CN117535211B - Culture medium combination of diphtheria bacillus and preparation method of diphtheria toxoid - Google Patents

Culture medium combination of diphtheria bacillus and preparation method of diphtheria toxoid Download PDF

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CN117535211B
CN117535211B CN202410027123.8A CN202410027123A CN117535211B CN 117535211 B CN117535211 B CN 117535211B CN 202410027123 A CN202410027123 A CN 202410027123A CN 117535211 B CN117535211 B CN 117535211B
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CN117535211A (en
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温嘉纳
杨净思
王虓宇
梁疆莉
廖宏玮
姬光
高娜
顾琴
蔡路奎
马艳
胡文著
赵婷
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Institute of Medical Biology of CAMS and PUMC
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Abstract

The invention relates to a culture medium combination of diphtheria bacillus and a preparation method of diphtheria toxoid, belonging to the technical field of microbial culture, wherein the culture medium combination comprises a resuscitating culture medium, a subculture medium, a toxigenic culture medium, a growth liquid I and a growth liquid II; the preparation method of diphtheria toxoid comprises resuscitating culture, second generation culture, third generation culture, toxigenic culture and toxin toxoid. The culture medium prepared by the invention has the advantages of readily available raw materials, clear sources, reliable tracing, simple operation, convenient production and high toxin yield and purity of diphtheria toxoid.

Description

Culture medium combination of diphtheria bacillus and preparation method of diphtheria toxoid
Technical Field
The invention relates to the technical field of microorganism culture, in particular to a culture medium combination of diphtheria bacillus and a preparation method of diphtheria toxoid.
Background
Diphtheria (diphtheria) is an acute upper respiratory infectious disease caused by diphtheria bacillus (Corynebacterium diphtheriae), and is mainly clinically manifested by upper respiratory inflammation, usually symptoms of pharynx, posterior nasal cavity, larynx and trachea, or damage of cardiac muscle and other tissues such as peripheral nerves. Part of diphtheria bacterial strain can produce exotoxin with extremely strong toxicity, and can cause damage to films and organs in a large range. Prior to the introduction of diphtheria immunity, diphtheria is one of the main causes of death in children.
Diphtheria bacillus is an aerobic or facultative anaerobic gram positive bacterium, which is arranged in a V, L, Y fence-like manner on a dyed specimen, one end or two ends of the thallus are round and expanded into a bar shape, different-dyed particles with deep coloration are arranged in the thallus, and the optimal culture temperature is 34-35 ℃. Diphtheria toxin is a polypeptide chain secreted by diphtheria and contains no sugar and prosthetic groups, and has a total of 560 amino acids and a molecular weight of 58-62 kDa, and comprises A, B fragments, wherein fragment B is a receptor binding and transmembrane fragment, fragment A is a toxic fragment, and finally causes cell death by repressing the activity of the relevant enzyme in protein synthesis. Studies show that because the toxin-producing genes of the diphtheria bacillus are carried by beta-phage, after the beta-phage infects the nontoxic diphtheria bacillus, phage DNA and bacterial DNA are integrated to form lysogenic diphtheria bacillus, so that the lysogenic diphtheria bacillus can obtain the toxin-producing capability, and therefore, toxins produced by different toxin-producing diphtheria bacillus strains are the same.
The forest culture medium is considered to be the most suitable culture medium for diphtheria bacillus toxigenic because of being rich in animal proteins and blood group substances for promoting bacterial growth and having higher toxin titers. However, the culture medium Cheng Linshi has the problems of low traceability, high exogenous pollution risk, difficulty in realizing standardized production and the like because of the conditions of unknown meat source, difficulty in accurate control of enzyme hydrolysis, unclear hydrolysate content, difficulty in standardization of blood group substances and the like in the preparation process. In addition, the culture medium for diphtheria growth and toxigenic is mainly developed by taking peptone, but single animal-derived peptone cannot provide enough nutrient components and a buffer system for supporting bacterial growth, and plant-derived peptone can generate substances such as saponin for inhibiting diphtheria toxigenic, so that bacterial toxigenic amount is difficult to meet the requirement of 150Lf/mL in Chinese pharmacopoeia.
Disclosure of Invention
The invention aims to provide a culture medium combination of diphtheria bacillus and a preparation method of diphtheria toxoid, and aims to solve the problems that a meat-based basic material in a culture medium in the prior art is not clear in source and is not strong in traceability, and the toxin yield and purity of diphtheria toxoid are difficult to meet the requirements.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a culture medium combination of diphtheria bacillus, which comprises a resuscitating culture medium, a subculture medium and a toxigenic culture medium;
The resuscitation medium comprises mixed liquor and calf serum; the mixed solution takes water as a solvent and comprises the following components in concentration: 2-20 g/L of polypeptone, 1-10 g/L of beef extract powder, 2-20 g/L of glucose and 1-10 g/L of sodium chloride; the volume ratio of the mixed solution to calf serum is 1:2-4;
The subculture medium takes water as a solvent and comprises the following components in concentration: 720-780 mL/L of tryptone digestive juice, 0.1-2 g/L of yeast extract powder, 1-5 g/L of maltose, 5-10 g/L of sodium chloride, 1-5 mL/L of lactic acid, 1-3 g/L of disodium hydrogen phosphate dodecahydrate, 1-10 mL/L of calcium chloride solution, 5-20 mL/L of potassium dihydrogen phosphate solution, 1-10 mL/L of growth solution I and 1-5 mL/L of growth solution II;
The toxigenic culture medium takes water as a solvent and comprises the following components in concentration: 720-780 mL/L of polypeptone pancreatin digestion solution, 0.1-2 g/L of yeast extract powder, 1-5 g/L of maltose, 5-10 g/L of sodium chloride, 1-5 mL/L of lactic acid, 1-3 g/L of disodium hydrogen phosphate dodecahydrate, 1-10 mL/L of calcium chloride solution, 5-20 mL/L of potassium dihydrogen phosphate solution, 40-60 mL/L of calf serum pancreatin digestion solution, 1-10 mL/L of growth solution I and 1-5 mL/L of growth solution II;
The growth liquid I takes water as a solvent and comprises the following components in concentration: 50-150 g/L of L-cystine and 50-150 mL/L of hydrochloric acid;
The growth solution II takes water as a solvent and comprises the following components in concentration: 200-250 g/L of magnesium sulfate, 1-5 g/L of beta-alanine, 0.1-1 g/L of pimelic acid, 0.1-1 g/L of zinc sulfate heptahydrate, 0.1-1 g/L of copper sulfate pentahydrate, 0.1-1 g/L of manganese chloride tetrahydrate, 1-5 g/L of nicotinic acid and 20-40 mL/L of hydrochloric acid.
Preferably, the polypeptone pancreatin digestion solution takes water as a solvent, and comprises the following components in concentration: 20-60 g/L of polypeptone, 1-10 g/L of beef extract powder, 5-20 g/L of pancreatin and 10-20 mL/L of acetic acid;
The calf serum pancreatin digestion solution takes water as a solvent and comprises the following components in concentration: 40-60 mL/L calf serum, 5-20 g/L pancreatin, 1-3 g/L disodium hydrogen phosphate dodecahydrate and 1-10 mL/L calcium chloride solution.
Preferably, the initial concentration of the calcium chloride solution is 20-30wt%;
The initial concentration of the potassium dihydrogen phosphate solution is 0.5-2wt%;
The initial concentration of the lactic acid is 80-90 wt%;
the initial concentration of the hydrochloric acid is 30-40 wt%.
The invention also provides application of the culture medium combination in preparation of diphtheria toxoid.
The invention also provides a preparation method of diphtheria toxoid, which comprises the following steps:
(1) Inoculating diphtheria bacillus strain into a resuscitating culture medium, and standing and culturing for 46-48 hours to obtain a resuscitating strain;
(2) Inoculating the resuscitated strain into a subculture medium, and standing and culturing for 22-24 hours to obtain a second-generation strain;
(3) Inoculating the second-generation strain into a subculture medium, and performing static culture for 22-24 hours, and then performing shake culture for 22-24 hours to obtain a third-generation strain;
(4) Inoculating the third-generation strain into a toxigenic culture medium, and fermenting and culturing to obtain diphtheria bacillus fermentation liquor containing diphtheria toxin;
(5) Poisoning, salting out and detoxication the diphtheria bacillus fermentation broth to obtain diphtheria bacillus toxoid;
the resuscitating medium in step (1) is the resuscitating medium in the medium combination;
The subculture medium in the steps (2) to (3) is the subculture medium in the culture medium combination;
the toxigenic medium in step (4) is the toxigenic medium in the medium combination.
Preferably, in the step (1), the volume ratio of the diphtheria bacillus strain to the resuscitation medium is 1:60-100;
The temperature of the stationary culture in the step (1) is 30-40 ℃.
Preferably, the temperature of the stationary culture in the step (2) is 30-40 ℃.
Preferably, the temperature of the stationary culture in the step (3) is 30-40 ℃;
the temperature of the shake culture in the step (3) is 30-40 ℃, and the rotating speed of the shake culture is 100-200 rpm.
Preferably, the volume ratio of the third-generation strain to the toxigenic culture medium in the step (4) is 1:10-30;
the temperature of the fermentation culture in the step (4) is 30-40 ℃, the time of the fermentation culture is 55-70 h, the rotating speed of the fermentation culture is 100-300 rpm, the ventilation amount of the fermentation culture is 2-3L/min, and the pH of the fermentation liquor of the fermentation culture is 7-8.
Preferably, the reagent used for the poisoning in the step (5) is formaldehyde solution; the initial concentration of the formaldehyde solution is 36.5-38 vt; the addition amount of the formaldehyde solution is 1.9-2.0 mL;
The salting-out temperature in the step (5) is 2-8 ℃, and the salting-out time is 24-48 hours;
the detoxification temperature in the step (5) is 35-40 ℃, and the detoxification time is 30-40 d.
The invention has the following technical effects and advantages:
The culture medium combination of the diphtheria bacillus is based on the polypeptone with clear sources and definite components, macromolecular proteins and peptide chains are further digested through the pancreatin treatment, and beef extract powder and calf serum treated by the pancreatin are added at the same time, so that the culture medium combination can be used for resuscitation, passage and toxigenic culture of the diphtheria bacillus, and the toxigenic amount of the diphtheria bacillus can reach the standard that the toxigenic amount is more than 150Lf/mL specified in Chinese pharmacopoeia;
The preparation method of diphtheria toxoid has the advantages of simple operation, convenient production, easy material obtaining and the like, and the purity of the prepared diphtheria toxoid can reach the standard that the purity is more than 1500Lf/mg specified in Chinese pharmacopoeia.
Drawings
FIG. 1 shows the OD 600 of diphtheria bacillus cultured with the combination of culture media prepared in example 1 in relation to the amount of toxin produced;
FIG. 2 shows the result of 10% SDS-PAGE of diphtheria toxin and diphtheria toxoid obtained by culturing diphtheria bacillus using the combination of media prepared in example 1 and the modified forest culture medium (control) prepared in comparative example 2; lane 1 shows an electrophoresis pattern of diphtheria toxin yield of 150Lf/mL after 28h incubation of diphtheria bacillus in control medium; lane 2 shows an electrophoresis pattern of diphtheria toxin yield of 10Lf/mL after 20h incubation of diphtheria bacillus in the medium combination; lane 3 shows an electrophoresis pattern of diphtheria toxin yield of 40Lf/mL after 30h incubation of diphtheria bacillus in the medium combination; lane 4 shows an electrophoresis pattern of diphtheria toxin yield of 60Lf/mL after 40h incubation of diphtheria bacillus in the medium combination; lane 5 shows an electrophoresis pattern of diphtheria toxin yield of 140Lf/mL after 50h incubation of diphtheria bacillus in the medium combination; lane 6 shows an electrophoresis pattern of diphtheria toxin yield of 160Lf/mL after 60h incubation of diphtheria bacillus in the medium combination; lane 7 is TIANGEN MP205,205 double color pre-stained protein molecular weight standard electrophoresis pattern; lane 8 shows an electrophoresis pattern of diphtheria toxoid yield 160Lf/mL after culture of diphtheria bacillus in control medium; lane 9 shows an electrophoresis pattern of diphtheria toxoid produced by the culture broth combination of diphtheria bacillus at 200 Lf/mL.
Detailed Description
The invention provides a culture medium combination of diphtheria bacillus, which comprises a resuscitating culture medium, a subculture medium and a toxigenic culture medium;
the resuscitation medium comprises mixed liquor and calf serum; the mixed solution takes water as a solvent and comprises the following components in concentration: 2-20 g/L, preferably 10g/L, of polypeptone; 1-10 g/L of beef extract powder, preferably 5g/L; glucose 2-20 g/L, preferably 10g/L; 1-10 g/L sodium chloride, preferably 5g/L sodium chloride; the volume ratio of the mixed solution to calf serum is 1:2-4, preferably 1:3;
the subculture medium takes water as a solvent and comprises the following components in concentration: 720-780 mL/L, preferably 750mL/L, of polypeptone pancreatin digestion solution; 0.1-2 g/L yeast extract powder, preferably 1g/L yeast extract powder; maltose 1-5 g/L, preferably 3g/L; 5 to 10g/L of sodium chloride, preferably 8.5g/L; lactic acid 1-5 mL/L, preferably 2.5mL/L; 1 to 3g/L of disodium hydrogen phosphate dodecahydrate, preferably 1.5g/L; 1-10 mL/L, preferably 5mL/L, of calcium chloride solution; 5-20 mL/L, preferably 10mL/L, of potassium dihydrogen phosphate solution; 1-10 mL/L, preferably 5mL/L, of growth solution I; 1-5 mL/L, preferably 2mL/L, of growth solution II;
The toxigenic culture medium takes water as a solvent and comprises the following components in concentration: 720-780 mL/L, preferably 750mL/L, of polypeptone pancreatin digestion solution; 0.1-2 g/L yeast extract powder, preferably 1g/L yeast extract powder; maltose 1-5 g/L, preferably 3g/L; 5 to 10g/L of sodium chloride, preferably 8.5g/L; lactic acid 1-5 mL/L, preferably 2.5mL/L; 1 to 3g/L of disodium hydrogen phosphate dodecahydrate, preferably 1.5g/L; 1-10 mL/L, preferably 5mL/L, of calcium chloride solution; 5-20 mL/L, preferably 10mL/L, of potassium dihydrogen phosphate solution; 40-60 mL/L calf serum pancreatin digestion solution, preferably 50mL/L; 1-10 mL/L, preferably 5mL/L, of growth solution I; 1-5 mL/L, preferably 2mL/L, of growth solution II;
the growth liquid I takes water as a solvent and comprises the following components in concentration: 50-150 g/L, preferably 100g/L, of L-cystine; hydrochloric acid 50-150 mL/L, preferably 100mL/L;
The growth solution II takes water as a solvent and comprises the following components in concentration: 200-250 g/L magnesium sulfate, preferably 225g/L; beta-alanine 1-5 g/L, preferably 2.3g/L; pimelic acid 0.1-1 g/L, preferably 0.15g/L; 0.1 to 1g/L of zinc sulfate heptahydrate, preferably 0.71g/L; 0.1 to 1g/L of copper sulfate pentahydrate, preferably 0.78g/L; manganese chloride tetrahydrate 0.1-1 g/L, preferably 0.24g/L; nicotinic acid 1-5 g/L, preferably 4.6g/L; hydrochloric acid 20-40 mL/L, preferably 30mL/L.
In the invention, the polypeptone pancreatin digestion solution takes water as a solvent and comprises the following components in concentration: 20-60 g/L, preferably 40g/L, of polypeptone; 1-10 g/L of beef extract powder, preferably 5g/L; pancreatic enzyme 5-20 g/L, preferably 10g/L; acetic acid 10-20 mL/L, preferably 12mL/L;
The calf serum pancreatin digestion solution takes water as a solvent and comprises the following components in concentration: calf serum 40-60 mL/L, preferably 50mL/L; pancreatic enzyme 5-20 g/L, preferably 10g/L; 1 to 3g/L of disodium hydrogen phosphate dodecahydrate, preferably 1.5g/L; the concentration of the calcium chloride solution is 1-10 mL/L, preferably 5mL/L.
In the present invention, the initial concentration of the calcium chloride solution is 20 to 30wt%, preferably 25wt%;
the initial concentration of the potassium dihydrogen phosphate solution is 0.5-2 wt%, preferably 1wt%;
the initial concentration of lactic acid is 80-90 wt%, preferably 85wt%;
the initial concentration of the hydrochloric acid is 30 to 40wt%, preferably 37wt%.
The invention also provides application of the culture medium combination in preparation of diphtheria toxoid.
The invention also provides a preparation method of diphtheria toxoid, which comprises the following steps:
(1) Inoculating diphtheria bacillus strain into a resuscitating culture medium, and standing and culturing for 46-48 hours, preferably 48 hours to obtain a resuscitating strain;
(2) Inoculating the resuscitated strain into a subculture medium, and standing and culturing for 22-24 hours, preferably 24 hours to obtain a second-generation strain;
(3) Inoculating the second-generation strain into a subculture medium, standing for 22-24 hours, preferably 24 hours, and performing shake culture for 22-24 hours, preferably 24 hours to obtain a third-generation strain;
(4) Inoculating the third-generation strain into a toxigenic culture medium, and fermenting and culturing to obtain diphtheria bacillus fermentation liquor containing diphtheria toxin;
(5) Poisoning, salting out and detoxication the diphtheria bacillus fermentation broth to obtain diphtheria bacillus toxoid;
the resuscitating medium in step (1) is the resuscitating medium in the medium combination;
The subculture medium in the steps (2) to (3) is the subculture medium in the culture medium combination;
the toxigenic medium in step (4) is the toxigenic medium in the medium combination.
In the invention, the diphtheria bacillus strain is PW8 strain, and is derived from Romania PW.8 (PW.: werssevsee), the Chinese food and drug inspection institute receives and prepares the diphtheria corynebacterium original strain for preservation on the 8 th day of 1955, the preservation name is diphtheria corynebacterium PW8 strain (CMCC 38007), the Chinese medical science sciences medical biology institute is in 2009 to enter into a 'pertussis, diphtheria and tetanus strain' technical service contract with the Chinese food and drug inspection institute, the diphtheria corynebacterium original strain is obtained, and a main seed strain library and a working seed strain library are established through joint vaccine research laboratory subculture of the Chinese medical science sciences, and liquid nitrogen at-80 ℃ is preserved for standby. The diphtheria bacillus strains used in the invention are diphtheria bacillus working seed strains.
In the invention, the volume ratio of the diphtheria bacillus strain to the resuscitation medium in the step (1) is 1:60-100, preferably 1:80;
the stationary culture in the step (1) is carried out at a temperature of 30 to 40℃and preferably 35 ℃.
In the present invention, the stationary culture in the step (2) is carried out at a temperature of 30 to 40℃and preferably 35 ℃.
In the present invention, the stationary culture in step (3) is performed at a temperature of 30 to 40 ℃, preferably 35 ℃;
The temperature of the shake culture in the step (3) is 30-40 ℃, preferably 35 ℃; the rotation speed of the shaking culture is 100-200 rpm, preferably 140rpm.
In the invention, the volume ratio of the third-generation strain to the toxigenic medium in the step (4) is 1:10-30, preferably 1:20;
The temperature of the fermentation culture in the step (4) is 30-40 ℃, preferably 35 ℃; the fermentation culture time is 55-70 h, preferably 70h; the rotation speed of the fermentation culture is 100-300 rpm, preferably 300rpm; the aeration rate of the fermentation culture is 2-3L/min, preferably 2.5L/min; the pH of the fermentation culture is 7 to 8, preferably 7.8.
In the invention, fermentation maintenance liquid, glutamic acid solution and malt flour solution are also added in the fermentation culture in the step (4);
The volume ratio of the fermentation maintenance liquid to the toxigenic culture medium is 4-6:70, preferably 4.2:70; the volume ratio of the glutamic acid solution to the toxigenic culture medium is 1-1.5:70, preferably 1.26:70; the volume ratio of the malt flour liquid to the toxigenic culture medium is 18-20:70, preferably 20:70;
The fermentation maintenance liquid takes water as a solvent and comprises the following components in concentration: 50-150 g/L of beef extract powder, preferably 100g/L; yeast extract powder 10-50 g/L, preferably 20g/L; 1to 3g/L of disodium hydrogen phosphate dodecahydrate, preferably 1.5g/L; 1-10 mL/L, preferably 5mL/L, of calcium chloride solution; 10-50 mL/L, preferably 20mL/L, of growth solution I;
the glutamic acid solution takes water as a solvent and comprises the following components in concentration: glutamic acid 150-250 g/L, preferably 200g/L; ammonia water 100-150 mL/L, preferably 120mL/L;
The malt flour liquid takes water as a solvent and comprises the following components in concentration: 400-500 g/L malt flour, preferably 450g/L malt flour; 1 to 3g/L of disodium hydrogen phosphate dodecahydrate, preferably 1.5g/L; 1-10 mL/L, preferably 5mL/L, of calcium chloride solution;
the initial concentration of the aqueous ammonia is 20 to 30wt%, preferably 28wt%.
In the invention, the reagent used for the poisoning in the step (5) is formaldehyde solution; the initial concentration of the formaldehyde solution is 36.5-38 vt, preferably 37 vt; the addition amount of the formaldehyde solution is 1.9-2.0 mL, preferably 1.96mL;
The salting-out temperature in the step (5) is 2-8 ℃, preferably 4 ℃; the salting-out time is 24-48 h, preferably 48h;
The detoxification temperature in the step (5) is 35-40 ℃, preferably 37 ℃; the detoxification time is 30-40 d, preferably 35d.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1: culture medium combination of diphtheria bacillus and diphtheria toxoid preparation
1. Culture medium combination and auxiliary liquid preparation
Preparing a tryptone pancreatin digestion solution: mixing 40g of polypeptone and 5g of beef extract powder with a proper amount of water for injection with the temperature of 50 ℃, regulating the pH value to 8.0 by using a 20wt% NaOH solution, adding 10g of pancreatin, digesting for 3 hours in a water bath with the temperature of 50 ℃, regulating the pH value to 8.0 by using the 20wt% NaOH solution every 30min, adding 12mL of acetic acid after digestion is finished, boiling for 5min, fixing the volume to 1L by using the water for injection, filtering, and obtaining the tryptone pancreatin digestion solution with the amino nitrogen content of 2.5mg/mL, and standing at the temperature of 4 ℃ for later use.
Calf serum pancreatin digest preparation: mixing 50mL of qualified calf serum with a proper amount of water for injection with the temperature of 50 ℃, adjusting the pH to 8.0 by using a 20wt% NaOH solution, adding 10g of pancreatin, digesting for 3 hours in a water bath with the temperature of 50 ℃, wherein the pH is adjusted to 8.0 by using the 20wt% NaOH solution every 30min, adding 1.5g of disodium hydrogen phosphate dodecahydrate and 5mL of 25wt% calcium chloride solution after digestion is finished, boiling for 5min, fixing the volume to 1L by using the water for injection, filtering to obtain calf serum pancreatin digestion solution, and standing at the temperature of 4 ℃ for later use.
And (3) preparing a growth solution I: taking 100g L-cystine and 100mL 37wt% hydrochloric acid to fix volume in 1L injection water, filtering to obtain growth liquid I, and standing at normal temperature for standby.
And II, preparing a growth solution: mixing 4.6g of nicotinic acid with a proper amount of water for injection, adding 30mL of 37wt% hydrochloric acid, mixing until the mixture is completely dissolved, adding 225g of magnesium sulfate, 2.3g of beta-alanine, 0.15g of pimelic acid, 0.71g of zinc sulfate heptahydrate, 0.78g of copper sulfate pentahydrate and 0.24g of manganese chloride tetrahydrate, fixing the volume to 1L by the water for injection, filtering to obtain a No. II growth solution, and standing at normal temperature for standby.
25Wt% calcium chloride solution preparation: 250g of anhydrous calcium chloride was taken and fixed in 1L of water for injection, and filtered to obtain a 25wt% calcium chloride solution.
1Wt% potassium dihydrogen phosphate solution: 10g of potassium dihydrogen phosphate was taken and fixed in 1L of water for injection, and filtered to obtain a 1wt% potassium dihydrogen phosphate solution.
Preparation of resuscitation medium: taking 10g of polypeptone, 5g of beef extract powder, 10g of glucose and 5g of sodium chloride to fix volume in 1L of water for injection, adjusting the pH to 7.2 by using 20wt% NaOH solution, and sterilizing for 20min at 115 ℃; adding 3L of qualified calf serum, mixing, subpackaging 40mL per test tube, placing on an inclined plane, intermittently sterilizing, sterilizing at 85deg.C for 60min per day, and continuously sterilizing for 3 days to obtain resuscitating culture medium.
Preparation of a subculture medium: mixing 750mL of polypeptone pancreatin digestion solution, 1g of yeast extract powder, 3g of maltose, 8.5g of sodium chloride, 2.5mL of 85wt% lactic acid and 1.5g of disodium hydrogen phosphate dodecahydrate with a proper amount of water for injection, heating to 50 ℃, regulating the pH to 8.3 by using a 20wt% NaOH solution, adding 5mL of 25wt% calcium chloride solution, boiling for 5min, filtering, cooling to 35 ℃, adding 5mL of growth solution I, 2mL of growth solution II and 10mL of 1wt% potassium dihydrogen phosphate solution, regulating the pH to 7.8 by using acetic acid after the volume is regulated to 1L by using water for injection, and sterilizing for 20min at 115 ℃ to obtain a passage culture medium with the amino nitrogen content of 1.88 mg/mL.
Preparation of a toxigenic culture medium: 750mL of polypeptone pancreatin digestion solution, 1g of yeast extract powder, 3g of maltose, 8.5g of sodium chloride, 2.5mL of 85wt% lactic acid and 1.5g of disodium hydrogen phosphate dodecahydrate are mixed with a proper amount of water for injection, the pH is adjusted to 8.3 by using a 20wt% NaOH solution after heating to 50 ℃, 5mL of 25wt% calcium chloride solution is added, boiling is carried out for 5min, filtering is carried out, 5mL of growth solution I, 2mL of growth solution II, 10mL of 1wt% potassium dihydrogen phosphate solution and 50mL of calf serum pancreatin digestion solution are added after cooling to 35 ℃, the pH is adjusted to 7.8 by using acetic acid after the volume is fixed to 1L by using water for injection, and sterilization is carried out for 20min at 115 ℃, thus obtaining the toxigenic culture medium.
Preparing a fermentation maintenance solution: mixing 100g of beef extract powder, 20g of yeast extract powder, 1.5g of disodium hydrogen phosphate dodecahydrate with a proper amount of water for injection, heating to 50 ℃, regulating the pH to 8.3 by using a 20wt% NaOH solution, adding 5mL of 25wt% calcium chloride solution, boiling for 5min, filtering, cooling to 35 ℃, adding 20mL of No. I growth solution, regulating the pH to 7.5 by using acetic acid after the volume of the solution is fixed to 1L by using the water for injection, and sterilizing at 115 ℃ for 20min to obtain fermentation maintenance liquid.
Preparation of glutamic acid solution: mixing 200g of glutamic acid with a proper amount of water for injection, slowly adding 120mL of 28wt% ammonia water until the glutamic acid is completely dissolved, fixing the volume to 1L by using the water for injection, adjusting the pH to 7.5 by using glutamic acid or 28wt% ammonia water, filtering, and sterilizing at 115 ℃ for 20min to obtain a glutamic acid solution.
Malt flour liquid preparation: mixing 450g malt flour, 1.5g disodium hydrogen phosphate dodecahydrate and a proper amount of water for injection, adding 5mL 25wt% calcium chloride solution, boiling for 5min, fixing the volume to 1L with water for injection, filtering, and sterilizing at 115 ℃ for 20min to obtain malt flour liquid.
2. Diphtheria toxoid preparation
(1) And (3) strain recovery: inoculating 500 mu L of diphtheria bacillus strain into a test tube filled with 40mL of resuscitating medium, and standing and culturing at 35 ℃ for 48 hours until colonies grow out on the surface of the resuscitating medium to obtain resuscitating strain;
(2) And (3) secondary culture: scraping the resuscitating strain in the step (1) by using an inoculating loop, inoculating the resuscitating strain into a small square bottle filled with 20mL of subculture medium, and standing and culturing for 24 hours at 35 ℃ until a strain film layer grows on the liquid surface of the subculture medium, thus obtaining a second-generation strain;
(3) And (3) carrying out third generation culture: scraping the second-generation strain in the step (2) by using an inoculating loop, inoculating the second-generation strain into a triangular flask filled with 100mL of subculture medium, standing and culturing for 24 hours at 35 ℃, performing shake culture for 24 hours in a constant-temperature shaking table, setting the culture temperature to 35 ℃ and the rotating speed to 140rpm until bacteria aggregation particles exist in the subculture medium, and obtaining the third-generation strain;
(4) Fermentation culture: inoculating 350mL of the third-generation strain in the step (3) into a Sartorius 15L fermentation tank filled with 7L of toxigenic medium, performing co-fermentation culture at 35 ℃ for 70h, setting ventilation capacity to be 2.5L/min, setting the rotating speed to be 300 rpm, and using 2L of malt powder liquid to maintain the pH value of the fermentation liquid to be 7.8; starting from 10h of fermentation culture, adding 35mL of fermentation maintenance liquid into a fermentation tank every 2h until the fermentation culture is stopped for 34h, and adding 420mL of fermentation maintenance liquid; starting from 34h of fermentation culture, adding 14mL of glutamic acid solution into a fermentation tank every 4h, and adding 126mL of glutamic acid solution when the fermentation culture is finished; obtaining diphtheria bacillus fermentation liquor after fermentation culture;
(5) Diphtheria bacillus toxin extraction and toxoid: after the fermentation broth of the diphtheria bacillus in the step (4) is cooled to 25 ℃, 50mL of 37vt percent formaldehyde solution is added, stirring is carried out for 30min at 300rpm, the diphtheria bacillus is killed, after bacterial liquid is collected, centrifugation is carried out for 30min at 5000rpm, supernatant fluid is taken for 10 times concentration, 4.1g of sodium bicarbonate, 205g of ammonium sulfate and 5.74g of active carbon are added into the concentrated solution, and standing is carried out for 24h at 4 ℃ for one-stage salting out; centrifuging at 8000rpm for 25min, collecting supernatant, adding 150.5g ammonium sulfate, standing at 4deg.C for 48 hr for two-stage salting-out, centrifuging at 8000rpm for 25min, collecting precipitate, dissolving in physiological saline, removing ammonium sulfate with ultrafiltration membrane with molecular weight cut-off not lower than 10kD, adding 1.96mL 37vt% formaldehyde solution and 13mL 9.125wt% lysine solution, and removing toxic substance at 37deg.C for 35d to obtain diphtheria toxoid.
Comparative example 1: culture medium combination of diphtheria bacillus without calf serum pancreatin digestive juice and diphtheria toxoid preparation
1. Culture medium combination without calf serum pancreatin digestion solution and auxiliary solution preparation
A polypeptone tryptic digest, a growth solution No. I, a growth solution No. II, a 25wt% calcium chloride solution, a 1wt% potassium dihydrogen phosphate solution, a resuscitator medium, a subculture medium, a toxigenic medium, a fermentation maintenance solution, a glutamic acid solution and a malt meal solution were prepared by the preparation method described in example 1. In contrast, in this example, calf serum pancreatin digestate was not contained in the toxigenic medium.
2. Diphtheria toxoid preparation
Diphtheria toxoid was prepared using the preparation method described in example 1.
Comparative example 2: improved forest culture medium and diphtheria toxoid preparation
1. Improved forest culture medium and auxiliary liquid preparation
The preparation method described in example 1 was used to prepare a growth solution I, a 25wt% calcium chloride solution, and a glutamic acid solution.
Preparing beef pancreatin digestion solution: taking 1000g of fresh beef with fat and tendons removed, mincing, adding 2.5L of water for injection, uniformly mixing, heating to 90 ℃, adding 7.5L of water for injection, cooling to 50 ℃, regulating the pH value to 8.0 by using 20wt% NaOH solution, adding 8g of pancreatin, carrying out water bath digestion for 3 hours at 50 ℃, regulating the pH value to 8.0 by using 20wt% NaOH solution every 10 minutes, adding 8g of pancreatin to digest for 1 hour after digestion, adding 12mL of acetic acid, boiling for 5 minutes, sterilizing, filtering, and obtaining beef pancreatin digestion liquid with the amino nitrogen content of 3.5mg/mL, and standing at 4 ℃ for later use.
And II, preparing a growth solution: mixing 1.15g of nicotinic acid with a proper amount of water for injection, adding 30mL of 37wt% hydrochloric acid, mixing until the mixture is completely dissolved, adding 1.15g of beta-alanine, 0.075g of pimelic acid, 0.4g of zinc sulfate heptahydrate, 0.5g of copper sulfate pentahydrate and 0.15g of manganese chloride tetrahydrate, fixing the volume to 1L by the water for injection, filtering to obtain a growth solution II, and standing at normal temperature for standby.
Preparation of improved forest subculture medium: mixing 412mL of beef pancreatin digestion solution, 3g of yeast extract powder, 2.5mL of 85wt% lactic acid and 0.8g of disodium hydrogen phosphate dodecahydrate with a proper amount of water for injection, adding 3.2mL of 25wt% calcium chloride solution, heating to 60 ℃, adjusting the pH to 8.3 with 20wt% NaOH solution, boiling for 5min, cooling to 60 ℃, filtering, adding 2mL of II growth solution, fixing the volume to 1L with water for injection, adjusting the pH to 7.8 with acetic acid, and sterilizing for 20min at 115 ℃ to obtain the diphtheria culture medium with the amino nitrogen content of 1.44 mg/mL.
Preparation of an improved forest toxic culture medium: 537mL of beef pancreatin digestion solution, 3g of yeast extract powder, 2.5mL of 85wt% lactic acid and 0.8g of disodium hydrogen phosphate dodecahydrate are mixed with a proper amount of water for injection, 3.2mL of 25wt% calcium chloride solution is added, then the mixture is heated to 60 ℃, the pH is regulated to 8.3 by 20wt% NaOH solution, the mixture is boiled for 5min and cooled to 60 ℃, the mixture is filtered, 5mL of I growth solution is added to 1L of water for injection, the pH is regulated to 7.8 by acetic acid, and the mixture is sterilized for 20min at 115 ℃, so as to obtain the diphtheria culture medium with the amino nitrogen content of 1.88 mg/mL.
Preparing an iron-removing maltose solution: culturing 500g barley until germination, adding into 1000g steamed Oryza Glutinosa, adding 4L injectable water, mixing to obtain homogenate, maintaining at 55deg.C, digesting for 5 hr, and removing residues to obtain sugar solution; then, 4g of disodium hydrogen phosphate dodecahydrate and 16mL of 25wt% calcium chloride solution were added for iron removal, the supernatant was concentrated to 2L after filtration, and sterilized at 115℃for 20 minutes to obtain an iron-removed maltose solution.
2. Diphtheria toxoid preparation
(1) And (3) strain recovery: inoculating 500 mu L of diphtheria bacillus strain into a test tube filled with 5mL of modified forest subculture medium, and standing and culturing at 35 ℃ for 48 hours until bacterial colonies grow on the surface of the modified forest subculture medium, thus obtaining resuscitated strain;
(2) And (3) secondary culture: secondary culture was performed using the preparation method described in example 1, except that in this example, the medium was a modified woods subculture medium;
(3) And (3) carrying out third generation culture: the preparation method described in example 1 was used for the third generation culture, except that in this example, the medium was a modified woods subculture medium;
(4) Fermentation culture: inoculating 350mL of the third-generation strain in the step (3) into a Sartorius 15L fermentation tank filled with 7L of modified forest toxigenic medium, performing co-fermentation culture at 35 ℃ for 48h, setting ventilation capacity to be 2.5L/min and rotating at 300rpm, and using 2L of iron-removing maltose liquid to maintain the pH value of the fermentation liquid to be 7.8. In the fermentation culture process, 70mL of growth solution I is added for 12h, 14mL of growth solution II is added for 14h, and from 16h of fermentation culture, 14mL of glutamic acid solution is added into the fermentation tank every 4h, and 112mL of glutamic acid solution is added at the end of fermentation culture. Obtaining diphtheria bacillus fermentation liquor after fermentation culture;
(5) Diphtheria bacillus toxin extraction and toxoid: diphtheria toxin extraction and toxoid were performed using the preparation method described in example 1, except that in this example, the amount of ammonium sulfate added to the supernatant was 395g.
Comparative example 3: tryptone culture medium and diphtheria toxoid preparation
1. Tryptone culture medium and auxiliary liquid preparation
A growth solution No. I, a growth solution No. II, a 25wt% calcium chloride solution, a 1wt% potassium dihydrogen phosphate solution, a fermentation maintenance solution, a glutamic acid solution and a malt flour solution were prepared by the preparation method described in example 1.
Preparation of tryptone culture medium: mixing 40g of tryptone, 5g of yeast extract, 1g of yeast extract, 3g of maltose, 8.5g of sodium chloride, 2.5mL of 85wt% lactic acid and 1.5g of disodium hydrogen phosphate dodecahydrate with a proper amount of water for injection, heating to 50 ℃, regulating the pH to 8.3 by using a 20wt% NaOH solution, adding 5mL of 25wt% calcium chloride solution, boiling for 5min, filtering, cooling to 35 ℃, adding 5mL of growth solution I, 2mL of growth solution II and 10mL of 1wt% potassium dihydrogen phosphate solution, regulating the pH to 7.8 by using acetic acid after the volume is regulated to 1L by using water for injection, and sterilizing for 20min at 115 ℃ to obtain the tryptone culture medium.
2. Diphtheria toxoid preparation
(1) And (3) strain recovery: inoculating 500 mu L of diphtheria bacillus strain into a test tube filled with 5mL of tryptone culture medium, and standing and culturing at 35 ℃ for 48 hours until colonies grow on the surface of the tryptone culture medium, thus obtaining a resuscitated strain;
(2) And (3) secondary culture: the second generation culture was performed by the preparation method described in example 1, except that in this example, the medium was tryptone medium;
(3) And (3) carrying out third generation culture: the preparation method described in example 1 was used for the third generation culture, except that in this example, the medium was tryptone medium;
(4) Fermentation culture: fermentation culture was performed using the preparation method described in example 1, except that in this example, the medium was tryptone;
(5) Diphtheria bacillus toxin extraction and toxoid: diphtheria toxin extraction and toxoid were performed using the preparation method described in example 1.
Comparative example 4: polypeptone culture medium and diphtheria toxoid preparation
1. Preparation of polypeptone culture medium and auxiliary liquid
A growth solution No. I, a growth solution No. II, a 25wt% calcium chloride solution, a 1wt% potassium dihydrogen phosphate solution, a fermentation maintenance solution, a glutamic acid solution and a malt flour solution were prepared by the preparation method described in example 1.
A polypeptone medium was prepared by the preparation method described in comparative example 3, except that in this example, peptone was polypeptone.
2. Diphtheria toxoid preparation
(1) And (3) strain recovery: inoculating 500 mu L of diphtheria bacillus strain into a test tube filled with 5mL of polypeptone culture medium, and standing and culturing at 35 ℃ for 48 hours until colonies grow on the surface of the resuscitating culture medium to obtain resuscitating strain;
(2) And (3) secondary culture: the second generation culture was performed by the preparation method described in example 1, except that in this example, the medium was polypeptone medium;
(3) And (3) carrying out third generation culture: the preparation method described in example 1 was used for the third generation culture, except that in this example, the medium was polypeptone medium;
(4) Fermentation culture: fermentation culture was performed using the preparation method described in example 1, except that in this example, the medium was polypeptone medium;
(5) Diphtheria bacillus toxin extraction and toxoid: diphtheria toxin extraction and toxoid were performed using the preparation method described in example 1.
Experimental example 1: diphtheria bacillus growth and toxin production condition
Diphtheria toxoid was produced by culturing diphtheria bacillus according to the diphtheria toxoid production method described in example 1 and comparative examples 1 to 4, and the growth and toxigenic status of diphtheria bacillus in example 1 and comparative examples 1 to 4 were observed, and the results are shown in table 1.
TABLE 1 growth and toxigenic status of diphtheria bacilli in different media
The results show that the diphtheria bacillus is cultivated by adopting the culture medium combination prepared in the embodiment 1, the diphtheria bacillus grows rapidly and is in a short rod shape, the form is well maintained in the late cultivation period, the toxin yield of 55 hours of cultivation is 150Lf/mg, and the final toxin yield is 180Lf/mg; culturing the diphtheria bacillus by adopting the culture medium combination without calf serum pancreatin digestive juice prepared in the comparative example 1, wherein the diphtheria bacillus grows rapidly and takes a short bar shape, the shape of the diphtheria bacillus is tiny in the late stage of culture, the toxin yield of the diphtheria bacillus is 150Lf/mg after 55h of culture, the toxin yield of the diphtheria bacillus is 160Lf/mg after 60h of culture, and the diphtheria bacillus does not produce toxin any more in the late stage; the modified forest culture medium prepared in the comparative example 2 is adopted to culture the diphtheria bacillus, the diphtheria bacillus grows rapidly and takes a short rod shape, the form is well maintained in the late culture period, the toxin production is rapid, the toxin production amount of the culture is 150Lf/mg in 28h, and the toxin production amount of the culture is 190Lf/mg in 48 h; culturing the diphtheria bacillus by adopting the tryptone culture medium prepared in the comparative example 3, wherein the diphtheria bacillus grows rapidly and takes a short rod shape, the shape of the diphtheria bacillus in the late culture period is fine, the toxin yield is low, and the final toxin yield is 100Lf/mg; the polypeptone culture medium prepared in the comparative example 4 is used for culturing the diphtheria bacillus, the diphtheria bacillus grows rapidly and takes a short rod shape, the shape of the diphtheria bacillus in the later period of culture is fine, the toxin yield is low, and the final toxin yield is 80Lf/mg. It can be seen that the combination of the culture medium prepared in example 1 is close to the modified forest culture medium in the toxicity production effect, and the toxicity production amount of diphtheria bacillus and the purity of diphtheria toxoid reach the standards specified in Chinese pharmacopoeia.
Experimental example 2: diphtheria bacillus growth and toxigenic dynamic change
Diphtheria toxoid preparation method according to example 1 is used for culturing diphtheria bacillus and preparing diphtheria toxoid, sampling is carried out at intervals continuously for 5 hours to obtain bacterial liquid samples, OD 600 value and toxin yield of the diphtheria bacillus in each bacterial liquid sample are measured, and the results are shown in table 2 and figure 1.
TABLE 2 diphtheria bacillus growth and toxigenic dynamics
The result shows that with the increase of the culture time, the diphtheria bacillus can keep a better growth and metabolism state in the culture medium combination prepared in the embodiment 1, and can produce toxin after 20 hours of culture, and the toxin production amount of the diphtheria bacillus after 55 hours of culture reaches the toxin collection requirement of Chinese pharmacopoeia.
Experimental example 3: diphtheria toxoid purity detection
Diphtheria toxoid was prepared by culturing diphtheria bacillus according to the diphtheria toxoid preparation method described in example 1, and SDS-PAGE electrophoresis was performed on the diphtheria toxoid and diphtheria toxoid prepared by using the diphtheria toxoid preparation method described in comparative example 2 as a control, and the results are shown in FIG. 2.
The results show that the bands of diphtheria toxin produced using the medium combination prepared in example 1 and diphtheria toxin produced by the control are all located around 60kD of the standard protein band; the diphtheria toxoid poisoned by formaldehyde is in a dispersed form; the diphtheria toxin prepared in example 1 has more hetero protein than the control, but the hetero protein content is reduced after purification and toxoid treatment, and the produced diphtheria toxoid has higher purity.
As can be seen from the above examples, the present invention provides a culture medium composition for diphtheria bacillus and a method for preparing diphtheria toxoid. The production of diphtheria bacillus by the culture medium combination culture prepared according to the invention can reach the standard that the production of toxin is more than 150Lf/mL specified in Chinese pharmacopoeia; the diphtheria toxoid prepared by the diphtheria toxoid preparation method provided by the invention has the purity reaching the standard of purity more than 1500Lf/mg specified in Chinese pharmacopoeia, and has the advantages of clear sources of culture medium components, simple and convenient preparation method, high toxin yield and purity of diphtheria toxoid and the like.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (5)

1. A diphtheria bacillus culture medium combination, which is characterized by comprising a resuscitating culture medium, a subculture culture medium and a toxigenic culture medium;
The resuscitation medium comprises mixed liquor and calf serum; the mixed solution takes water as a solvent and comprises the following components in concentration: 2-20 g/L of polypeptone, 1-10 g/L of beef extract powder, 2-20 g/L of glucose and 1-10 g/L of sodium chloride; the volume ratio of the mixed solution to calf serum is 1:2-4;
The subculture medium takes water as a solvent and comprises the following components in concentration: 720-780 mL/L of tryptone digestive juice, 0.1-2 g/L of yeast extract powder, 1-5 g/L of maltose, 5-10 g/L of sodium chloride, 1-5 mL/L of lactic acid, 1-3 g/L of disodium hydrogen phosphate dodecahydrate, 1-10 mL/L of calcium chloride solution, 5-20 mL/L of potassium dihydrogen phosphate solution, 1-10 mL/L of growth solution I and 1-5 mL/L of growth solution II;
The toxigenic culture medium takes water as a solvent and comprises the following components in concentration: 720-780 mL/L of polypeptone pancreatin digestion solution, 0.1-2 g/L of yeast extract powder, 1-5 g/L of maltose, 5-10 g/L of sodium chloride, 1-5 mL/L of lactic acid, 1-3 g/L of disodium hydrogen phosphate dodecahydrate, 1-10 mL/L of calcium chloride solution, 5-20 mL/L of potassium dihydrogen phosphate solution, 40-60 mL/L of calf serum pancreatin digestion solution, 1-10 mL/L of growth solution I and 1-5 mL/L of growth solution II;
The growth liquid I takes water as a solvent and comprises the following components in concentration: 50-150 g/L of L-cystine and 50-150 mL/L of hydrochloric acid;
The growth solution II takes water as a solvent and comprises the following components in concentration: 200-250 g/L of magnesium sulfate, 1-5 g/L of beta-alanine, 0.1-1 g/L of pimelic acid, 0.1-1 g/L of zinc sulfate heptahydrate, 0.1-1 g/L of copper sulfate pentahydrate, 0.1-1 g/L of manganese chloride tetrahydrate, 1-5 g/L of nicotinic acid and 20-40 mL/L of hydrochloric acid;
the polypeptone pancreatin digestion solution takes water as a solvent and comprises the following components in concentration: 20-60 g/L of polypeptone, 1-10 g/L of beef extract powder, 5-20 g/L of pancreatin and 10-20 mL/L of acetic acid;
The calf serum pancreatin digestion solution takes water as a solvent and comprises the following components in concentration: 40-60 mL/L of calf serum, 5-20 g/L of pancreatin, 1-3 g/L of disodium hydrogen phosphate dodecahydrate and 1-10 mL/L of calcium chloride solution;
the initial concentration of the calcium chloride solution is 20-30wt%;
The initial concentration of the potassium dihydrogen phosphate solution is 0.5-2wt%;
the initial concentration of the lactic acid is 80-90 wt%;
the initial concentration of the hydrochloric acid is 30-40 wt%.
2. Use of the combination of media of claim 1 for the preparation of diphtheria toxoid.
3. A method for preparing diphtheria toxoid, comprising the steps of:
(1) Inoculating diphtheria bacillus strain into a resuscitating culture medium, and standing and culturing for 46-48 hours to obtain a resuscitating strain;
(2) Inoculating the resuscitated strain into a subculture medium, and standing and culturing for 22-24 hours to obtain a second-generation strain;
(3) Inoculating the second-generation strain into a subculture medium, and performing static culture for 22-24 hours, and then performing shake culture for 22-24 hours to obtain a third-generation strain;
(4) Inoculating the third-generation strain into a toxigenic culture medium, and fermenting and culturing to obtain diphtheria bacillus fermentation liquor containing diphtheria toxin;
(5) Poisoning, salting out and detoxication the diphtheria bacillus fermentation broth to obtain diphtheria bacillus toxoid;
the resuscitation medium of step (1) is the resuscitation medium of the combination of media of claim 1;
the subculture medium in the steps (2) to (3) is the subculture medium in the combination of media as claimed in claim 1;
the toxigenic medium in step (4) is the toxigenic medium of the combination of media of claim 1.
4. The method according to claim 3, wherein the stationary culture in the step (2) is carried out at a temperature of 30 to 40 ℃.
5. The method according to claim 4, wherein the stationary culture in the step (3) is carried out at a temperature of 30 to 40 ℃;
the temperature of the shake culture in the step (3) is 30-40 ℃, and the rotating speed of the shake culture is 100-200 rpm.
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A群脑膜炎球菌多糖-白喉毒素无毒变异体CRM197结合物的制备及其初步评价;张营营等;中国生物制品学杂志;20121017(第10期);1248-1253 *
Macrophage activation by bacterial cell walls and related synthetic compounds;H Takada等;ASM Journals;19790701;第25卷(第1期);摘要 *
Monitoring the enzyme expression in a respiratory chain of Corynebacterium glutamicum in a copper ion-supplemented culture medium;Tomoichirou Kusumoto等;Biochemistry & Molecular Biology;20141023;第79卷;223-229 *
重组白喉毒素的制备及体外抑瘤效果的研究;张英波;中国硕士学位论文全文数据库;20070315;摘要 *

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