CN112961221A - Preparation method of varicella-zoster virus glycoprotein E extracellular domain protein - Google Patents
Preparation method of varicella-zoster virus glycoprotein E extracellular domain protein Download PDFInfo
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Abstract
The invention discloses a preparation method of varicella-zoster virus glycoprotein E extracellular domain protein, which comprises the following steps: s1, constructing a recombinant plasmid vector of the varicella-zoster virus glycoprotein E extracellular region gene carrying the preferred codon of the escherichia coli; s2, transforming the recombinant plasmid vector into a transformant obtained by competent Escherichia coli; s3, culturing the bacterial cells by using a culture medium containing a nonionic surfactant and a magnesium salt; s4, cleaning and collecting thalli; s5, extracting and dissolving inclusion bodies; s6, inclusion body renaturation; and S7, purifying the protein. The preparation method of the invention has simple operation and high protein expression, and the obtained target protein has immunogenicity, can stimulate effective cellular immunity and humoral immunity, and can be used as a candidate antigen for vaccine development. The protein preparation method disclosed by the invention can be applied to industrial mass acquisition of target proteins and has great advantages in the aspects of vaccine and detection method development.
Description
Technical Field
The invention belongs to the technical field of vaccines, and particularly relates to a preparation method of an extracellular domain protein of varicella-zoster virus glycoprotein E.
Background
Varicella-zoster virus (VZV) is the causative agent of chickenpox and shingles. The Kurarin Schker uses CHO cell to express gE glucoprotein of VZV, and is supplemented with AS04 adjuvant to prepare a new generation of herpes zoster vaccine, which can effectively stimulate the cellular immunity and humoral immunity of the vaccinee. According to the analysis of the phase III clinical results provided by the traditional Chinese medicine, the effective rate is more than 90%, and the effect is better.
The prior patent application (CN201611119488.5 a preparation method of the extracellular domain protein of varicella-zoster virus glycoprotein E) of the researchers of the invention focuses on the construction of a recombinant plasmid vector, in particular on the preferred codon of Escherichia coli of the extracellular domain gene of varicella-zoster virus glycoprotein E.
In actual production, the influence of culture conditions on protein expression, namely the yield of inclusion bodies, is researched, so that the method has great economic value.
Disclosure of Invention
The invention further studies the influence of culture conditions on the generation of inclusion bodies by strains on the basis of the prior patent application (CN201611119488.5 a preparation method of the extracellular region protein of varicella-zoster virus glycoprotein E).
The invention aims to disclose a preparation method of a varicella-zoster virus glycoprotein E extracellular domain protein, which comprises the following steps:
s1, constructing a recombinant plasmid vector of the varicella-zoster virus glycoprotein E extracellular region gene carrying the preferred codon of the escherichia coli;
s2, transforming the recombinant plasmid vector into a transformant obtained by competent Escherichia coli;
s3, culturing the bacterial cells by using a culture medium containing a nonionic surfactant and a magnesium salt;
s4, cleaning and collecting thalli;
s5, extracting and dissolving inclusion bodies;
s6, inclusion body renaturation;
and S7, purifying the protein.
In some embodiments of the present invention, in the step S1, the varicella-zoster virus glycoprotein E extracellular region gene of the E.coli preferred codon is ligated with a prokaryotic expression vector (e.g., pET series vector, etc.) to construct the recombinant plasmid vector.
In some embodiments of the present invention, in the S3 step, the nonionic surfactant is a polyether type nonionic surfactant, preferably with a molecular weight of 1200-1600.
In some embodiments of the present invention, in the step of S3, the magnesium salt is selected from one or more of magnesium chloride, magnesium nitrate, and magnesium sulfate.
In some embodiments of the present invention, in the step S3, the concentration of the nonionic surfactant in the medium is 0.5 to 5mM, preferably 1 to 3 mM.
In some embodiments of the invention, the Mg 3 step2+The concentration in the medium is 1-10mM, preferably 5-8 mM.
In some embodiments of the present invention, in the step S3, the medium is composed of lactose 5-10g/L, tryptone 5-10g/L, yeast powder 10-15g/L, sodium chloride 5-8g/L, polyether type nonionic surfactant with molecular weight of 1200-1600 mM, Mg2+5-8mM。
In some embodiments of the invention, in the step S3, the medium further comprises 0.1-10mM IPTG.
In some preferred embodiments of the invention, in the step S3, the medium further comprises 1-5mM IPTG.
In some embodiments of the invention, the step of S4, wherein the washing and collecting of the bacterial cells comprises a step of ultrasonic washing with a solution of 1-2 times the concentration of sodium chloride in the culture medium.
In some embodiments of the present invention, in the step of S5, the extraction method of the inclusion bodies is ultrasonication and centrifugation.
In some embodiments of the present invention, in the step S5, the solution of inclusion bodies comprises 8M urea or 6M guanidine hydrochloride.
In some preferred embodiments of the present invention, the solution of inclusion bodies is one of the following solutions:
(1)10-50mM PB +8M urea;
(2)10-50mM PB +8M urea;
(3)10-50mM PB +6M guanidine hydrochloride;
(4)10-50mM PB +6M guanidine hydrochloride;
the pH of the solution is between 7 and 8.
In some embodiments of the present invention, in step S6, the inclusion body renaturation is dilution renaturation, that is, the inclusion body solubilization solution is gradually added into the renaturation solution; preferably, the ratio of the inclusion body dissolving solution to the renaturation solution is 1:10-1: 20.
In some embodiments of the invention, the protein purification process in step S7 comprises at least two steps of protein purification process, anion chromatography and molecular sieve (size exclusion) chromatography.
In some preferred embodiments of the present invention, in the step S7, the anion chromatography packing material used is a protein purification packing material containing diethylaminoethyl as a ligand, such as diethylaminoethyl or a packing material containing an equivalent binding principle ligand (e.g., sepherose DEAE).
In some preferred embodiments of the present invention, in the step S7, the molecular sieve chromatographic packing used is a purification packing having a separation range of 10kDa to 100kDa, for example superdex 200pg and superose 12pg are commonly used.
In some preferred embodiments of the present invention, in the S7 step, the anion-chromatography protein eluent is one of the following solutions:
10-50mM PB +1-10mM EDTA + 200-; further preferably the NaCl content is 300-350 mM;
10-50mM tris +1-10mM EDTA + 200-; further preferably, the NaCl content is 300-350 mM.
In some preferred embodiments of the present invention, in the step of S7, the elution solution of the molecular sieve chromatography protein is one of the following solutions:
10-50mM PB+1-10mM EDTA+100-150mM NaCl；
10-50mM tris+1-10mM EDTA+100-150mM NaCl。
in some embodiments of the present invention, in step S3, the concentration of magnesium salt in the medium is determined by:
s11, determining a first candidate interval for the concentration of magnesium salt in the medium to be (C1, C2), wherein C1 is 0.1mM and C2 is 20 mM;
s22, preparing a culture medium with the concentration of magnesium salt of C3 and C4,
s23, respectively culturing the transformed escherichia coli, comparing the content of the induced protein, and if the content of the induced protein obtained by the culture medium with the concentration of C3 is more than C4, determining that the second candidate interval is (C4, C2); otherwise determining a second candidate interval (C1, C3);
s24, repeating S22 and S23 three times to obtain the concentration C of magnesium salt in the culture mediumopt。
In some embodiments of the present invention, the step S3 of testing the culture medium sterilized by moist heat to determine whether the osmotic pressure after sterilization is acceptable comprises the following steps:
s21, preparing a culture medium, and performing moist heat sterilization at 121 ℃ for 15-25min in a batch, wherein a is more than or equal to 10;
s22, the medium of each batch was tested for osmotic pressure before and after sterilization, respectively, as indicated by vectors X1 and X2, respectively;
s23, calculating the stability of the vector using the following formula:
the vector X1 has a stability of
The vector X2 has a stability of
Wherein the content of the first and second substances,
s24, if the stability of either vector X1 or X2 is less than-1.33, judging that the osmotic pressure of the culture medium is unqualified, and shortening the moist heat sterilization time until the stability of both vector X1 and vector X2 is more than-1.33.
The beneficial technical effects of the invention are as follows:
according to the preparation method of the extracellular domain protein of the varicella-zoster virus glycoprotein E, the culture medium is added with the nonionic surfactant and the magnesium salt, so that the total amount of the inducible expression protein in the cultured thalli and the proportion of the inducible expression protein in the whole mycoprotein can be obviously improved.
The preparation method of the invention has simple operation and high protein expression, and the obtained target protein has immunogenicity, can stimulate effective cellular immunity and humoral immunity, and can be used as a candidate antigen for vaccine development. The protein preparation method disclosed by the invention can be applied to industrial mass acquisition of target proteins and has great advantages in the aspects of vaccine and detection method development.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Unless otherwise indicated, the examples and comparative examples employ the same treatment methods and procedures.
The polyether type nonionic surfactant is an ethylene oxide adduct of polypropylene glycol. The SDS-PAGE method comprises the steps of taking 1mL of culture solution by 12.5% separation gel and 6% concentration gel, centrifuging and resuspending, heating wastewater for 20min, and loading 10 uL.
In the following examples and comparative examples, unless otherwise specified, parallel tests were conducted with the same operating procedures and parameters.
Example 1
The preparation method of the extracellular domain protein of the varicella-zoster virus glycoprotein E comprises the following steps:
e.coli BL21(DE3) positive expressing bacteria were obtained according to the method of the prior patent application (CN201611119488.5 a method for the preparation of the extracellular domain protein of varicella zoster virus glycoprotein E).
Coli BL21(DE3) was first cultured to OD at 37 ℃ in growth medium6001.0, and then inducing in an induction medium at 24-26 ℃ for 12 h.
The growth medium consists of: 8g/L of lactose, 8g/L of tryptone, 10g/L of yeast powder, 6g/L of sodium chloride, 1mM of polyether type nonionic surfactant with molecular weight of 1200-1600, and Mg2+5mM。
The induction medium consists of: 8g/L of lactose, 8g/L of tryptone, 10g/L of yeast powder, 6g/L of sodium chloride, 1mM of polyether type nonionic surfactant with molecular weight of 1200-1600, and Mg2+5mM，IPTG 3mM。
The magnesium salt is magnesium chloride.
The cultured bacterial liquid is firstly centrifuged to obtain thalli, suspended by water containing 9g/L of sodium chloride and then ultrasonically treated for 5s at 100W, and the process is repeated for 3 times to obtain the thalli with the culture medium, particularly the surfactant in the thalli, and the cleaning and the collection of the thalli are completed.
In the research, the activity of the protein obtained in the subsequent steps is very low by the thalli obtained by the common method of directly suspending by clear water and then centrifugally cleaning. This may be associated with residual surfactant, which is significantly enhanced by sonication with sodium chloride solution. The concentration of the treated sodium chloride solution is preferably 1 to 2 times the concentration of sodium chloride in the culture medium. The concentration of less than 0.5 times and more than 2.5 times are not ideal, and the effect of improving the activity of the protein is not ideal.
Extracting and dissolving the collected thallus with conventional method, namely the method of the prior patent application (CN201611119488.5 a preparation method of the extracellular domain protein of varicella-zoster virus glycoprotein E) to obtain inclusion body; renaturation of inclusion bodies; and purifying the protein to obtain the target protein.
And (4) investigating the results:
the ratio of the total amount of the protein induced to be expressed and the amount of the protein induced to be expressed to the whole bacterial protein was examined by SDS-PAGE.
Example 2
The differences from implementation class 1 are:
the growth medium consists of: 8g/L of lactose, 8g/L of tryptone, 10g/L of yeast powder, 6g/L of sodium chloride, 1mM of polyether type nonionic surfactant with molecular weight of 1200-1600, and Mg2+5mM。
The induction medium consists of: 8g/L of lactose, 8g/L of tryptone, 10g/L of yeast powder, 6g/L of sodium chloride, 1mM of polyether type nonionic surfactant with molecular weight of 1200-1600, and Mg2+5mM，IPTG 3mM。
The magnesium salt is magnesium sulfate.
Compared with example 1, the total amount of the protein for inducing expression and the amount of the protein for inducing expression are reduced by 1-2% in the proportion of the whole mycoprotein.
Example 3
The differences from implementation class 1 are:
the growth medium consists of: 8g/L of lactose, 8g/L of tryptone, 10g/L of yeast powder, 6g/L of sodium chloride, 3mM of polyether type nonionic surfactant with molecular weight of 1200-1600, and Mg2+8mM。
The induction medium consists of: lactose 8g/L, tryptone 8g/L, yeast powder 10 g-L, sodium chloride 6g/L, molecular weight 1200-1600 polyether type nonionic surfactant 3mM, Mg2+8mM，IPTG 3mM。
The magnesium salt is magnesium chloride.
Compared with example 1, the total amount of the protein for inducing expression and the amount of the protein for inducing expression are increased by 2-3% in the proportion of the whole mycoprotein.
Example 4
The differences from implementation class 1 are:
the growth medium consists of: 8g/L of lactose, 8g/L of tryptone, 10g/L of yeast powder, 6g/L of sodium chloride, 2mM of polyether type nonionic surfactant with molecular weight of 1200-1600, and Mg2+6mM。
The induction medium consists of: 8g/L of lactose, 8g/L of tryptone, 10g/L of yeast powder, 6g/L of sodium chloride, 2mM of polyether type nonionic surfactant with molecular weight of 1200-1600, and Mg2+6mM，IPTG 3mM。
The magnesium salt is magnesium chloride.
Compared with example 1, the total amount of the protein for inducing expression and the amount of the protein for inducing expression are increased by 7-8% in the proportion of the whole mycoprotein.
Example 5
The differences from implementation class 1 are:
the growth medium consists of: 8g/L of lactose, 8g/L of tryptone, 10g/L of yeast powder, 6g/L of sodium chloride, 1mM of polyether type nonionic surfactant with molecular weight of 1000-1200-2+5mM。
The induction medium consists of: 8g/L of lactose, 8g/L of tryptone, 10g/L of yeast powder, 6g/L of sodium chloride, 1mM of polyether type nonionic surfactant with molecular weight of 1000-1200-2+5mM，IPTG 3mM。
The magnesium salt is magnesium chloride.
Compared with example 1, the total amount of the protein for inducing expression and the proportion of the protein for inducing expression in the whole mycoprotein are reduced by about 2-3%.
Example 6
The difference from the implementation class 1;
the growth medium consists of: the content of the lactose is 8g/L,8g/L of tryptone, 10g/L of yeast powder, 6g/L of sodium chloride, 1mM of polyether type nonionic surfactant with molecular weight of 1800-2000-2+5mM。
The induction medium consists of: 8g/L of lactose, 8g/L of tryptone, 10g/L of yeast powder, 6g/L of sodium chloride, 1mM of polyether type nonionic surfactant with molecular weight of 1800-2000-2+5mM，IPTG 3mM。
The magnesium salt is magnesium chloride.
Compared with example 1, the total amount of the protein for inducing expression and the proportion of the protein for inducing expression in the whole mycoprotein are reduced by about 3-4%.
Example 7
The differences from implementation class 1 are:
the growth medium consists of: 8g/L lactose, 8g/L tryptone, 10g/L yeast powder, 6g/L sodium chloride, 801 mM Tween and Mg2+5mM。
The induction medium consists of: 8g/L lactose, 8g/L tryptone, 10g/L yeast powder, 6g/L sodium chloride, 801 mM Tween and Mg2+5mM，IPTG 3mM。
The magnesium salt is magnesium chloride.
Compared with example 1, the total amount of the protein for inducing expression and the proportion of the protein for inducing expression in the whole mycoprotein are reduced by more than 10%.
Example 8
The differences from implementation class 1 are:
in step S3, the concentration of magnesium salts in the medium is determined by the following method:
s11, determining a first candidate interval for the concentration of magnesium salt in the medium to be (C1, C2), wherein C1 is 0.1mM and C2 is 20 mM;
s22, preparing a culture medium with the concentration of magnesium salt of C3 and C4,
s23, respectively culturing the transformed escherichia coli, comparing the content of the induced protein, and if the content of the induced protein obtained by the culture medium with the concentration of C3 is more than C4, determining that the second candidate interval is (C4, C2); otherwise determining a second candidate interval (C1, C3);
s24, repeating S22 and S23 three times to obtain the concentration C of magnesium salt in the culture mediumopt。
The concentration of magnesium salts in the medium is an important factor affecting the amount of induced protein, and the method of this example is used to determine the appropriate concentration of magnesium salts in the medium. The invention can quickly determine the concentration of the magnesium salt under the condition of determining other components in the culture.
Example 9
The differences from implementation class 1 are:
in the step S3, the method of inspecting the culture medium subjected to moist heat sterilization to determine whether or not the osmotic pressure after sterilization is acceptable includes the following steps:
s21, preparing a culture medium, and performing moist heat sterilization at 121 ℃ for 15-25min in a batch, wherein a is more than or equal to 10;
s22, the medium of each batch was tested for osmotic pressure before and after sterilization, respectively, as indicated by vectors X1 and X2, respectively;
s23, calculating the stability of the vector using the following formula:
the vector X1 has a stability of
The vector X2 has a stability of
Wherein the content of the first and second substances,
s24, if the stability of either vector X1 or X2 is less than-1.33, judging that the osmotic pressure of the culture medium is unqualified, and shortening the moist heat sterilization time until the stability of both vector X1 and vector X2 is more than-1.33.
The osmotic pressure of the culture medium may change in the process of moist heat sterilization, and the method can be used for detecting whether the osmotic pressure of the culture medium after moist heat sterilization is qualified or not, and can also be used for assisting in determining the sterilization time which does not influence the osmotic pressure as much as possible under the requirement of composite sterilization.
Example 10
The difference from example 1 is that:
in the step S5, the extraction method of the inclusion body comprises ultrasonic crushing and centrifugation;
in step S5, the solution of inclusion bodies comprises 8M urea or 6M guanidine hydrochloride, preferably one of the following solutions:
(1)10-50mM PB +8M urea;
(2)10-50mM PB +8M urea;
(3)10-50mM PB +6M guanidine hydrochloride;
(4)10-50mM PB +6M guanidine hydrochloride;
the pH of the solution is between 7 and 8;
s6, the inclusion body renaturation is dilution renaturation, that is, the inclusion body dissolving solution is gradually added into the renaturation solution; preferably, the ratio of the inclusion body dissolving solution to the renaturation solution is 1:10-1: 20;
in the step S7, the protein purification process at least comprises two steps of protein purification processes of anion chromatography and molecular sieve chromatography;
preferably, the anion chromatographic packing used is a packing for protein purification, the ligand of which is diethylaminoethyl;
preferably, the molecular sieve chromatographic packing is used for purification with the separation range of 10KDa-100 KDa;
preferably, the anion chromatography protein eluent is one of the following solutions:
1. 10-50mM PB +1-10mM EDTA + 200-; further preferably the NaCl content is 300-350 mM;
2. 10-50mM tris +1-10mM EDTA + 200-; further preferably the NaCl content is 300-350 mM;
preferably, the molecular sieve chromatography protein eluent is one of the following solutions:
1、10-50mM PB+1-10mM EDTA+100-150mM NaCl；
2、10-50mM tris+1-10mM EDTA+100-150mM NaC。
comparative example 1
The differences from implementation class 1 are:
the growth medium consists of: 8g/L lactose, 8g/L tryptone, 10g/L yeast powder, 6g/L sodium chloride and Mg2+5mM。
The induction medium consists of: 8g/L lactose, 8g/L tryptone, 10g/L yeast powder, 6g/L sodium chloride and Mg2+5mM，IPTG 3mM。
The magnesium salt is magnesium chloride.
Compared with example 1, the total amount of the protein for inducing expression and the proportion of the protein for inducing expression in the whole mycoprotein are reduced by more than 15%.
While the preferred embodiments and examples of the present invention have been described in detail, the present invention is not limited to the embodiments and examples, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.
Claims (10)
1. A preparation method of varicella-zoster virus glycoprotein E extracellular domain protein comprises the following steps:
s1, constructing a recombinant plasmid vector of the varicella-zoster virus glycoprotein E extracellular region gene carrying the preferred codon of the escherichia coli;
s2, transforming the recombinant plasmid vector into a transformant obtained by competent Escherichia coli;
s3, culturing the bacterial cells by using a culture medium containing a nonionic surfactant and a magnesium salt;
s4, cleaning and collecting thalli;
s5, extracting and dissolving inclusion bodies;
s6, inclusion body renaturation;
and S7, purifying the protein.
2. The method according to claim 1, wherein the recombinant plasmid vector is constructed by ligating the extracellular region gene of varicella-zoster virus glycoprotein E having codon preference of Escherichia coli to a prokaryotic expression vector in step S1.
3. The method as claimed in claim 1 or 2, wherein in the step S3, the nonionic surfactant is polyether type nonionic surfactant, preferably with molecular weight of 1200-1600;
and/or, in the step S3, the magnesium salt is selected from one or more of magnesium chloride, magnesium nitrate and magnesium sulfate.
4. The method according to any one of claims 1 to 3, wherein the concentration of the nonionic surfactant in the medium in the S3 step is 0.5 to 5mM, preferably 1 to 3 mM;
and/or, in the step S3, the Mg2+The concentration in the medium is 1-10mM, preferably 5-8 mM.
5. The method according to any one of claims 1 to 4, wherein the medium composition in the step S3 is lactose 5-10g/L, tryptone 5-10g/L, yeast powder 10-15g/L, sodium chloride 5-8g/L, polyether type nonionic surfactant with molecular weight of 1200-1600 (M-K) 1-3mM, Mg2+5-8mM。
6. The method according to any one of claims 1 to 5, wherein in the step S3, the medium further comprises 0.1 to 10mM IPTG, preferably 1 to 5mM IPTG.
7. The method according to any one of claims 1 to 6, wherein the step of S4, wherein the washing and collecting of the microbial cells comprises a step of ultrasonic washing with a solution having a concentration of 1 to 2 times the concentration of sodium chloride in the culture medium.
8. The method according to any one of claims 1 to 7, wherein in the step S5, the inclusion bodies are extracted by ultrasonication and centrifugation;
and/or, in the step S5, the solution of the inclusion body comprises 8M urea or 6M guanidine hydrochloride, preferably one of the following solutions:
(1)10-50mM PB +8M urea;
(2)10-50mM PB +8M urea;
(3)10-50mM PB +6M guanidine hydrochloride;
(4)10-50mM PB +6M guanidine hydrochloride;
the pH of the solution is between 7 and 8;
and/or, in the step S6, the inclusion body renaturation is dilution renaturation, namely, the inclusion body dissolving solution is gradually added into the renaturation solution; preferably, the ratio of the inclusion body dissolving solution to the renaturation solution is 1:10-1: 20;
and/or, in the step S7, the protein purification process at least comprises two steps of protein purification processes of anion chromatography and molecular sieve chromatography;
preferably, the anion chromatographic packing used is a packing for protein purification, the ligand of which is diethylaminoethyl;
preferably, the molecular sieve chromatographic packing is used for purification with the separation range of 10KDa-100 KDa;
preferably, the anion chromatography protein eluent is one of the following solutions:
10-50mM PB +1-10mM EDTA + 200-; further preferably the NaCl content is 300-350 mM;
10-50mM tris +1-10mM EDTA + 200-; further preferably the NaCl content is 300-350 mM;
preferably, the molecular sieve chromatography protein eluent is selected from the following solutions:
10-50mM PB+1-10mM EDTA+100-150mM NaCl；
10-50mM tris+1-10mM EDTA+100-150mM NaCl；
the pH of the solution is between 7 and 8.
9. The method according to any one of claims 1 to 8, wherein in the step S3, the concentration of magnesium salt in the medium is determined by:
s11, determining a first candidate interval for the concentration of magnesium salt in the medium to be (C1, C2), wherein C1 is 0.1mM and C2 is 20 mM;
s22, preparing a culture medium with the concentration of magnesium salt of C3 and C4,
s23, respectively culturing the transformed escherichia coli, comparing the content of the induced protein, and if the content of the induced protein obtained by the culture medium with the concentration of C3 is more than C4, determining that the second candidate interval is (C4, C2); otherwise determining a second candidate interval (C1, C3);
s24, repeating S22 and S23 three times to obtain the concentration C of magnesium salt in the culture mediumopt。
10. The method according to any one of claims 1 to 9, wherein the step of S3, in which the culture medium sterilized by moist heat is examined to determine whether or not the osmotic pressure after sterilization is acceptable, comprises the steps of:
s21, preparing a culture medium, and performing moist heat sterilization at 121 ℃ for 15-25min in a batch, wherein a is more than or equal to 10;
s22, the medium of each batch was tested for osmotic pressure before and after sterilization, respectively, as indicated by vectors X1 and X2, respectively;
s23, calculating the stability of the vector using the following formula:
the vector X1 has a stability of
The vector X2 has a stability of
Wherein the content of the first and second substances,
s24, if the stability of either vector X1 or X2 is less than-1.33, judging that the osmotic pressure of the culture medium is unqualified, and shortening the moist heat sterilization time until the stability of both vector X1 and vector X2 is more than-1.33.
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