CN118006534A - Cultivation method of escherichia coli recombinant strain with surface expression of African swine fever IrrE protein - Google Patents
Cultivation method of escherichia coli recombinant strain with surface expression of African swine fever IrrE protein Download PDFInfo
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- CN118006534A CN118006534A CN202410204556.6A CN202410204556A CN118006534A CN 118006534 A CN118006534 A CN 118006534A CN 202410204556 A CN202410204556 A CN 202410204556A CN 118006534 A CN118006534 A CN 118006534A
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Abstract
The invention discloses a method for cultivating escherichia coli recombinant bacteria with surface expression of African swine fever IrrE protein, which comprises the following steps: s1, preparing materials: irrE recombinant E.coli material, restriction enzymes; t4 ligase, kanamycin, protein electrophoresis main solution, protein loading buffer solution and culture medium; s2, preparing a culture medium: a LB culture medium is adopted, 1.0% of refined peptone, 0.5% of yeast extract powder and 1.0% of sodium chloride are arranged in the LB liquid culture medium, and then the culture medium is stirred to be completely dissolved.
Description
Technical Field
The invention relates to the technical field of bacterial cultivation, in particular to a method for cultivating escherichia coli recombinant bacteria with surface expression of African swine fever IrrE protein.
Background
African swine fever is an acute infectious disease of pigs caused by African swine fever virus, the death rate can reach 100 percent, african Swine Fever Virus (ASFV) is a large double-stranded DNA virus with a capsule, is the only member of the African swine fever virus family which is named recently, is also the only arbovirus DNA virus, the whole length of a virus genome is 17 Okb-190 kb, the center of the virus genome is provided with a conservation region of about 125kb, the two ends of the virus genome are provided with variable regions, and the addition or deletion of the repeated sequences is the main reason for the difference of the genome length of different isolates [31 ]. The virulence of most strains of African swine fever virus is strong, but the immunogenicity is low, and only a few proteins have immunogenicity, so that the prior study shows that the corresponding epitope of P72 antibody induced by ASFV strains in different areas is quite conservative, the P72 capsid protein accounts for 32 percent of the total protein of the whole virus particle, the antigenicity is quite stable and is often used as serological detection and immune preparation, epidemiological study shows that the high lethality and high infectivity of ASFV are not effective at present, so that the guideline of treating ASFV is strictly checked and slaughtered in a large scale, so that the establishment of a set of efficient and safe diagnosis methods is indispensable, the prior study is to clone the P72 gene (1938 bp) of the African swine fever virus into a plasmid vector by utilizing a gene recombination technology, express the capsid protein P72 in E.coli, utilize the 6 histidine sequence of pET-30c (+) vector and further purify the fused protein by a nickel column, thereby laying a foundation for the diagnosis method of African swine fever antigen with recombinant protein as detection antigen.
The escherichia coli expression system is commonly used for vaccine research work, and is beneficial to developing high-efficiency vaccines aiming at African swine fever. However, in the process of recombining and cultivating the existing escherichia coli recombinant bacteria, the nutrient solution components are relatively single, the agar is mainly used for providing the nutrient components for the escherichia coli recombinant bacteria, more reference carriers are needed for research work of the african swine fever vaccine, a large number of the escherichia coli recombinant bacteria are required to be propagated, and when the escherichia coli recombinant bacteria are propagated in a large quantity, the single nutrient solution components cannot meet the requirements of the escherichia coli recombinant bacteria, so that the problem that the local escherichia coli recombinant bacteria are insufficient in nutrient content, death and stop growing and breeding occur, the quality of experimental finished products is influenced finally, a series of experiments are influenced, and the research work of the african swine fever vaccine is not facilitated.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for cultivating escherichia coli recombinant bacteria with surface expression of african swine fever IrrE protein, which solves the problems of insufficient nutrition content, death and growth stopping of local escherichia coli recombinant bacteria caused by single nutrient solution component of the escherichia coli recombinant bacteria in the process of recombinant cultivation, thereby improving the quality of experimental finished products and being beneficial to the development work of subsequent african swine fever vaccines.
In order to achieve the above purpose, the present invention provides the following technical solutions: a method for cultivating escherichia coli recombinant bacteria with surface expression of African swine fever IrrE protein comprises the following steps:
S1, preparing materials: irrE recombinant E.coli material, restriction enzymes; t4 ligase, kanamycin, protein electrophoresis main solution, protein loading buffer solution and culture medium;
S2, preparing a culture medium: adopting LB culture medium, wherein 1.0% of refined peptone, 0.5% of yeast extract powder and 1.0% of sodium chloride are arranged in the LB liquid culture medium, then stirring to completely dissolve, adjusting pH to 7.0 by 5molNaOH (about0.2 ml/1000ml culture medium), and sterilizing under high pressure for 20min (120 ℃ 0.1 Mpa);
S3, bacterial culture: activating IrrE recombinant escherichia coli, thawing IrrE recombinant escherichia coli glycerol bacteria stored at-80 ℃ at room temperature, inoculating 500ul of bacteria into 50ml of LB culture medium, adding corresponding screening resistance, and culturing at 37 ℃ for 7h; then loading the strain into a tank, inoculating 50ml of the IrrE recombinant escherichia coli activated in the previous step into a 3L fermentation medium, setting temperature, air flow and pressure values, and monitoring DO and PH values;
S4, preparing a nutrient solution: one part of peptone, one part of lactose, one part of dipotassium hydrogen phosphate, one part of vitamin and one part of amino acid, one part of glucose and fructose and one part of trace elements (including iron, copper, manganese, zinc, cobalt, molybdenum, chromium, nickel, vanadium, fluorine, selenium, iodine, silicon and tin) are added into the mixture, five parts of pure sterile water is finally added, and then all nutrient materials are fully stirred and mixed by adopting a sterilized stirring rod until a viscous substance is formed, at the moment, one third of the total materials are gradually added according to the growth condition of IrrE recombinant escherichia coli observed by naked eyes until the total addition of the materials is finished;
s5, detection and later storage: when the IrrE recombinant escherichia coli is observed by naked eyes and basically fully distributed on the inner bottom end surface of the culture medium, new nutrients can be stopped from being added, meanwhile, sampling is carried out every 1 hour from 4 hours of fermentation to detect OD, and when the OD value is more than or equal to 40, induction is started; inducer: IPTG (1 mol/L, filtration and sterilization), adding 4ml to a final concentration of 1mmol/L, controlling the temperature at the induction stage to be 35 ℃ again, and inducing for 10 hours under other conditions; after the induction is finished, the mixture is placed in a tank, centrifuged at 5000rpm, and the thalli are collected and stored in a refrigerator at the temperature of minus 80 ℃.
S6, data recording: irrE recombinant E.coli was selected and inoculated into 50mL of LB liquid medium containing kanamycin (50. Mu.g/mL), shaking overnight culture was performed at 37℃at 180r/min, then transferred into fresh LB liquid medium containing 2mmol/LIPTG and 50. Mu.g/mL kanamycin at an inoculum size of 0.5%, shaking culture was performed at 37℃at 180r/min, sampling measurement A600 was performed every 2h, and a growth curve was drawn.
Preferably, in step 4, the vitamins include vitamin B1, vitamin B2, vitamin B6, and vitamin B12, and the amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, and asparagine.
Preferably, in step 5, the expression of the target protein can be induced by different concentrations of IPTG, and when the concentration of the IPTG reaches 2mmol/L and the temperature is 37 ℃ for 6 hours, the soluble expression of the target protein is most obvious, so that the optimal induction condition of the IrrE protein can be set to be that the final concentration of the IPTG is 2mmol/L, the induction temperature is 37 ℃ and the induction time is 6 hours.
The invention has the following beneficial effects:
(1) According to the method for cultivating the escherichia coli recombinant strain with the surface expressing the african swine fever IrrE protein, new nutrients are continuously added in the growth of the IrrE recombinant escherichia coli, so that the normal and rapid growth and propagation of the IrrE recombinant escherichia coli are ensured, the problems that the nutrient solution component of the existing escherichia coli recombinant strain is single, the content of the nutrients ingested by the local escherichia coli recombinant strain is insufficient, and a subsequent series of experiments are influenced in the process of the recombinant cultivation of the escherichia coli recombinant strain are solved, the quality of experimental finished products is improved, and the development work of subsequent african swine fever vaccines is facilitated.
(2) According to the cultivation method of the escherichia coli recombinant strain with the surface expressing the African swine fever IrrE protein, when the IrrE recombinant escherichia coli is basically fully distributed on the inner bottom end surface of the culture medium, new nutrients can be stopped from being added, sampling detection is carried out every 1 hour from 4 hours of fermentation, so that experimenters can know the propagation condition and the growth quality of the escherichia coli in real time, subsequent nutrient supply is facilitated, and the controllability of the escherichia coli cultivation condition is improved.
Detailed Description
All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a method for cultivating escherichia coli recombinant bacteria with surface expression of African swine fever IrrE protein, which comprises the following steps: a method for cultivating escherichia coli recombinant bacteria with surface expression of African swine fever IrrE protein comprises the following steps:
S1, preparing materials: irrE recombinant E.coli material, restriction enzymes; t4 ligase, kanamycin, protein electrophoresis main solution, protein loading buffer solution and culture medium;
S2, preparing a culture medium: adopting LB culture medium, wherein 1.0% of refined peptone, 0.5% of yeast extract powder and 1.0% of sodium chloride are arranged in the LB liquid culture medium, then stirring to completely dissolve, adjusting pH to 7.0 by 5molNaOH (about0.2 ml/1000ml culture medium), and sterilizing under high pressure for 20min (120 ℃ 0.1 Mpa);
S3, bacterial culture: activating IrrE recombinant escherichia coli, thawing IrrE recombinant escherichia coli glycerol bacteria stored at-80 ℃ at room temperature, inoculating 500ul of bacteria into 50ml of LB culture medium, adding corresponding screening resistance, and culturing at 37 ℃ for 7h; then loading the strain into a tank, inoculating 50ml of the IrrE recombinant escherichia coli activated in the previous step into a 3L fermentation medium, setting temperature, air flow and pressure values, and monitoring DO and PH values;
S4, preparing a nutrient solution: one part of peptone, one part of lactose, one part of dipotassium hydrogen phosphate, one part of vitamin and one part of amino acid, one part of glucose and fructose and one part of trace elements (including iron, copper, manganese, zinc, cobalt, molybdenum, chromium, nickel, vanadium, fluorine, selenium, iodine, silicon and tin) are added into the mixture, five parts of pure sterile water is finally added, and then all nutrient materials are fully stirred and mixed by adopting a sterilized stirring rod until a viscous substance is formed, at the moment, one third of the total materials are gradually added according to the growth condition of IrrE recombinant escherichia coli observed by naked eyes until the total addition of the materials is finished;
S5, detection and later storage: when the IrrE recombinant escherichia coli is observed by naked eyes and basically fully distributed on the inner bottom end surface of the culture medium, new nutrients can be stopped from being added, meanwhile, sampling is carried out every 1 hour from 4 hours of fermentation to detect OD, and when the OD value is more than or equal to 40, induction is started; inducer: IPTG (1 mol/L, filtration and sterilization), adding 4ml to a final concentration of 1mmol/L, controlling the temperature at the induction stage to be 35 ℃ again, and inducing for 10 hours under other conditions; after the induction is finished, placing the strain in a tank, centrifuging at 5000rpm, collecting thalli, and storing in a refrigerator at the temperature of minus 80 ℃;
s6, data recording: irrE recombinant E.coli was selected and inoculated into 50mL of LB liquid medium containing kanamycin (50. Mu.g/mL), shaking overnight culture was performed at 37℃at 180r/min, then transferred into fresh LB liquid medium containing 2mmol/LIPTG and 50. Mu.g/mL kanamycin at an inoculum size of 0.5%, shaking culture was performed at 37℃at 180r/min, sampling measurement A600 was performed every 2h, and a growth curve was drawn.
Wherein in step 4, the vitamins comprise vitamin B1, vitamin B2, vitamin B6 and vitamin B12, the amino acids comprise glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine and asparagine, in step 5, the expression of the target protein can be induced by different concentrations of IPTG, and when the concentration of the IPTG reaches 2mmol/L and the concentration of the IPTG is cultured for 6 hours at 37 ℃, the soluble expression of the target protein is most obvious, therefore, the optimal induction condition of the IrrE protein can be set to be the final concentration of the IPTG of 2mmol/L, the induction temperature of 37 ℃ and the induction time of 6 hours.
In summary, according to the cultivation method of the escherichia coli recombinant bacteria with the surface expressing the african swine fever IrrE protein, new nutrients are continuously added in the growth of the IrrE recombinant escherichia coli, so that the normal and rapid growth and reproduction of the IrrE recombinant escherichia coli are ensured, the limitation that the prior art mainly relies on agar to provide nutrients for the escherichia coli recombinant bacteria is improved, the problems that the nutrition liquid components of the existing escherichia coli recombinant bacteria are single in the recombination cultivation process, the requirements of the existing escherichia coli recombinant bacteria cannot be met when the escherichia coli recombinant bacteria are subjected to subsequent mass reproduction, the nutrition content of the local escherichia coli recombinant bacteria is insufficient, death occurs and the growth and reproduction stops are solved, the quality of experimental finished products is improved, and the development and the work of subsequent african swine fever vaccines are facilitated.
It is noted that relational terms such as first and second, and the like are 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. Moreover, 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.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (3)
1. A method for cultivating escherichia coli recombinant bacteria with surface expression of African swine fever IrrE protein is characterized by comprising the following steps: the method comprises the following steps:
S1, preparing materials: irrE recombinant E.coli material, restriction enzymes; t4 ligase, kanamycin, protein electrophoresis main solution, protein loading buffer solution and culture medium;
S2, preparing a culture medium: adopting LB culture medium, wherein 1.0% of refined peptone, 0.5% of yeast extract powder and 1.0% of sodium chloride are arranged in the LB liquid culture medium, then stirring to completely dissolve, adjusting pH to 7.0 by 5molNaOH (about0.2 ml/1000ml culture medium), and sterilizing under high pressure for 20min (120 ℃ 0.1 Mpa);
S3, bacterial culture: activating IrrE recombinant escherichia coli, thawing IrrE recombinant escherichia coli glycerol bacteria stored at-80 ℃ at room temperature, inoculating 500ul of bacteria into 50ml of LB culture medium, adding corresponding screening resistance, and culturing at 37 ℃ for 7h; then loading the strain into a tank, inoculating 50ml of the IrrE recombinant escherichia coli activated in the previous step into a 3L fermentation medium, setting temperature, air flow and pressure values, and monitoring DO and PH values;
S4, preparing a nutrient solution: one part of peptone, one part of lactose, one part of dipotassium hydrogen phosphate, one part of vitamin and one part of amino acid, one part of glucose and fructose and one part of trace elements (including iron, copper, manganese, zinc, cobalt, molybdenum, chromium, nickel, vanadium, fluorine, selenium, iodine, silicon and tin) are added into the mixture, five parts of pure sterile water is finally added, and then all nutrient materials are fully stirred and mixed by adopting a sterilized stirring rod until a viscous substance is formed, at the moment, one third of the total materials are gradually added according to the growth condition of IrrE recombinant escherichia coli observed by naked eyes until the total addition of the materials is finished;
S5, detection and later storage: when the IrrE recombinant escherichia coli is observed by naked eyes and basically fully distributed on the inner bottom end surface of the culture medium, new nutrients can be stopped from being added, meanwhile, sampling is carried out every 1 hour from 4 hours of fermentation to detect OD, and when the OD value is more than or equal to 40, induction is started; inducer: IPTG (1 mol/L, filtration and sterilization), adding 4ml to a final concentration of 1mmol/L, controlling the temperature at the induction stage to be 35 ℃ again, and inducing for 10 hours under other conditions; after the induction is finished, placing the strain in a tank, centrifuging at 5000rpm, collecting thalli, and storing in a refrigerator at the temperature of minus 80 ℃;
s6, data recording: irrE recombinant E.coli was selected and inoculated into 50mL of LB liquid medium containing kanamycin (50. Mu.g/mL), shaking overnight culture was performed at 37℃at 180r/min, then transferred into fresh LB liquid medium containing 2mmol/LIPTG and 50. Mu.g/mL kanamycin at 0.5% of the inoculum size, shaking culture was performed at 37℃at 180r/min, A600 was measured by sampling every 2h, and a growth curve was drawn.
2. The method for cultivating recombinant escherichia coli with surface expression of african swine fever IrrE protein as set forth in claim 1, wherein the method comprises the steps of: wherein in step 4, the vitamins include vitamin B1, vitamin B2, vitamin B6, and vitamin B12, and the amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, and asparagine.
3. The method for cultivating recombinant escherichia coli with surface expression of african swine fever IrrE protein as set forth in claim 1, wherein the method comprises the steps of: in the step 5, the expression of the target protein can be induced by different concentrations of IPTG, and when the concentration of the IPTG reaches 2mmol/L and the temperature of 37 ℃ is cultured for 6 hours, the soluble expression of the target protein is most obvious, so that the optimal induction condition of the IrrE protein can be set to be that the final concentration of the IPTG is 2mmol/L, the induction temperature is 37 ℃ and the induction time is 6 hours.
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