CN110819582B - Fermentation medium and culture method for expressing foot-and-mouth disease virus-like particle antigen by using escherichia coli - Google Patents
Fermentation medium and culture method for expressing foot-and-mouth disease virus-like particle antigen by using escherichia coli Download PDFInfo
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Abstract
The invention discloses a fermentation culture medium and a culture method for expressing foot-and-mouth disease virus-like particle antigen by escherichia coli, wherein the fermentation culture medium comprises a basal culture medium, a culture medium for supplementing and a culture medium for inducing expression, and the basal culture medium can shorten the lag phase of escherichia coli strains for production and enable the strains to enter logarithmic phase as soon as possible; the feed medium can improve the multiplication capacity of escherichia coli in the logarithmic growth phase and ensure that escherichia coli has higher bacteria number in the high-density fermentation process; the culture medium for induction expression can maintain the platform phase of escherichia coli so as to improve the expression quantity of the virus-like particle antigen, and meanwhile, the components in culture can promote the antigen to be expressed in a soluble form. The fermentation culture method comprises the following steps: the fermentation medium and the culture method can greatly improve the antigen expression quantity of the virus-like particles and can simultaneously make the antigen expressed in a soluble form to the maximum extent.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a fermentation medium and a culture method for expressing a foot-and-mouth disease virus-like particle antigen by using escherichia coli.
Background
Foot-and-Mouth Disease is an acute, hot and highly contagious animal epidemic Disease mainly caused by Foot-and-Mouth Disease Virus (FMDV) and mainly caused by artiodactyl diseases. The disease is a virulent infectious disease which currently has the greatest threat to the development of animal husbandry worldwide, and therefore the world animal health Organization (OIE) lists the infectious disease which must be reported. Attenuated vaccines have played a great role in controlling the pandemic of european foot and mouth disease, but have been discontinued due to the virulent return of vaccine strains that have caused the outbreak of foot and mouth disease. Although the inactivated vaccine used at present is safe, the inactivated vaccine still has the risk of causing epidemic outbreak if the inactivated vaccine is not completely inactivated, and cannot adapt to the current international trade situation.
Virus-like particles (virus-like particles VLPs) are hollow particles that self-assemble in vivo or in vitro with one or more structural proteins of a virus, and because they do not contain viral genetic material, they cannot replicate and amplify and do not have the ability to infect disease. VLPs are similar to natural virus particles in morphological structure, well simulate antigen epitopes of natural viruses, and the tens of nanometers of scale and highly ordered epitope structure are also beneficial to phagocytosis and antigen processing of dendritic cells, so that immune protection reaction on viruses is induced to be generated collectively. Compared with the traditional vaccine, the VLPs have equivalent immunogenicity and better safety, can also be used as carrier proteins to carry foreign antigens or intervene non-protein antigens through chemical coupling, and are important components involved in the vaccine.
To remove impurities sufficiently, and in particular to avoid viral gene inclusion, VLPs are typically prepared by in vitro assembly following exogenous expression in an expression system. Expression systems can be divided into eukaryotic expression systems and prokaryotic expression systems. The eukaryotic system is faced with the problems of long culture period, high cost, low expression level, difficult purification and the like, and restricts the large-scale production of VLPs vaccines. Compared with eukaryotic expression systems, the Escherichia coli expression system is earlier and more widely applied to the field of biological medicine, and is a well-known expression system with high efficiency, low cost and good safety. Because the structure and assembly of the particle-type antigen are complex, the expression amount and the expression form of the particle-type antigen have important influence on the structure of the antigen and subsequent purification and in vitro assembly, and the in vitro assembly efficiency is a key factor for determining the quality of the virus-like particle product. When an escherichia coli system is used for antigen expression, the antigen expression amount is too low, so that the production cost is greatly increased while the productivity is reduced; the antigen expression amount is too high, the possibility that the antigen is expressed in an inclusion body form is greatly increased, the subsequent purification difficulty and cost are increased, and the in vitro assembly efficiency is influenced.
Disclosure of Invention
The invention aims to: aiming at the problems in the prior art, the high-density large-scale fermentation culture medium and the large-scale fermentation culture method capable of producing the virus-like particle antigen in a large scale are provided, the high-density fermentation culture process is used for improving the expression quantity of the antigen, and simultaneously ensuring that the antigen is expressed in a soluble form, so that the effects of reducing the production cost and improving the quality of the final virus-like particle are achieved.
The technical scheme adopted by the invention is as follows:
a fermentation culture medium for expressing foot-and-mouth disease virus-like particle antigen by escherichia coli comprises a basal culture medium and a supplemented culture medium for cultivating the escherichia coli, and a culture medium for inducing expression during inducing expression;
the basal medium per liter comprises: 6-12 g of monopotassium phosphate, 3-8 g of diammonium phosphate, 1-5 g of sodium chloride, 5-10 g of yeast extract, 8-15 g of tryptone, 15-30 g of glycerol, 0.01-0.02 wt% of defoaming agent, 0.4-0.6 vt% of trace element solution, 1-3 vt% of antibiotic solution and 1-3 vt% of magnesium sulfate solution;
the medium for feeding comprises per liter: 5-10 g of yeast extract, 8-15 g of tryptone, 0.4-0.6 vt% of trace element solution, 1-3 vt% of antibiotic solution and 1-3 vt% of magnesium sulfate solution;
the culture medium for inducing expression comprises the following components in each liter: 5-10 g of yeast extract, 8-15 g of tryptone, 50-100 g of glycerol, 0.05-0.1 vt% of isopropyl thiogalactoside solution and 1-3 vt% of magnesium sulfate solution.
Further, the basal medium is obtained by: dissolving potassium dihydrogen phosphate, diammonium hydrogen phosphate, sodium chloride, yeast extract, tryptone, glycerol and a defoaming agent in water to obtain a mixture, adjusting the pH value of the mixture to 7.0-7.2, carrying out autoclaving at 116 ℃ for 25-30 min, cooling to 50 ℃ or below, adding a trace element solution, an antibiotic solution and a magnesium sulfate solution, and uniformly mixing.
Further, the medium for feeding is obtained by: dissolving yeast extract and tryptone in water to obtain a mixture, adjusting the pH value of the mixture to 7.0-7.2, carrying out autoclaving at 116 ℃ for 25-30 min, then cooling to 50 ℃ or below, adding a trace element solution, an antibiotic solution and a magnesium sulfate solution, and uniformly mixing to obtain the yeast extract.
Further, the medium for inducing expression was obtained by the following procedure: dissolving yeast extract, tryptone and glycerol in water to obtain a mixture, adjusting the pH value of the mixture to 7.0-7.2, carrying out autoclaving at 116 ℃ for 25-30 min, then cooling to 50 ℃ or below, adding an isopropyl thiogalactoside solution and a magnesium sulfate solution, and uniformly mixing to obtain the product.
Furthermore, each liter of the trace element solution comprises 150-200 g of citric acid, 0.5-1.0 g of ethylene diamine tetraacetic acid, 0.1-0.4 g of cobalt chloride, 2-5 g of calcium chloride, 0.05-0.3 g of copper chloride, 0.2-0.5 g of boric acid, 0.8-2.5 g of manganese chloride, 0.1-0.4 g of sodium molybdate, 0.5-2.0 g of zinc acetate and 5-12 g of ferric citrate; each liter of the antibiotic solution comprises 50-150 g of ampicillin, 50-100 g of kanamycin and 25-60 g of chloramphenicol; the trace element solution and the antibiotic solution comprise the components but are not limited to the components, and the trace elements and the antibiotic required by the fungus in the fermentation culture are provided, and in the preparation process, the components are dissolved and then filtered by a 0.22 mu m filter; the concentration of the isopropylthiogalactoside solution is 240 g/L235-; the isopropyl thiogalactoside solution is prepared by dissolving and then filtering through a 0.22 mu m filter.
The fermentation medium is applied to antigen production and vaccine preparation.
The method for performing fermentation culture by adopting the fermentation culture medium for expressing the foot-and-mouth disease virus-like particle antigen by using the escherichia coli comprises the following steps:
s1, inoculating escherichia coli expressing foot-and-mouth disease virus-like particle antigen into a basal culture medium according to the proportion of 2-5 vt%, culturing for 6-12 h at 37 ℃, and then performing expanded culture;
s2, determining OD of bacterial liquid600nmWhen the value is 5-10, feeding the culture medium for feeding at the feeding speed of 0.5-2 ml/L per hour, and continuing to culture;
s3, determining OD of bacterial liquid600nmWhen the value reaches 30 and above, cooling to 15-20 ℃, and when the temperature of the bacterial liquid is reduced to 20 ℃, continuing culturing for 0.5 h;
s4, adding a culture medium for inducible expression into the bacterial liquid, wherein the adding speed of the culture medium is 1ml/L per hour, and the inducible expression is carried out for 12-16 hours;
s5, centrifuging the bacteria liquid after induction expression for 10-15min, and collecting bacteria mud.
Further, in the fermentation culture process, fermentation parameters are recorded at certain intervals, and bacteria liquid OD is measured by aseptic sampling600nmThe value is obtained.
Further, the specific conditions of the expanded bacteria culture in step S1 and the continued culture in step S2 are both: the culture temperature is 36.8-37.2 ℃, the dissolved oxygen rate is 30-50%, and the pH value is regulated by using ammonia water to be 6.8-7.2.
Further, the specific conditions for continuing the culture in step S3 are: the culture temperature is 15-20 ℃, the dissolved oxygen rate is 20-40%, and the pH value is regulated by using ammonia water to be 7.2-7.4.
Further, the specific conditions for inducing expression in step S4 are: the temperature is 15-20 ℃, the dissolved oxygen rate is 20-40%, and the pH value is regulated by using ammonia water to be 7.0-7.2.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. according to the invention, the culture medium is divided into 3 culture media, and fermentation processes such as low-temperature induced expression are matched, so that on the basis of not additionally increasing process procedures, high expression quantity and high soluble antigen proportion in the large-scale production of the foot-and-mouth disease virus-like particle antigen can be realized, the production cost of the large-scale virus-like particle antigen is reduced, and a foundation is laid for the subsequent assembly of the virus-like particles and the preparation of the virus-like particle vaccine;
2. in the invention, the basic culture medium can shorten the slow phase of the strain for production and enter the logarithmic phase earlier; the culture medium for supplementing the materials can improve the multiplication capacity of escherichia coli in a logarithmic growth phase and improve the total bacterial count of the escherichia coli in a high-density fermentation process; the culture medium for induction expression can maintain the platform phase of escherichia coli so as to improve the expression quantity of the virus-like particle antigen, and meanwhile, the components in culture can promote the antigen to be expressed in a soluble form;
3. the invention adopts a large-scale fermentation culture process, wherein an induction expression mode of continuously adding a small amount of inducer at low temperature is adopted in the step of induction expression, so that the large-scale fermentation culture process can greatly improve the antigen expression quantity of the virus-like particles, and simultaneously can express the antigen in a soluble form to the maximum extent, thereby effectively reducing the cost of large-scale production of the foot-and-mouth disease virus-like particle antigen by an escherichia coli expression system and laying a good foundation for the subsequent purification of the antigen and the in-vitro assembly of the virus-like particles;
4. the culture medium and the fermentation culture method can be used for large-scale production and obtaining the foot-and-mouth disease virus-like particle antigen with high expression quantity and high solubility, can also be used for producing virus-like particle antigens of other viruses, and can also obviously improve the total expression quantity of the antigen and the proportion of the soluble antigen.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a graph of seed growth for production;
FIG. 2 is a growth diagram of the expanded bacteria culture;
FIG. 3 is a graph of feed growth;
FIG. 4 is a bacterial profile of different induced expression patterns;
FIG. 5 is a graph showing the expression levels of different inducible expression patterns;
FIG. 6 is a graph showing the amount of antigen expressed at different induction expression times;
FIG. 7 is a diagram showing the expression levels of antigens and the ratio of soluble antigens at different induced expression temperatures;
FIG. 8 is a diagram showing the expression level and soluble antigen ratio of virus-like particle antigens of other viruses expressed in Escherichia coli by using the medium and fermentation culture method of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
It is noted that relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The invention provides a fermentation medium and a culture method for expressing foot-and-mouth disease virus-like particle antigen by escherichia coli, which comprises a basal medium and a supplementary medium for cultivating the escherichia coli, and a medium for inducing expression during inducing expression;
the basic medium is obtained by the following method: dissolving potassium dihydrogen phosphate, diammonium hydrogen phosphate, sodium chloride, yeast extract, tryptone, glycerol and a defoaming agent in water, wherein the concentration of each component is as follows: 6g/L potassium dihydrogen phosphate, 3g/L diammonium hydrogen phosphate, 1g/L sodium chloride, 5g/L yeast extract, 8g/L tryptone, 15g/L glycerol and 0.01 wt% defoaming agent, adjusting the pH value to 7.0, carrying out autoclaving at 116 ℃ for 30min, then cooling to below 50 ℃, adding 0.5 vt% trace element solution, 2 vt% antibiotic solution and 2 vt% magnesium sulfate solution, and uniformly mixing to obtain the product;
the medium for feeding was obtained in the following manner: dissolving yeast extract and tryptone in water, wherein the concentration of each component is as follows: 5g/L of yeast extract and 8g/L of tryptone, adjusting the pH value to 7.0, carrying out autoclaving at 116 ℃ for 30min, then cooling to below 50 ℃, adding 0.5 vt% of trace element solution, 2 vt% of antibiotic solution and 2 vt% of magnesium sulfate solution, and uniformly mixing to obtain the yeast extract;
the medium for inducing expression was obtained in the following manner: dissolving yeast extract, tryptone and glycerol in water, wherein the concentration of each component is as follows: 5g/L yeast extract, 8g/L tryptone and 50g/L glycerol, adjusting pH to 7.0, autoclaving at 116 deg.C for 30min, cooling to below 50 deg.C, adding 0.05 vt% isopropyl thiogalactoside solution with concentration of 238g/L and 2 vt% magnesium sulfate solution, and mixing.
Wherein the microelement solution comprises 150g/L of citric acid, 0.5g/L of disodium ethylene diamine tetraacetate, 0.1g/L of cobalt chloride, 2g/L of calcium chloride, 0.05g/L of copper chloride, 0.2g/L of boric acid, 0.8g/L of manganese chloride, 0.1g/L of sodium molybdate, 0.5g/L of zinc acetate and 5g/L of ferric citrate; the antibiotic solution comprises 50g/L ampicillin, 50g/L kanamycin and 25g/L chloramphenicol.
The method for performing fermentation culture by adopting the fermentation culture medium for expressing the foot-and-mouth disease virus-like particle antigen by using the escherichia coli comprises the following steps:
s1, inoculating escherichia coli expressing foot-and-mouth disease virus-like particle antigen into a basic culture medium according to the proportion of 2 vt%, culturing for 6h at 37 ℃, then regulating and controlling the pH value by using ammonia water at the culture temperature of 36.8 ℃ and the dissolved oxygen rate of 30% to ensure that the pH value is 6.8, and carrying out expanded culture;
s2, determining OD of bacterial liquid600nmWhen the value is 5-10, feeding with a feeding medium at a feeding speed of 0.5ml/L per hour, controlling the pH value to be 6.8 by using ammonia water at a culture temperature of 36.8 ℃ and a dissolved oxygen rate of 30%, and continuing the culture;
s3, determining OD of bacterial liquid600nmWhen the value reaches 30 or above, cooling to 15 ℃, controlling the dissolved oxygen rate to be 20%, regulating and controlling the pH value by using ammonia water to enable the pH value to be 7.2, and continuously culturing for 0.5 h;
s4, adding a culture medium for inducible expression into the bacterial liquid, wherein the adding speed of the culture medium is 1ml/L per hour, the dissolved oxygen rate is 20% at the temperature of 15 ℃, regulating and controlling the pH value by using ammonia water to enable the pH value to be 7.0, and carrying out inducible expression for 12 hours;
s5, centrifuging the bacteria liquid after induction expression for 10min at 9000r/min, collecting bacteria mud, and storing at-20 ℃.
Example 2
The invention provides a fermentation medium and a culture method for expressing foot-and-mouth disease virus-like particle antigen by escherichia coli, which comprises a basal medium and a supplementary medium for cultivating the escherichia coli, and a medium for inducing expression during inducing expression;
the basic medium is obtained by the following method: dissolving potassium dihydrogen phosphate, diammonium hydrogen phosphate, sodium chloride, yeast extract, tryptone, glycerol and a defoaming agent in water, wherein the concentration of each component is as follows: the yeast extract is 10g/L, the tryptone is 15g/L, the glycerol is 30g/L, the antifoaming agent is 0.02 wt%, the pH value is adjusted to 7.2, the yeast extract is sterilized at 116 ℃ under high pressure for 30min, then the temperature is reduced to below 50 ℃, 0.5 vt% trace element solution, 2 vt% antibiotic solution and 2 vt% magnesium sulfate solution are added into the yeast extract, and the yeast extract is prepared by uniformly mixing;
the medium for feeding was obtained in the following manner: dissolving yeast extract and tryptone in water, wherein the concentration of each component is as follows:
adjusting the pH value to 7.2, carrying out autoclaving at 116 ℃ for 30min, then cooling to below 50 ℃, adding 0.5 vt% of trace element solution, 2 vt% of antibiotic solution and 2 vt% of magnesium sulfate solution, and uniformly mixing to obtain the yeast extract and the tryptone, wherein the concentration of the yeast extract is 10 g/L;
the medium for inducing expression was obtained in the following manner: dissolving yeast extract, tryptone and glycerol in water, wherein the concentration of each component is as follows: 10g/L yeast extract, 15g/L tryptone and 100g/L glycerol, adjusting pH to 7.2, autoclaving at 116 deg.C for 30min, cooling to below 50 deg.C, adding 0.1 vt% isopropyl thiogalactoside solution and 2 vt% magnesium sulfate solution, and mixing.
Wherein the microelement solution comprises 200g/L of citric acid, 1.0g/L of disodium ethylene diamine tetraacetate, 0.4g/L of cobalt chloride, 5g/L of calcium chloride, 0.3g/L of copper chloride, 0.5g/L of boric acid, 2.5g/L of manganese chloride, 0.4g/L of sodium molybdate, 2.0g/L of zinc acetate and 12g/L of ferric citrate; the antibiotic solution comprises 150g/L ampicillin, 100g/L kanamycin and 60g/L chloramphenicol.
The method for performing fermentation culture by adopting the fermentation culture medium for expressing the foot-and-mouth disease virus-like particle antigen by using the escherichia coli comprises the following steps:
s1, inoculating escherichia coli expressing foot-and-mouth disease virus-like particle antigen into a basic culture medium according to the proportion of 2 vt%, culturing for 12h at 37 ℃, then regulating and controlling the pH value by using ammonia water at the culture temperature of 37.2 ℃ and the dissolved oxygen rate of 50% to ensure that the pH value is 7.2, and carrying out expanded culture;
s2, determining OD of bacterial liquid600nmWhen the value is 5-10, feeding with a feeding medium at a feeding speed of 0.5ml/L per hour, controlling the pH value to be 7.2 by using ammonia water at a culture temperature of 37.2 ℃ and a dissolved oxygen rate of 50%, and continuing the culture;
s3, determining OD of bacterial liquid600nmWhen the value reaches 30 or above, cooling to 20 ℃, controlling the pH value to be 7.4 by using ammonia water with the dissolved oxygen rate of 40 percent, and continuously culturing for 0.5 h;
s4, adding a culture medium for inducible expression into the bacterial liquid, wherein the adding speed of the culture medium is 1ml/L per hour, the dissolved oxygen rate is 40% at the temperature of 20 ℃, regulating and controlling the pH value by using ammonia water to enable the pH value to be 7.2, and carrying out inducible expression for 16 hours;
s5, centrifuging the bacteria liquid after induction expression for 10min at 9000r/min, collecting bacteria mud, and storing at-20 ℃.
Example 3
The invention provides a fermentation medium and a culture method for expressing foot-and-mouth disease virus-like particle antigen by escherichia coli, which comprises a basal medium and a supplementary medium for cultivating the escherichia coli, and a medium for inducing expression during inducing expression;
the basic medium is obtained by the following method: dissolving potassium dihydrogen phosphate, diammonium hydrogen phosphate, sodium chloride, yeast extract, tryptone, glycerol and a defoaming agent in water, wherein the concentration of each component is as follows: 10g/L potassium dihydrogen phosphate, 5g/L diammonium hydrogen phosphate, 2g/L sodium chloride, 8g/L yeast extract, 10g/L tryptone, 20g/L glycerol and 0.015 wt% defoaming agent, adjusting the pH value to 7.1, sterilizing at 116 ℃ for 30min under high pressure, cooling to below 50 ℃, adding 0.5 vt% trace element solution, 2 vt% antibiotic solution and 2 vt% magnesium sulfate solution, and uniformly mixing to obtain the product;
the medium for feeding was obtained in the following manner: dissolving yeast extract and tryptone in water, wherein the concentration of each component is as follows:
adjusting the pH value to 7.1, carrying out autoclaving at 116 ℃ for 30min, then cooling to below 50 ℃, adding 0.5 vt% of trace element solution, 2 vt% of antibiotic solution and 2 vt% of magnesium sulfate solution, and uniformly mixing to obtain the yeast extract and the tryptone, wherein the concentration of the yeast extract is 8 g/L;
the medium for inducing expression was obtained in the following manner: dissolving yeast extract, tryptone and glycerol in water, wherein the concentration of each component is as follows: yeast extract 8g/L, tryptone 10g/L, glycerol 80g/L, adjusting pH to 7.1, autoclaving at 116 deg.C for 30min, cooling to below 50 deg.C, adding 0.08 vt% isopropyl thiogalactoside solution 238g/L and 2 vt% magnesium sulfate solution, and mixing.
Wherein the microelement solution comprises 180g/L of citric acid, 0.8g/L of disodium ethylene diamine tetraacetate, 0.2g/L of cobalt chloride, 3g/L of calcium chloride, 0.15g/L of copper chloride, 0.3g/L of boric acid, 1.2g/L of manganese chloride, 0.3g/L of sodium molybdate, 1.0g/L of zinc acetate and 8g/L of ferric citrate; the antibiotic solution comprises 100g/L ampicillin, 80g/L kanamycin and 45g/L chloramphenicol.
The method for performing fermentation culture by adopting the fermentation culture medium for expressing the foot-and-mouth disease virus-like particle antigen by using the escherichia coli comprises the following steps:
s1, inoculating escherichia coli expressing foot-and-mouth disease virus-like particle antigen into a basic culture medium according to the proportion of 2 vt%, culturing for 6h at 37 ℃, then regulating and controlling the pH value by using ammonia water at the culture temperature of 37.0 ℃ and the dissolved oxygen rate of 40% to ensure that the pH value is 7.0, and carrying out expanded culture;
s2, determining OD of bacterial liquid600nmFeeding with a feeding medium at a rate of 5-10 hr0.5ml/L, at the culture temperature of 37.0 ℃, the dissolved oxygen rate of 40%, regulating and controlling the pH value by using ammonia water to ensure that the pH value is 7.0, and continuing the culture;
s3, determining OD of bacterial liquid600nmWhen the value reaches 30 or above, cooling to 17 ℃, controlling the dissolved oxygen rate to be 30%, regulating and controlling the pH value by using ammonia water to enable the pH value to be 7.3, and continuously culturing for 0.5 h;
s4, adding a culture medium for inducible expression into the bacterial liquid, wherein the adding speed of the culture medium is 1ml/L per hour, the dissolved oxygen rate is 30% at the temperature of 17 ℃, regulating and controlling the pH value by using ammonia water to enable the pH value to be 7.1, and carrying out inducible expression for 15 hours;
s5, centrifuging the bacteria liquid after induction expression for 10min at 9000r/min, collecting bacteria mud, and storing at-20 ℃.
Examples of the experiments
1. The seed for production of escherichia coli expressing foot-and-mouth disease virus-like particles was cultured using the basal medium and LB medium of example 3, respectively, and the growth curve of the producing strain was plotted.
As shown in FIG. 1, it is understood from the graph that when the production strain is cultured in the LB medium using the basic medium of the present invention, the production strain is in the logarithmic growth phase for 6 to 12 hours, the time taken for the production strain to enter the logarithmic growth phase is shortened by about 2 hours as compared with the LB medium, and the OD of the production strain to enter the logarithmic growth phase and the plateau phase is also determined600nmThe value is higher.
2. Escherichia coli expressing the foot-and-mouth disease virus-like particle antigen was subjected to amplification culture using the basal medium and LB medium of example 3, respectively.
As shown in FIG. 2, it is understood from the graph that the time taken for the cells to enter the logarithmic growth phase is shortened and OD is decreased in the case of the expanded cells using the basal medium of the present invention, as compared with the case of using the LB medium600nmThe value is much higher than that of LB culture medium.
3. After the aftosa virus-like particle antigen-expressing escherichia coli entered the logarithmic growth phase, the basal medium and the supplemented medium of example 3 were used for feeding, respectively, and as a result, when the escherichia coli was fed using the supplemented medium, the escherichia coli was fed using the basal medium alone, as compared with the supplemented medium aloneOD of logarithmic growth phase600nmThe values are significantly higher. The results are shown in FIG. 3.
4. Respectively adopting one-time addition of sufficient IPTG inducer and continuous small-amount addition of IPTG-containing culture medium for inducible expression, and carrying out inducible expression on Escherichia coli expressing the foot-and-mouth disease virus-like particle antigen, the results are shown in figure 5 and figure 6.
As can be seen, the OD of the bacteria was measured after adding IPTG in one portion600nmThe value was significantly decreased and then slowly increased again, and when the medium for inducible expression of example 3 was used for inducible expression, the bacterial OD was decreased600nmIs always in a stable state. Comparing the total induction expression amount of the foot-and-mouth disease virus-like particle antigen by adopting two induction expression modes, the result shows that the total induction expression amount of the culture medium for induction expression is obviously higher than the total induction expression amount of the IPTG inducer added at one time, and the expression proportion of the soluble antigen is higher when the induction expression culture medium is used.
5. The results of inducible expression using the medium for inducible expression of example 3 and measurement of the amount of expression of the foot-and-mouth disease virus-like particle antigen at various times after initiation of induction are shown in fig. 6. The result shows that the expression quantity of the antigen is the highest 12-16 h after induction.
6. The foot-and-mouth disease virus-like particle antigen is induced and expressed under different temperature conditions, and the result is shown in figure 7, when the induced expression temperature is 15-20 ℃, the induced expression is carried out under the temperature condition, the expression level of the foot-and-mouth disease virus-like particle antigen is high, and the expression level of the soluble antigen is highest.
7. The results of using the culture medium and the fermentation culture method of the invention in example 3 to express the virus-like particle antigens of other viruses to escherichia coli are shown in fig. 8, and the results show that the expression amount and/or the ratio of soluble antigens are higher in the culture medium and the method of the invention compared with the traditional culture medium and fermentation medium.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. A fermentation culture medium for expressing foot-and-mouth disease virus-like particle antigen by escherichia coli is characterized by comprising a basal culture medium and a supplemented culture medium for cultivating the escherichia coli, and a culture medium for induced expression during induced expression;
the basal medium comprises per liter: 6-12 g of monopotassium phosphate, 3-8 g of diammonium phosphate, 1-5 g of sodium chloride, 5-10 g of yeast extract, 8-15 g of tryptone, 15-30 g of glycerol, 0.01-0.02 wt% of defoaming agent, 0.4-0.6 vt% of trace element solution, 1-3 vt% of antibiotic solution and 1-3 vt% of magnesium sulfate solution;
the medium for feeding comprises per liter: 5-10 g of yeast extract, 8-15 g of tryptone, 0.4-0.6 vt% of trace element solution, 1-3 vt% of antibiotic solution and 1-3 vt% of magnesium sulfate solution;
each liter of the trace element solution comprises 150-200 g of citric acid, 0.5-1.0 g of disodium ethylene diamine tetraacetate, 0.1-0.4 g of cobalt chloride, 2-5 g of calcium chloride, 0.05-0.3 g of copper chloride, 0.2-0.5 g of boric acid, 0.8-2.5 g of manganese chloride, 0.1-0.4 g of sodium molybdate, 0.5-2.0 g of zinc acetate and 5-12 g of ferric citrate; each liter of the antibiotic solution comprises 50-150 g of ampicillin, 50-100 g of kanamycin and 25-60 g of chloramphenicol; the concentration of the isopropylthiogalactoside solution is 240 g/L235-;
the culture medium for inducing expression comprises the following components in each liter: 5-10 g of yeast extract, 8-15 g of tryptone, 50-100 g of glycerol, 0.05-0.1 vt% of isopropyl thiogalactoside solution and 1-3 vt% of magnesium sulfate solution.
2. The fermentation medium for expressing the foot-and-mouth disease virus-like particle antigen of escherichia coli as claimed in claim 1, wherein the basal medium is obtained by the following operations: dissolving potassium dihydrogen phosphate, diammonium hydrogen phosphate, sodium chloride, yeast extract, tryptone, glycerol and a defoaming agent in water to obtain a mixture, adjusting the pH value of the mixture to 7.0-7.2, carrying out autoclaving at 116 ℃ for 25-30 min, cooling to 50 ℃ or below, adding a trace element solution, an antibiotic solution and a magnesium sulfate solution, and uniformly mixing.
3. The fermentation medium for expressing aftosa-like particle antigen in escherichia coli according to claim 1, wherein the medium for feeding is obtained by the following steps: dissolving yeast extract and tryptone in water to obtain a mixture, adjusting the pH value of the mixture to 7.0-7.2, carrying out autoclaving at 116 ℃ for 25-30 min, then cooling to 50 ℃ or below, adding a trace element solution, an antibiotic solution and a magnesium sulfate solution, and uniformly mixing to obtain the yeast extract.
4. The fermentation medium for expressing aftosa-like particle antigen in escherichia coli according to claim 1, wherein the medium for inducing expression is obtained by: dissolving yeast extract, tryptone and glycerol in water to obtain a mixture, adjusting the pH value of the mixture to 7.0-7.2, carrying out autoclaving at 116 ℃ for 25-30 min, then cooling to 50 ℃ or below, adding an isopropyl thiogalactoside solution and a magnesium sulfate solution, and uniformly mixing to obtain the product.
5. Use of the fermentation medium of any one of claims 1-4 for antigen production and vaccine preparation.
6. The method for fermentation culture by using the fermentation culture medium for expressing the foot-and-mouth disease virus-like particle antigen of the escherichia coli as defined in any one of claims 1 to 4, which is characterized by comprising the following steps:
s1, inoculating escherichia coli expressing foot-and-mouth disease virus-like particle antigen into a basal culture medium according to the proportion of 2-5 vt%, culturing for 6-12 h at 37 ℃, and then performing expanded culture;
s2, determining OD of bacterial liquid600nmWhen the value is 5-10, feeding the culture medium for feeding at the feeding speed of 0.5-2 ml/L per hour, and continuing to culture;
s3, determining OD of bacterial liquid600nmA value of up to 30 andwhen the temperature is higher than the above range, the temperature is reduced to 15-20 ℃, and when the temperature of the bacterial liquid is reduced to 20 ℃, the bacterial liquid is continuously cultured for 0.5 h;
s4, adding a culture medium for inducible expression into the bacterial liquid, wherein the adding speed of the culture medium is 1ml/L per hour, and the inducible expression is carried out for 12-16 hours;
s5, centrifuging the bacteria liquid after induction expression for 10-15min, and collecting bacteria mud.
7. The method for fermentation culture of the fermentation medium for expression of foot-and-mouth disease virus-like particle antigen of Escherichia coli according to claim 6, wherein the specific conditions of the expanded culture in step S1 and the continuous culture in step S2 are as follows: the culture temperature is 36.8-37.2 ℃, the dissolved oxygen rate is 30-50%, and the pH value is regulated by using ammonia water to be 6.8-7.2.
8. The method for fermentation culture of the fermentation medium for expressing the foot-and-mouth disease virus-like particle antigen of Escherichia coli according to claim 6, wherein the specific conditions for the continuous culture in step S3 are as follows: the culture temperature is 15-20 ℃, the dissolved oxygen rate is 20-40%, and the pH value is regulated by using ammonia water to be 7.2-7.4.
9. The method for fermentation culture of the fermentation medium for expressing the foot-and-mouth disease virus-like particle antigen of Escherichia coli according to claim 6, wherein the specific conditions for inducing expression in step S4 are as follows: the temperature is 15-20 ℃, the dissolved oxygen rate is 20-40%, and the pH value is regulated by using ammonia water to be 7.0-7.2.
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