CN112877246B - Method for efficiently preparing vibrio cholerae ghost - Google Patents
Method for efficiently preparing vibrio cholerae ghost Download PDFInfo
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Abstract
The invention provides a method for efficiently preparing vibrio cholerae ghost, which comprises the following steps: adding arabinose for induction in the culture process of the vibrio cholerae engineering bacteria, and after 1-3 h of induction culture, adding arabinose again for secondary induction: in the induction stage, when the dissolved oxygen value is detected to be suddenly reduced, adding kanamycin, and continuously culturing for 0.5-1.5 h until the number of viable bacteria is 0; after the induction culture is carried out for 4-6 h, the bacterial ghost forming rate reaches more than 99%. The method utilizes a secondary induction strategy, shortens the induction time, improves the bacterial ghost obtained by harvesting by more than 16 percent, reduces the endotoxin content to below 1/5 of the initial process, obviously improves the production efficiency and reduces the production cost; the vibrio cholerae ghost prepared by the method is uniform and complete in shape, the number of the live bacteria is 0, inactivation treatment is not needed, and the safety is higher.
Description
Technical Field
The invention belongs to the technical field of microbial culture, and particularly relates to a method for efficiently preparing a vibrio cholerae ghost.
Background
The Austrian scholars Lubitz W in the 80 th of the 20 th century developed a novel Vaccine, the bacterin Ghost Vaccine (Vaccine of Bacterial Ghost). The Bacterial Ghost (BG) is a complete Bacterial empty shell without cytoplasmic components, and the basic principle is that the E protein of bacteriophage PhiXl74 can crack gram-negative bacteria, and the cracked gram-negative bacteria form a transmembrane pore structure on a cell membrane, so that cytoplasmic contents in the bacteria are discharged from the pore. The immunogenicity of the inactivated bacterial empty shell as a vaccine is better than that of the traditional inactivated vaccine. Vibrio Cholerae Ghost (VCG) can be used as a presentation system, and the VCG carrier has adjuvant property, and can generate good humoral and cellular immune response. The application of VCG in vaccine production not only can effectively stimulate the immune response of an organism and resist the infection of pathogens, but also can generate immunological memory and resist the re-infection of the pathogens, and simultaneously has the advantages of no need of adjuvant, no pathogenicity, good safety, long-term stable existence, capability of being used as a vector to construct a multivalent recombinant vaccine and the like.
In our previous studies (CN109355221B), high-density fermentation of Vibrio cholerae ghost was achieved by medium component screening, induction time determination, and induction condition optimization, etc. However, in the process, the induction time (the induction time is 11 hours when the bacterial ghost forming rate reaches more than 99%) is too long, the equipment utilization rate is reduced, and the working time of workers is increased. Meanwhile, the longer induction time leads to the crushing of partial bacterial ghost and the loss of a complete bacterial ghost structure on one hand, and leads to higher endotoxin content due to the crushing of the bacterial ghost on the other hand; the quality of the vibrio cholerae ghost used as a vaccine and a vaccine adjuvant can be influenced by the breakage of part of the ghost and the rise of endotoxin. In addition, although the bacterial ghost forming rate can reach more than 99 percent by utilizing the process, a few vibrio cholerae with lost plasmids still exist, the safety of the vibrio cholerae bacterial ghost as a vaccine is greatly influenced, and further inactivation treatment is required, for example, 0.4 percent formaldehyde is added for inactivation at 37 ℃ for 48 hours.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for efficiently preparing a vibrio cholerae ghost, and mainly establishes a brand-new ghost induction culture and preparation strategy.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for efficiently preparing vibrio cholerae ghost comprises the following steps:
s1, selecting a single colony of the vibrio cholerae engineering strain, inoculating the single colony into a seed culture medium, and culturing for 8-14 h to obtain a vibrio cholerae seed solution;
s2, inoculating the vibrio cholerae seed liquid into a fermentation culture medium, culturing until the absorbance value of the bacterial liquid is 4.5-5.5 at 600nm, and adding arabinose for induction;
s3, after induction culture is carried out for 1-3 h, arabinose is added again for secondary induction;
s4, in the induction stage, when the dissolved oxygen value is detected to suddenly drop (namely 'dissolved oxygen inflection point'), adding kanamycin, and continuously culturing for 0.5-1.5 h until the number of viable bacteria is 0;
s5, after induced culture for 4-6 h, the bacterial ghost forming rate reaches more than 99%.
Further, simultaneously with the induction culture, nutrients and antibiotics are supplemented according to a supplement rate of 1.0mL/min, wherein the nutrients and antibiotics comprise: 5.0-8.0 mL/L of glycerol, 50-80 g/L of yeast powder and 150-250 mg/L of ampicillin.
Preferably, the nutrients and antibiotics include: 5.0mL/L of glycerol, 50g/L of yeast powder and 200mg/L of ampicillin.
Preferably, in step S2, the inoculation amount of the Vibrio cholerae seed liquid is performed in a range of 5% to 10%.
Preferably, in step S2, the culturing is carried out at 33-35 ℃, the dissolved oxygen level is 20-30%, and the pH value is 7.30-7.50, and the culturing time is 5-8 h; the fermentation medium comprises the following components: 2.0-8.0 g/L glucose, 2.0-10.0 g/L yeast powder, 1.0-5.0 g/L peptone, 1.0-2.0 g/L, KCl 1.0.0-3.0 g/L, MgSO 4.7H 2O 1.0.0-3.0 g/L citric acid, (NH 4))2SO41.0-3.0 g/L, betaine 0.5-1.5 g/L, V B1 15～25mg/L、VH3-8 mg/L of ampicillin, 50-150 mg/L of ampicillin and 0.5-1.5 mL/L of choline chloride, and the pH value is 7.30-7.50.
Preferably, in the step S2, the addition amount of the arabinose is 1.0-4.0 g/L; more preferably, the arabinose is added in an amount of 2.0 g/L.
Preferably, in the step S3, the added amount of the arabinose is 1.0-4.0 g/L; more preferably, the arabinose is added in an amount of 2.0 g/L.
Preferably, kanamycin is added at the time of induction culture for 3.5 to 4.5 hours in step S4.
Preferably, the pH value of the induction stage is maintained at 7.30-7.50, the dissolved oxygen level is controlled at 20-30%, and the temperature is controlled at 28-30 ℃.
The invention has the beneficial effects that:
the method shortens the induction time by utilizing a secondary induction strategy, shortens the induction culture time required by the bacteria shadow formation rate of more than 99% from 11 hours before technical improvement to 4 hours, improves the yield of the harvested bacteria shadows by more than 16%, obviously improves the production efficiency and reduces the production cost;
the method creatively utilizes the kanamycin added at the dissolved oxygen inflection point of the induction stage, so that the number of live bacteria of the vibrio cholerae is reduced to 0, the safety of preparing the bacterial ghost of the vibrio cholerae is improved, and the influence of further inactivation treatment by adopting formaldehyde and the like on the safety of the bacterial ghost and the increase of heavy workload are avoided;
the process of the invention not only effectively shortens the induction culture time and efficiently prepares the vibrio cholerae ghost, but also reduces the endotoxin content to below 1/5 of the process before improvement, and the prepared ghost has uniform and complete shape, thereby reducing the production cost of the vibrio cholerae ghost and improving the safety and the effectiveness of the vibrio cholerae ghost.
Drawings
FIG. 1 a: the influence of arabinose addition concentration on the vibrio cholerae bacterial concentration during secondary induction;
FIG. 1 b: effect of arabinose addition concentration on viable count (resistant plates) at secondary induction;
FIG. 1 c: effect of arabinose addition concentration on viable count (non-resistant plates) at secondary induction;
FIG. 1 d: effect of arabinose addition concentration on ghost formation rate (calculated by number of resistant plates) at secondary induction;
FIG. 1 e: effect of arabinose addition concentration on ghost formation rate (calculated by number of non-resistant plates) at secondary induction;
FIG. 2 a: the influence of arabinose addition time on the vibrio cholerae bacterial concentration during secondary induction;
FIG. 2 b: effect of arabinose addition time on viable count (resistant plates) at secondary induction;
FIG. 2 c: effect of arabinose addition time on viable count (non-resistant plates) at secondary induction;
FIG. 2 d: effect of arabinose addition time on ghost formation rate (calculated by number of resistant plates) at secondary induction;
FIG. 2 e: effect of arabinose addition time on ghost formation rate (calculated by number of non-resistant plates) at secondary induction;
FIG. 3 a: inducing the change of the stirring speed in the culture stage;
FIG. 3 b: inducing the change of dissolved oxygen level in the culture stage
FIG. 4: the effect of antibiotic species on the number of viable bacteria;
FIG. 5: the effect of kanamycin addition on the number of viable bacteria;
FIG. 6 a: comparing the bacterial concentrations before and after the improvement of the preparation process of the vibrio cholerae bacterial ghost;
FIG. 6 b: comparing the number of live bacteria (resistant plate) before and after the improvement of the preparation process of the vibrio cholerae ghost;
FIG. 6 c: comparison of viable count (non-resistant plate) before and after the improvement of the preparation process of the vibrio cholerae ghost;
FIG. 6 d: comparing the bacterial ghost forming rates (calculated by the bacterial number of the resistant plate) before and after the preparation process of the vibrio cholerae bacterial ghost is improved;
FIG. 6 e: comparing the bacterial ghost forming rates (calculated by the number of non-resistant plates) before and after the vibrio cholerae bacterial ghost preparation process is improved;
FIG. 6 f: comparing the quality of the obtained bacterial sludge before and after the preparation process of the vibrio cholerae bacterial ghost is improved;
FIG. 7 a: the vibrio cholerae ghost shape prepared by the process is shown in the specification;
FIG. 7 b: improving the shape of the bacterial ghost of the vibrio cholerae prepared by the prior process;
FIG. 8: the influence of the preparation process of the bacterial ghost of the vibrio cholerae on the content of endotoxin.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
The term "inflection point of dissolved oxygen" as used herein refers to a time point when a sudden/sharp decrease in the value of dissolved oxygen in a fermentation broth is detected by an oxygen-dissolving electrode of a fermenter during a fermentation culture.
Example 1: determination of the ghost Induction strategy of Vibrio cholerae
1.1 materials
1.1.1 strains: the vibrio cholerae engineering strain is provided by Hochenne professor of animal medical college of Chinese agricultural university, the preparation method of the vibrio cholerae engineering strain is patented (the patent name is a preparation method of a vibrio cholerae ghost and the application of the vibrio cholerae ghost in poultry vaccine; the patent application number is 201711461847.X), and a single colony (containing a target plasmid pBAD-sE) which is constructed and identified to be positive by the method is the strain used by the invention.
1.1.2.1 Medium
1.1.2.2 seed culture Medium: LB medium (peptone 10.0g/L, yeast powder 5.0g/L, NaCl10.0g/L), adjusted to pH 7.50 with 4.0mol/L NaOH solution.
Fermentation medium: 5.0g/L glucose, 5.0g/L yeast powder, 2.0g/L peptone and 1.5g/L, KCl 2.0.0 g/L, MgSO citric acid4·7H2O 1.5g/L、(NH4)2SO42.0g/L, 1.0g/L betaine, 1.0mL/L, V choline chlorideB120.0mg/L、VH5.0 mg/L; the preparation method comprises the following steps: respectively weighing glucose, yeast powder, peptone, citric acid, KCl, and MgSO4·7H2O、(NH4)2SO4Betaine, VB1、VHMeasuring choline chloride, adding deionized water, fixing volume, shaking thoroughly for dissolving, adjusting pH to 7.50 with 4mol/L NaOH solution, and sterilizing at high temperature (121 deg.C, 15 min); after cooling, 100mg/L of ampicillin was added according to the aseptic requirements.
1.2 methods
1.2.1 seed culture: a single colony of the Vibrio cholerae engineering strain is picked up and inoculated into a 500mL triangular flask containing 200mL of seed culture medium, and the culture is carried out for 12h at 35 ℃ and 160 rpm.
1.2.2 fermentation culture: inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 5%, maintaining the culture temperature at 35 ℃, the pH at 7.50, controlling the dissolved oxygen level at 20-30%, culturing at 35 ℃ for 8h, and controlling the thallus concentration (OD)600) When the content is about 5.0, adding arabinose to the obtained product according to a certain induction strategy (the specific operation is shown in the result part of the embodiment 3) for induction; in the induction stage, the pH value is maintained at 7.50, the dissolved oxygen level is controlled at 20-30%, and the temperature is controlled at 30 ℃. Meanwhile, in the induction process, certain nutrients and antibiotics (5.0 mL/L of glycerol, 50g/L of yeast powder and 200mg/L of ampicillin) are supplemented at a supplement rate of 1.0 mL/min.
1.2.3 measurement method
1.2.3.1 temperature, pH and dissolved oxygen level: the measurement is carried out by utilizing a temperature electrode, a pH electrode and a dissolved oxygen electrode which are attached to the fermentation tank respectively.
1.2.3.2 cell concentration: the optical density of the fermentation broth was determined at 600nm using a spectrophotometer and recorded as OD600The measured value is between 0.2 and 0.9.
1.2.3.3 glucose concentration: measured using a biosensing analyzer (SBA-40E).
1.2.3.4 viable count: diluting the fermentation liquor by 10 times in a gradient manner, coating a flat plate, observing the number of colonies on the flat plate, and calculating the number of viable bacteria (CFU/mL) contained in the fermentation liquor.
1.2.3.5 plasmid stability: and respectively coating the diluted fermentation liquor on a resistant plate containing 100mg/L of ampicillin and a non-resistant plate containing no ampicillin, wherein the ratio of the viable bacteria number of the resistant plate to the non-resistant plate is plasmid stability (%).
1.2.3.6 bacterial ghost formation rate: taking fermentation liquor with induction time of 0h and fermentation liquor with different induction times, respectively counting live bacteria at different times, wherein the ratio of the number of the live bacteria at different induction times to the number of the live bacteria at induction time of 0h is the bacterial ghost formation rate (%) at the induction time.
1.3 results
1.3.1 arabinose addition concentration at Secondary Induction
When the vibrio cholerae is fermented and cultured for 8 hours according to the 1.2.2, adding 2.0g/L arabinose for induction; subsequently, when the induction was carried out for 3 hours, 1.0g/L, 2.0g/L, 3.0g/L and 4.0g/L of arabinose were again added to carry out the secondary induction, and the results are shown in FIG. 1. Wherein, a is the bacterial concentration, b is the viable bacteria number (resistant plate), c is the viable bacteria number (non-resistant plate), d is the bacterial ghost forming rate (calculated by the bacterial number of the resistant plate), and e is the bacterial ghost forming rate (calculated by the bacterial number of the non-resistant plate).
As shown in FIGS. 1(a, b, and d), when arabinose was added again for the second induction after 3 hours of induction, the induction time required for the bacterial ghost formation rate to reach 99% or more was significantly shortened as compared with the case of the process (patent No. ZL 201811313599.9) in which only one induction was carried out; when the concentration of arabinose added in the secondary induction is 1g/L, the bacterial ghost forming rate reaches more than 99 percent when the secondary induction is carried out for 7 hours (the induction time is counted from the time of adding arabinose for the first time, and the same is carried out if no special description is given below); when the concentration of the secondarily added arabinose is 2g/L or more than 2g/L, the bacterial ghost forming rate reaches more than 99 percent, and the required induction time is 6 hours; therefore, the concentration of arabinose added for the second time is preferably 2 g/L. Furthermore, as can be seen from FIGS. 1(c, e), the number of viable bacteria on the non-resistant plate gradually increased due to the loss of the growth of the plasmid Vibrio cholerae in the late induction period.
1.3.2 arabinose addition time at Secondary Induction
When the vibrio cholerae is fermented and cultured for 8 hours according to the 1.2.2, adding 2.0g/L arabinose for induction; subsequently, when the induction was performed for 1 hour, 2 hours, 3 hours, and 4 hours, respectively, the secondary induction was performed by adding 2.0g/L arabinose again, and the results are shown in FIG. 2. Wherein, a is the bacterial concentration, b is the viable bacteria number (resistant plate), c is the viable bacteria number (non-resistant plate), d is the bacterial ghost forming rate (calculated by the bacterial number of the resistant plate), and d is the bacterial ghost forming rate (calculated by the bacterial number of the non-resistant plate).
As can be seen from FIGS. 2(a, b, d), the timing of the second arabinose addition significantly affected the vibrio cholerae ghost formation; adding arabinose for secondary induction when the induction is carried out for 1h, 2h, 3h and 4h respectively, wherein the induction time required for the vibrio cholerae ghost formation rate to reach more than 99% is 5h, 4h, 6h and 7h respectively; therefore, it is preferable to perform secondary induction by adding 2g/L arabinose at the time of 2h induction. Furthermore, as shown in FIG. 2(c, e), the number of viable bacteria on the non-resistant plate gradually increased due to the growth of the lost plasmid, Vibrio cholerae engineering bacteria, at the late stage of induction.
1.4 summary
When the inducer is added to induce the expression of the foreign protein, the inducer is added for multiple times, so that the inhibition effect caused by adding the inducer with too high concentration at one time can be avoided, the expression of the promoter and the target gene can be stimulated for multiple times, and the expression level of the foreign gene is improved. When the vibrio cholerae ghost is induced to form, after arabinose is added for the first time, arabinose is added for the second time, so that the induction time required when the vibrio cholerae ghost forming rate reaches more than 99 percent can be obviously shortened; and by considering the concentration and the adding time of the secondary addition of the arabinose, the secondary induction is preferably carried out by adding 2g/L of arabinose after the primary addition of the arabinose is induced for 2 hours; after the induction culture for 4 hours, the bacterial ghost forming rate reaches more than 99 percent. In addition, although arabinose is induced when the Vibrio cholerae engineering bacteria have higher plasmid stability, Vibrio cholerae with lost plasmids still exists in the induction process and after the induction is finished, and the Vibrio cholerae engineering bacteria with lost plasmids gradually increase in the later culture period due to the existence of the Vibrio cholerae engineering bacteria with lost plasmids, thus seriously affecting the safety and quality of the vibrio cholerae ghost.
Example 2: addition of antibiotics during induction of vibrio cholerae ghost
2.1 materials
2.1.1 strains: engineered strain of Vibrio cholerae (same as example 1).
2.1.2 culture Medium
2.1.2.1 seed culture Medium: the same as in example 1.
2.1.2.2 fermentation medium: along with example 1.
2.2 methods
2.2.1 seed culture: the same as in example 1.
2.2.2 fermentation culture: inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 5%, maintaining the culture temperature at 35 ℃, the pH at 7.50, controlling the dissolved oxygen level at 20-30%, culturing at 35 ℃ for 8h, and controlling the thallus concentration (OD)600) When the concentration is about 5.0, adding 2.0g/L of arabinose for induction, and adding 2.0g/L of arabinose again for secondary induction after 2 hours; in the induction stage, the pH value is maintained at 7.50, the dissolved oxygen level is controlled at 20-30%, and the temperature is controlled at 30 ℃. Meanwhile, in the induction process, certain nutrients and antibiotics (5.0 mL/L of glycerol, 50g/L of yeast powder and 200mg/L of ampicillin) are supplemented at a supplement rate of 1.0 mL/min. In addition, according to the experimental requirements of this example, antibiotics were added at a certain concentration during the induction phase (see the results section of this example 3 for a specific operation).
2.2.3 measurement method
2.2.3.1 temperature, pH and dissolved oxygen level: the same as in example 1.
2.2.3.2 cell density: the same as in example 1.
2.2.3.3 glucose concentration: the same as in example 1.
2.2.3.5 plasmid stability: the same as in example 1.
2.2.3.6 bacterial ghost formation rate: the same as in example 1.
2.3 results
2.3.1 oxygen solubility Change in Vibrio cholerae engineering bacteria culture Process
In the induction stage of vibrio cholerae ghost, after arabinose is added for the first time, the ventilation volume and the tank pressure are respectively adjusted to 1.0m3And 0.06MPa, and further maintaining the dissolved oxygen level by adjusting the stirring speed, wherein the stirring speed and the dissolved oxygen level are shown in FIG. 3, wherein a is the stirring speed and b is the dissolved oxygen level.
As can be seen from FIG. 3, the oxygen consumption decreased gradually and the stirring speed decreased gradually to maintain the dissolved oxygen level at 20% to 30% up to 4.1 hours after the start of induction. When the induction is carried out for 4.1h, the dissolved oxygen level is suddenly reduced, which indicates that the oxygen consumption is increased; after 4.1h, the oxygen consumption is gradually increased, and the stirring speed is required to be continuously increased to maintain a certain dissolved oxygen level, which indicates that the vibrio cholerae engineering bacteria begin to propagate after 4.1h, so that the growth and propagation of the vibrio cholerae can be effectively inhibited by adding a proper antibiotic at the moment (namely an 'dissolved oxygen inflection point').
2.3.2 types of antibiotics added
At the inflection point of dissolved oxygen (4.1 hours), 100mg/L of penicillin, 50mg/L of kanamycin, and 10mg/L of polymyxin B were added, and the number of viable bacteria (non-resistant plate) at 0.5 hours and 1.0 hour after the addition was as shown in FIG. 4.
As can be seen from FIG. 4, the number of viable bacteria was significantly reduced by the addition of antibiotics at the late stage of induction. Kanamycin inhibits bacterial growth more effectively than penicillin and polymyxin B, and the number of viable bacteria is 0 within 0.9h after addition (i.e.: 5h after induction culture), so kanamycin is preferably added at the induction stage.
2.3.3 kanamycin addition concentration
As shown in FIG. 5, the numbers of viable bacteria (non-resistant plates) at 0.5h and 0.9h after addition of 20mg/L, 30mg/L, 50mg/L and 100mg/L kanamycin at the inflection point of dissolved oxygen (4.1h), respectively.
As can be seen from FIG. 5, the addition of kanamycin effectively inhibited bacterial growth; and when the kanamycin addition concentration is more than 30mg/L, the bacteriostatic effect is not obviously different. Therefore, preferably in the induction stage of the "dissolved oxygen inflection point" when adding 30mg/L kanamycin.
2.4 summary
The dissolved oxygen level is an important parameter of microbial fermentation, and both the dissolved oxygen level and the oxygen consumption rate can reflect the growth condition of bacteria. After the vibrio cholerae engineering bacteria are induced by the arabinose, the oxygen consumption of the vibrio cholerae engineering bacteria is gradually reduced because the vibrio cholerae engineering bacteria continuously form vibrio cholerae ghost, and the dissolved oxygen in the culture solution is gradually increased, so that the dissolved oxygen value measured by the dissolved oxygen electrode is gradually increased. In the process, the stirring speed, the ventilation quantity and the tank pressure are reduced to maintain the proper dissolved oxygen level; however, when the engineering bacteria of vibrio cholerae with lost plasmids begin to grow, the oxygen consumption is increased, the dissolved oxygen in the culture solution is reduced, and the dissolved oxygen value measured by the dissolved oxygen electrode is rapidly reduced; therefore, an inflection point appears in the dissolved oxygen value in the process. When the oxygen-dissolving inflection point is positioned, proper antibiotics are added to effectively inhibit the growth of the plasmid-lost vibrio cholerae; if antibiotics are added before the inflection point of the dissolved oxygen, the normal formation and shape of the bacterial ghost are affected; if antibiotics are added after this inflection point of dissolved oxygen, the inhibitory effect of the antibiotics on bacteria is reduced.
A variety of antibiotics can inhibit the growth of gram-negative bacteria, among which: beta-lactam (penicillins, cephalosporins) antibiotics inhibit bacterial growth by inhibiting the biosynthesis of the bacterial cell wall; the action mechanism of aminoglycoside (streptomycin, gentamicin, kanamycin, etc.) antibiotics is to inhibit the synthesis of bacterial proteins; the action mechanism of polymyxin antibiotics is that free amino with positive charges contained in drug molecules can be combined with phosphate radical with negative charges in phospholipid of gram-negative bacteria cell membrane, the polymyxin antibiotics are effective to bacteria which grow and grow in a propagation period and stop growing, and polymyxin B and polymyxin E are commonly used clinically.
Based on the change of the dissolved oxygen level after induction, antibiotics are added to inhibit the growth of bacteria when the 'dissolved oxygen inflection point' is determined, and the bacterial ghost forming rate of the vibrio cholerae reaches more than 99 percent. Meanwhile, by comparing different antibiotics and addition concentrations, 30mg/L kanamycin is preferably added, and the number of live bacteria is 0 when the induction culture is carried out for 5 hours, the safety of the vibrio cholerae ghost as a vaccine and an adjuvant is effectively guaranteed, and the operation of adding formaldehyde for inactivation is omitted.
Example 3: comparison before and after improvement of preparation process of vibrio cholerae ghost
3.1 materials
3.1.1 strains: engineered strain of Vibrio cholerae (same as example 1).
3.1.2 culture Medium
3.1.2.1 seed Medium: the same as in example 1.
3.1.2.2 fermentation medium: along with example 1.
3.2 methods
3.2.1 seed culture: the same as in example 1.
3.2.2 fermentation culture:
(1) the improved process of the invention is a preferred embodiment: inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 5%, maintaining the culture temperature at 35 ℃, the pH at 7.50, controlling the dissolved oxygen level at 20-30%, culturing at 35 ℃ for 8h, and controlling the thallus concentration (OD)600) When the concentration is about 5.0, adding 2.0g/L of arabinose for induction, and adding 2.0g/L of arabinose again for secondary induction after 2 hours; in the induction stage, the pH value is maintained at 7.50, the dissolved oxygen level is controlled at 20-30%, and the temperature is controlled at 30 ℃. Meanwhile, in the induction process, certain nutrients and antibiotics (5.0 mL/L of glycerol, 50g/L of yeast powder and 200mg/L of ampicillin) are supplemented at a supplement rate of 1.0 mL/min. In addition, in the induction phase, when the dissolved oxygen electrode detection dissolved oxygen value suddenly reduced (induced for 4.1h), adding 30mg/L kanamycin; and finishing fermentation when the induction time is 5 hours.
(2) Pre-improvement process (ex ZL 201811313599.9): inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 5%, maintaining the culture temperature at 35 ℃, the pH at 7.50, controlling the dissolved oxygen level at 20-30%, culturing at 35 ℃ for 8h, and controlling the thallus concentration (OD)600) When the concentration is about 5.0, adding 2.0g/L arabinose for induction; an induction phase, the pH being maintained at7.50, controlling the dissolved oxygen level at 20-30% and controlling the temperature at 30 ℃. Meanwhile, in the induction process, certain nutrient substances and antibiotics (5.0 mL/L of glycerol, 50g/L of yeast powder and 200mg/L of ampicillin) are supplemented at a supplement rate of 1.0 mL/min; when the fermentation is induced for 11 hours, the fermentation is finished.
3.2.3 measurement method
3.2.3.1 temperature, pH and dissolved oxygen level: the same as in example 1.
3.2.3.2 cell concentration: the same as in example 1.
3.2.3.3 glucose concentration: the same as in example 1.
3.2.3.5 plasmid stability: the same as in example 1.
3.2.3.6 bacterial ghost formation rate: the same as in example 1.
3.2.3.7 determination of endotoxin content: endotoxin content was determined according to the limulus kit (test tube quantitative chromogenic substrate method) using instructions.
3.3 results
3.3.1 fermentation Process comparison
The fermentation process of preparing the vibrio cholerae ghost by adopting the process and the process before improvement is shown in figure 6, wherein a is the bacterial concentration, b is the number of live bacteria (resistant plate), c is the number of live bacteria (non-resistant plate), d is the ghost forming rate (calculated by the number of the bacteria in the resistant plate), e is the ghost forming rate (calculated by the number of the bacteria in the non-resistant plate), and f is the quality of the harvested bacterial sludge.
As can be seen from FIGS. 6(a, b, d), when the bacterial ghost of Vibrio cholerae is prepared by the process of the present invention and the process before improvement, the bacterial ghost formation rate reaches more than 99% at 4h and 11h of induction, respectively. As shown in FIG. 6(b, c, e), the viable count was 0 after 5 hours of induction by the process of the present invention; adopting the pre-improvement process, when inducing for 11h and 12h, the viable count gradually increases, and when 12h, the viable count (non-resistant flat plate) is 9.0x108CFU/mL. As can be seen from FIG. 6(f), the wet weight of the harvested bacteria was 12.31g/L, which is 16.46% higher than that of the bacteria harvested by the process of the present invention (10.57 g/L).
3.3.2 Vibrio cholerae ghost morphology
The observation of the bacterial ghost forms of the vibrio cholerae prepared by different preparation processes by using a scanning electron microscope shows the result as shown in fig. 7, wherein a is the bacterial ghost form of the vibrio cholerae prepared by adopting the process of the invention, and b is the bacterial ghost form of the vibrio cholerae prepared by adopting the process before improvement.
As can be seen from FIG. 7, the Vibrio cholerae prepared by the process of the present invention has uniform ghost and complete morphology; the vibrio cholerae prepared by the improved pre-process has uneven ghost and partial ghost is broken. Therefore, the process provided by the invention is more beneficial to the efficient preparation of the vibrio cholerae ghost and can obviously improve the quality of the vibrio cholerae ghost.
3.3.3 endotoxin content
The endotoxin content was measured in the fermentation broth 5h after the induction of the process of the present invention and 11h after the induction of the process before the improvement, respectively, and the results are shown in FIG. 8.
As can be seen from FIG. 8, the endotoxin content was significantly affected by the Vibrio cholerae ghost preparation process. When the process and the pre-improvement process are adopted, the content of endotoxin is 152 ten thousand EU/mL and 783 ten thousand EU/mL respectively; the endotoxin content produced by the process of the present invention is below 1/5 of the prior art. As shown in the combined graph of FIG. 8, the process of the invention has short induction time, the bacterial ghost keeps complete shape and the content of endostatin is low; the prior process is improved, and the induction time is long, so that partial bacterial ghost is broken, and the endotoxin content is increased.
3.4 nodules
When the process is adopted for preparing the vibrio cholerae ghost, the induction period required by the ghost forming rate reaching more than 99 percent is shortened from 11h of the process before improvement to 4 h; and the number of live bacteria is 0 after 5h of induction, the operation that the process needs to be inactivated by formaldehyde and the like before improvement can be omitted; the bacterial ghost of the obtained vibrio cholerae is improved by more than 16 percent, the endotoxin content is reduced to be below 1/5 of the process before the improvement, and the bacterial ghost is formed uniformly and has complete shape. Therefore, the preparation method of the vibrio cholerae ghost has the advantages of short induction period, 0 viable bacteria number, low endotoxin content, complete ghost form and the like, obviously reduces the production cost of the vibrio cholerae ghost, improves the safety and the effectiveness of the vibrio cholerae ghost, and promotes the wide application of the vibrio cholerae ghost in the vaccine preparation industry.
Although the present invention has been disclosed in the form of preferred embodiments, it is not intended to limit the present invention, and those skilled in the art may make various changes, modifications, substitutions and alterations in form and detail without departing from the spirit and principle of the present invention, the scope of which is defined by the appended claims and their equivalents.
Claims (9)
1. A method for efficiently preparing a vibrio cholerae ghost is characterized by comprising the following steps:
s1, selecting a single colony of the vibrio cholerae engineering strain, inoculating the single colony into a seed culture medium, and culturing for 8-14 h to obtain a vibrio cholerae seed solution; the vibrio cholerae engineering strain is a strain containing a target plasmid pBAD-sE, wherein the plasmid pBAD-sE is a recombinant plasmid obtained by connecting a cleavage protein E gene fragment to a pBAD-24 plasmid;
s2, inoculating the vibrio cholerae seed liquid into a fermentation culture medium, culturing until the absorbance value of the liquid is 4.5-5.5 at 600nm, adding 1.0-4.0 g/L arabinose for induction, and controlling the dissolved oxygen level at 20-30%;
s3, after the induction culture is started for 1-3 h at S2, adding 1.0-4.0 g/L of arabinose again for secondary induction;
s4, when the dissolved oxygen value is detected to be reduced to be lower than 20% after the induction culture is started for 3.5-4.5 h at S2, adding kanamycin, and continuously culturing for 0.5-1.5 h until the number of viable bacteria is 0;
s5, finishing fermentation after the induction culture is started for 4-6 h at S2, wherein the bacterial ghost forming rate reaches more than 99%;
in step S2, the fermentation medium includes the following components: 2.0-8.0 g/L glucose, 2.0-10.0 g/L yeast powder, 1.0-5.0 g/L peptone, 1.0-2.0 g/L, KCl 1.0.0-3.0 g/L, MgSO 4.7H 2O 1.0.0-3.0 g/L citric acid, (NH 4))2SO41.0-3.0 g/L, betaine 0.5-1.5 g/L, VB1 15～25 mg/L、VH3-8 mg/L of ampicillin, 50-150 mg/L of ampicillin and 0.5-1.5 mL/L of choline chloride, and the pH value is 7.30-7.50.
2. The method for efficiently preparing a ghost of Vibrio cholerae according to claim 1, wherein nutrients and antibiotics are supplemented at a rate of 1.0mL/min during the induction culture, and the nutrients and antibiotics comprise: 5.0-8.0 mL/L of glycerol, 50-80 g/L of yeast powder and 150-250 mg/L of ampicillin.
3. The method for efficiently preparing the bacterial ghost of Vibrio cholerae according to claim 1, wherein the pH value of the induction stage is maintained at 7.30-7.50, the dissolved oxygen level is controlled at 20-30%, and the temperature is controlled at 28-30 ℃.
4. The method for efficiently producing a ghost of Vibrio cholerae according to claim 1, wherein the inoculating amount of the Vibrio cholerae seed solution is 5% to 10% in step S2.
5. The method for efficiently producing a ghost of Vibrio cholerae according to claim 1, wherein the culturing is performed at 33-35 ℃, 20-30% dissolved oxygen, and 7.30-7.50 pH for 5-8 h in step S2.
6. The method for efficiently producing a ghost of Vibrio cholerae according to claim 1, wherein the arabinose is added in an amount of 2.0g/L in step S2.
7. The method for preparing Vibrio cholerae ghost with high efficiency according to claim 1, wherein the arabinose is added again in an amount of 2.0g/L in step S3.
8. The method for efficiently producing a ghost of Vibrio cholerae according to claim 1, wherein the kanamycin is added in an amount of 30 to 100mg/L in step S4.
9. The method for efficiently preparing a ghost of Vibrio cholerae according to claim 1, comprising the steps of:
s1, selecting a single colony of the vibrio cholerae engineering strain, inoculating the single colony into a seed culture medium, and culturing for 12 hours to obtain a vibrio cholerae seed solution;
s2, inoculating the vibrio cholerae seed liquid into a fermentation culture medium according to the inoculation amount of 5%, and adding 2.0g/L arabinose for induction when the absorbance value of the bacterial liquid is about 5.0 at 600 nm; after induction is started, the pH value is maintained at 7.50, the dissolved oxygen level is controlled at 20-30%, the temperature is controlled at 30 ℃, and meanwhile, certain nutrients and antibiotics are supplemented at a supplement rate of 1.0mL/min, wherein the nutrients and antibiotics consist of: 5.0mL/L of glycerol, 50g/L of yeast powder and 200mg/L of ampicillin;
s3, after induction culture is started for 2 hours at S2, adding 2.0g/L arabinose again for secondary induction;
s4, detecting that the dissolved oxygen value is reduced to be lower than 20% when S2 starts to induce for 4.1h, and adding 30mg/L kanamycin;
s5, starting induction culture at S2 for 5h, and ending fermentation, wherein the bacterial ghost formation rate reaches more than 99%, and the number of viable bacteria is 0.
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