corynebacterium strain inoculation fermentor tanks, at the temperature of 30 ~ 38 DEG C, 42 ~ 48h is cultivated in aeration-agitation, wherein, air flow is 10 ~ 20L/min, stirring velocity is 300 ~ 400rpm, being 7.5 ~ 8.2 by adding the pH of maltose maintain liquid in fermentor cultivation, through fermentor cultivation, separation and purification, obtaining diphtheria toxoid.
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Cited By (2)
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| CN117535211A (en) * | 2024-01-09 | 2024-02-09 | 中国医学科学院医学生物学研究所 | Culture medium combination of diphtheria bacillus and preparation method of diphtheria toxoid |
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| CN117535211A (en) * | 2024-01-09 | 2024-02-09 | 中国医学科学院医学生物学研究所 | Culture medium combination of diphtheria bacillus and preparation method of diphtheria toxoid |
| CN117535211B (en) * | 2024-01-09 | 2024-04-16 | 中国医学科学院医学生物学研究所 | Culture medium combination of diphtheria bacillus and preparation method of diphtheria toxoid |
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