CN114292752A - Freeze-dried composition of recombinant escherichia coli strain and preparation method thereof - Google Patents
Freeze-dried composition of recombinant escherichia coli strain and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of strain preservation, and particularly relates to a freeze-dried composition of a recombinant escherichia coli strain and a preparation method thereof. The preservation composition consists of recombinant escherichia coli strains and a compound component, wherein the compound component comprises a freeze-drying protective agent, phosphate buffer salt or purified water. The composition has stable properties, simple preparation method and long preservation time, the foreign plasmid is easy to lose and remarkably reduces after the preserved strain is rejuvenated, and the survival rate of the strain can still be effectively maintained after long-term preservation.
Description
Technical Field
The invention belongs to the field of strain preservation, and particularly relates to a freeze-dried composition of a recombinant escherichia coli strain and a preparation method thereof.
Background
The strain preservation is to artificially create conditions to isolate the strain from the outside air. The principle of strain preservation is to reduce the metabolism of microorganism as much as possible to reduce variation, and generally, the strain can be in a 'semi-dormant' or 'dormant' state by various modes such as nutrition limitation, oxygen deficiency, drying, high temperature, low temperature and the like, so that the variation probability and death probability of the strain are reduced, and good genetic genes are kept.
The recombinant Escherichia coli is a genetic engineering bacterium, contains exogenous DNA fragments carried by vector plasmids and the like, and the fragments usually have genetic instability, easily lose exogenous plasmid replicons in the passage process, and easily and directly influence the related performance of the genetic engineering strains due to the loss of exogenous plasmids in the strain preservation and rejuvenation stages. The strain preservation of the recombinant escherichia coli usually adopts a glycerol tube-80 ℃ ultralow temperature or liquid nitrogen preservation mode, the equipment cost of the glycerol tube-80 ℃ ultralow temperature preservation is higher, the equipment cost is difficult to achieve by a general basic unit, and the strain survival rate is obviously reduced after the recombinant escherichia coli is preserved for a period of time; the preservation condition of liquid nitrogen preservation is strict, the preservation initial cost is high, the temperature is increased due to liquid nitrogen evaporation in the preservation process, the ice crystal state is changed, and the strains are easy to die, so that the residual amount of the liquid nitrogen needs to be frequently and closely concerned, the liquid nitrogen is periodically supplemented, the danger of explosion caused by exposure of the liquid nitrogen in the air exists during the supplement, and an operator needs to have high operation proficiency.
CN106867905A discloses a preservation solution for strain preservation and a high-density fermentation method of recombinant escherichia coli, wherein a working strain library is preserved for 332 days at 4-10 ℃, the survival rate of the strain is more than 81.2%, and the survival rate of the strain preserved for 538 days is more than 74.8%.
CN109294917A discloses a strain preservative and a method for cryopreservation of genetically engineered bacteria by using the preservative. The preservation solution is added with antibiotic for inhibiting microorganism, providing favorable growth selective pressure and inhibiting the deterioration of protecting group, but the preservation method still needs ultralow temperature condition at minus 80 ℃, and the antibiotic is added in the preservation solution, which is not beneficial to the survival rate maintenance of the strain for long-term (more than 5 years), for example, after the strain preservative and the preservation method are applied to a strain engineering bacterium for Escherichia coli with high pyruvic acid yield, the yield of pyruvic acid produced by the fermentation of the rejuvenated strain is reduced by 5 percent compared with that before preservation after the strain preservative is preserved for 1 year at minus 80 ℃.
In order to overcome the problems, a new strain preservation method needs to be researched and developed, the inventor tries to preserve the recombinant escherichia coli strain by using a freeze-drying method, however, the freeze-drying process is a complex phase change process, and in the freezing, freeze-thawing, drying and storing processes, various factors for inducing strain death, exogenous plasmid loss and strain viability reduction exist, and in addition, many factors such as the concentration of strain suspension, the age of freeze-dried strain materials, the type of culture medium, a protective agent and the like can also influence the freeze preservation of the strain, so that the suitable freeze-drying conditions and the suitable type and quantity of freeze-drying protective agent need to be selected according to the characteristics of the strain to ensure the long-term stable storage and production application of the strain.
Therefore, the research and development of the preservation method which is suitable for preserving the recombinant escherichia coli strains and has simple operation method and mild conditions, can ensure the stable performance of the strains and realize long-term preservation has great practical significance for the maintenance, the replication and the continuation of the excellent performance of the genetically engineered bacteria, and is more beneficial to the industrial stable production.
Disclosure of Invention
In view of the defects that in the prior art, even if a low-temperature protective agent is used or ultra-low temperature preservation is adopted, exogenous plasmids are easy to lose, the survival rate of the strains is remarkably reduced after long-term preservation, the operation of the preservation method is complex and the like in the preservation or rejuvenation process of the recombinant escherichia coli strains, the application obtains a freeze-dried composition suitable for long-term preservation of the recombinant escherichia coli strains and a preparation method thereof through research. After the recombinant escherichia coli strain with excellent performance is preserved by the freeze-drying composition and the freeze-drying method, the plasmid loss rate is reduced, and the survival rate and the thallus activity can be effectively maintained.
The technical scheme of the invention is as follows: a lyophilized composition of a recombinant Escherichia coli species, said composition consisting of a recombinant Escherichia coli species and a complex composition, said complex composition comprising a lyoprotectant, and phosphate buffer salt or purified water.
Experiments prove that most of Escherichia coli strains can use the method of the invention, and preferably, the recombinant Escherichia coli strains are polyethylene glycol recombinant human granulocyte colony stimulating factor engineering bacteria (P)ET-rhG-CSF/BL21(DE3) PlysS) and recombinant insulin glargine engineering bacterium (P)ET-Glargine/BL21(DE3) PlysS) or TNHH engineering bacterium (P)ET-TNHH/BL21(DE3)PlysS)。
Further preferably, the recombinant Escherichia coli strain is a pegylated recombinant human granulocyte colony stimulating factor engineering bacterium (P)ET-rhG-CSF/BL21(DE3) PlysS), the sequence of the engineering bacteria is shown in SEQ ID No.1 or SEQ ID No. 2.
Preferably, in the preparation ofWhen the Escherichia coli strain is recombined, the density OD of the strain liquid is600When the pH value reaches 1.0-1.5, using phosphate buffer solution or citric acid buffer solution to adjust the pH value of the bacterial liquid to 5.0-6.0, and continuing shaking culture.
Preferably, the final OD of the bacterial liquid of the recombinant Escherichia coli strain600The value is 2-3; more preferably 2.2 to 2.6.
Preferably, in the composition, the volume of the compound component is 0.2 to 0.8 times, more preferably 0.3 to 0.6 times of the volume of the seed liquid of the recombinant escherichia coli strain.
Preferably, in the composition, the lyoprotectant is one or more selected from skimmed milk powder, glycine and mannitol.
Preferably, in the composition, the mass volume concentration of the lyoprotectant in the complex is 5-25%, that is, 5-25 g of the lyoprotectant is contained in each 100mL of the solvent; more preferably 5 to 15%.
Preferably, in the composition, the complex component may further comprise a glycan.
Further preferably, the polysaccharide is selected from one or more of lactose, sucrose and trehalose.
Preferably, in the composition, the mass volume concentration of the glycan in the compound component is 0-35%, namely, 0-35 g of glycan is contained in each 100mL of solvent; more preferably, the mass volume concentration of the polysaccharide is 10 to 20%.
Preferably, the concentration of phosphate buffer salt in the composition is 10-50 mM.
Preferably, in the composition, the pH value of the phosphate buffer salt is 6.5-7.5; further preferably, the pH value is 6.8-7.2.
In a preferred embodiment, the freeze-dried composition of the recombinant escherichia coli strain comprises the recombinant escherichia coli strain and a compound component, wherein the volume ratio of a seed solution of the recombinant escherichia coli strain to the compound component is 1: 0.2-0.8, and the compound component is prepared by mixing a freeze-drying protective agent with the mass volume concentration of 5-25%, a glycan with the mass volume concentration of 0-35% and phosphate buffer salt or purified water, and fixing the volume; further preferably, the concentration of phosphate buffer salt in the composition is 10-50 mM; more preferably, the pH value of the phosphate buffer salt in the composition is 6.8-7.2.
In a second aspect of the invention, a method for preparing a lyophilized composition of recombinant escherichia coli species is provided: mixing the recombinant Escherichia coli strain with the compound components in the composition, packaging with freeze-drying tube, and freeze-drying.
Preferably, the preparation method comprises the following steps:
a. preparing a seed solution of a recombinant escherichia coli strain;
b. preparation of the complex ingredients in the composition: mixing the formula amount of the freeze-drying protective agent or the freeze-drying protective agent and the polysaccharide with phosphate buffer salt or purified water, and fixing the volume to obtain the freeze-drying protective agent;
c. taking the seed liquid obtained in the step a, centrifuging, removing supernatant, adding a compound component with the volume 0.2-0.8 times of that of the seed liquid, suspending by using a vortex oscillator until thalli are completely and uniformly suspended, subpackaging freeze-drying tubes, and carrying out freeze-drying treatment to obtain a freeze-dried composition of the recombinant escherichia coli strain, wherein a freeze-drying curve is as follows:
pre-freezing: firstly, fully cooling a composition solution to be lyophilized to-45 to-50 ℃ and maintaining for 2 to 3 hours;
sublimation drying: vacuumizing the freeze dryer and maintaining the vacuum pressure in the freeze dryer at 0.20 +/-0.02 mbar, heating the composition to-25 to-30 ℃ at the speed of 5 ℃/h, and maintaining the temperature for 2 to 5 h; heating to-15 to-20 ℃ at a speed of 5 ℃/h, and maintaining for 42 to 48 h;
and (3) resolving and drying: vacuumizing the freeze dryer and maintaining 0.10 +/-0.02 mbar, raising the temperature of the composition to 5-10 ℃, continuously drying for 2-4 h, then carrying out ultimate vacuum for 2h, carrying out plug pressing in a vacuum state, and taking out of the box.
In a preferred embodiment, a method of preparing a lyophilized composition of a recombinant E.coli species comprises the steps of:
1) inoculating the recombinant escherichia coli strain to a slant of a solid seed culture medium, and culturing for 12-15 h at 37.0 ℃, wherein the solid seed culture medium comprises the following components in percentage by weight (g/L): sterilizing yeast powder 5, peptone 10, NaCl 10 and agar powder 20 at 121 ℃ for 20 min;
2) then, the cultured colony of the seed slant is selected and inoculated in a liquid seed culture medium, and is cultured in a constant temperature oscillator at 37.0 ℃, wherein the liquid seed culture medium comprises the following components in percentage by weight (g/L): sterilizing yeast powder 5, peptone 10 and NaCl 10 at 121 ℃ for 20 min; when the density of bacterial liquid OD600When the pH value reaches 1.0-1.5, adjusting the pH value of the bacterial liquid to 5.0-6.0 by using 0.1mol/L phosphate buffer solution or citric acid buffer solution, and continuing shaking culture until the OD of the bacterial liquid is6002-3, obtaining a seed solution of the recombinant escherichia coli strain;
3) preparation of the complex ingredients in the composition: mixing the formula amount of the freeze-drying protective agent or the freeze-drying protective agent and the polysaccharide with phosphate buffer salt, adjusting the pH value of the compound component to 6.5-7.5 by using sodium hydroxide or hydrochloric acid, and fixing the volume to obtain the compound;
4) taking the seed liquid obtained in the step 2), centrifuging, removing supernatant, adding a compound component with the volume 0.2-0.8 times of that of the seed liquid, suspending by using a vortex oscillator until thalli are completely and uniformly suspended, subpackaging freeze-drying tubes, and carrying out freeze-drying treatment to obtain a freeze-dried composition of the recombinant escherichia coli strain, wherein the freeze-drying curve is as follows:
pre-freezing: firstly, fully cooling a composition solution to be lyophilized to-45 to-50 ℃ and maintaining for 2 to 3 hours;
sublimation drying: vacuumizing the freeze dryer and maintaining the vacuum pressure in the freeze dryer at 0.20 +/-0.02 mbar, heating the composition to-25 to-30 ℃ at the speed of 5 ℃/h, and maintaining the temperature for 2 to 5 h; heating to-15 to-20 ℃ at a speed of 5 ℃/h, and maintaining for 42 to 48 h;
and (3) resolving and drying: vacuumizing the freeze dryer and maintaining 0.10 +/-0.02 mbar, raising the temperature of the composition to 5-10 ℃, continuously drying for 2-4 h, then carrying out ultimate vacuum for 2h, carrying out plug pressing in a vacuum state, and taking out of the box.
The invention is not limited to other culture steps of the seed liquid, and any existing solid culture medium, liquid seed culture medium and culture method thereof for seed liquid culture can be used for the invention.
Compared with the prior art, the invention has the following outstanding advantages:
1. the invention provides a jellyDry composition in bacterial liquid Density OD600When the pH value reaches 1.0-1.5, inducing the strain by using a buffer solution with the pH value of 5.0-6.0 to obtain a recombinant escherichia coli strain, adding a freeze-drying composition with specific components, and performing freeze drying to obtain a strain freeze-drying composition capable of being stored for a long time;
2. the freeze-dried composition of the recombinant escherichia coli strain is obtained by adopting lower pH induction, and the loss of exogenous plasmids of the strain to be preserved is obviously reduced by controlling a freeze-drying curve, and the survival rate of the strain can be effectively maintained after long-term preservation;
3. the freeze-dried composition of the recombinant escherichia coli strain can realize long-term stable preservation at 2-8 ℃, avoids-80 ℃ ultra-low temperature preservation of the traditional glycerin tube, is simple in preparation method, and overcomes the defects that the operation of periodically supplementing liquid nitrogen during liquid nitrogen preservation is complicated and the like;
4. the freeze-dried composition of the recombinant escherichia coli strain can be quickly rejuvenated, and can obtain higher thallus growth density and higher target protein expression quantity.
Detailed Description
The present invention is further described by the following embodiments, which do not limit the scope of the present invention in any way, and those skilled in the art can make various modifications or improvements based on the basic idea of the present invention, but within the scope of the present invention, as long as they do not depart from the basic idea of the present invention.
The method of the invention is suitable for preservation of most recombinant Escherichia coli strains, such as the PEGylated recombinant human granulocyte colony stimulating factor engineering bacteria (P)ET-G-CSF/BL21(DE3) PlysS) and insulin glargine engineering bacteria (P)ET-Glargine/BL21(DE3) PlysS) or TNHH engineering bacterium (P)ET-TNHH/BL21(DE3) PlysS), and the like. To fully illustrate the technical solution of the present invention, the following examples use PET-G-CSF/BL21(DE3) PlysS is exemplified.
Example 1
Recombinant DNAPreparing enterobacter strain seed liquid: will PET-G-CSF/BL21(DE3) The PlysS strain is subjected to amplification culture step by step in a seed culture medium to obtain a seed solution, and the specific operation steps are as follows:
(1) will PET-G-CSF/BL21(DE3) Inoculating a PlysS strain to a slant of a solid seed culture medium, and culturing at 37.0 ℃ for 12-15 h, wherein the solid seed culture medium comprises the following components (g/L): sterilizing yeast powder 5, peptone 10, NaCl 10 and agar powder 20 at 121 ℃ for 20 min;
(2) then, the cultured colony of the seed slant is selected and inoculated in a liquid seed culture medium, and is cultured in a constant temperature oscillator at 37.0 ℃, wherein the liquid seed culture medium comprises the following components in percentage by weight (g/L): sterilizing yeast powder 5, peptone 10 and NaCl 10 at 121 ℃ for 20 min; when the density of bacterial liquid OD600When the pH value reaches 1.0-1.5, adjusting the pH value of the bacterial liquid to 5.0-6.0 by using 0.1mol/L phosphate buffer solution or citric acid buffer solution, and continuing shaking culture until the OD of the bacterial liquid is600And (2) 2-3) obtaining a seed solution of the recombinant escherichia coli strain.
Example 2
Preparing recombinant escherichia coli strain seed liquid: will PET-G-CSF/BL21(DE3) The PlysS strain is subjected to amplification culture step by step in a seed culture medium to obtain a seed solution, and the specific operation steps are as follows:
(1) will PET-G-CSF/BL21(DE3) Inoculating a PlysS strain to a slant of a solid seed culture medium, and culturing at 37.0 ℃ for 12-15 h, wherein the solid seed culture medium comprises the following components (g/L): sterilizing yeast powder 5, peptone 10, NaCl 10 and agar powder 20 at 121 ℃ for 20 min;
(2) then, the cultured colony of the seed slant is selected and inoculated in a liquid seed culture medium, and is cultured in a constant temperature oscillator at 37.0 ℃, wherein the liquid seed culture medium comprises the following components in percentage by weight (g/L): sterilizing yeast powder 5, peptone 10 and NaCl 10 at 121 ℃ for 20 min; when the density of bacterial liquid OD600When the pH value reaches 1.0-1.5, adjusting the pH value of the bacterial liquid to 5.0-6.0 by using 0.1mol/L phosphate buffer solution or citric acid buffer solution, and continuing shaking culture until the OD of the bacterial liquid is600To 3.5, recombinant Escherichia coli was obtainedSeed liquid of the strain.
Example 3
Preparing recombinant escherichia coli strain seed liquid: will PET-G-CSF/BL21(DE3) The PlysS strain is subjected to amplification culture step by step in a seed culture medium to obtain a seed solution, and the specific operation steps are as follows:
(1) will PET-G-CSF/BL21(DE3) Inoculating a PlysS strain to a slant of a solid seed culture medium, and culturing at 37.0 ℃ for 12-15 h, wherein the solid seed culture medium comprises the following components (g/L): sterilizing yeast powder 5, peptone 10, NaCl 10 and agar powder 20 at 121 ℃ for 20 min;
(2) then, the cultured colony of the seed slant is selected and inoculated in a liquid seed culture medium, and is cultured in a constant temperature oscillator at 37.0 ℃, wherein the liquid seed culture medium comprises the following components in percentage by weight (g/L): sterilizing yeast powder 5, peptone 10 and NaCl 10 at 121 ℃ for 20 min; when the density of bacterial liquid OD600When the pH value reaches 1.0-1.5, adjusting the pH value of the bacterial liquid to 7.0 by using 0.1mol/L phosphate buffer solution or citric acid buffer solution, and continuing shaking the flask to culture until the OD of the bacterial liquid600And (2) 2-3) obtaining a seed solution of the recombinant escherichia coli strain.
Example 4
(A) Components of the composition
(II) the preparation method of the composition comprises the following steps:
preparation of the complex ingredients in the composition: weighing 50g of skimmed milk powder and 200g of trehalose, adding into 10mM phosphate buffer salt with pH value of 6.8, adjusting pH value of the compound component to 6.8 with sodium hydroxide or hydrochloric acid, and diluting to 1L with phosphate buffer solution.
The cultured P obtained in example 1 was collectedET-G-CSF/BL21(DE3) Shaking the PlysS strain seed liquid, and adding 15mL of seed liquid into 9 sterile centrifuge tubes respectively by using a pipettor in a clean bench; placing the centrifugal tube into a table centrifuge, setting the rotation speed to be 4000rpm, and centrifuging for 10 min; taking off and centrifugingDiscarding the supernatant in a superclean workbench of the finished centrifuge tube, adding 4.5mL of compound components into 9 aseptic centrifuge tubes respectively by using a pipettor, and uniformly mixing to form a bacterial suspension; sucking 500 mu L of the bacterial suspension by using a pipettor, subpackaging the bacterial suspension into a tear-drop freeze-drying tube, carrying out freeze-drying treatment according to the following freeze-drying curve, and storing the freeze-drying tube of the obtained preservation composition at 2-8 ℃:
pre-freezing: firstly, fully cooling a composition solution to be lyophilized to-45 ℃ and maintaining for 2.5 hours;
sublimation drying: vacuumizing the freeze dryer and maintaining the vacuum pressure at 0.20 +/-0.02 mbar, heating the composition to-28 ℃ at the speed of 5 ℃/h, and maintaining the temperature for 3.5 h; heating to-18 ℃ at a speed of 5 ℃/h, and maintaining for 45 h;
and (3) resolving and drying: vacuumizing the freeze-drying machine and maintaining 0.10 +/-0.02 mbar, raising the temperature of the composition to 8 ℃, continuously drying for 3 hours, carrying out ultimate vacuum for 2 hours, pressing and plugging under the vacuum state, and discharging from the box.
Example 5
(A) Components of the composition
(II) the preparation method of the composition comprises the following steps:
preparation of the complex ingredients in the composition: 150g of glycine and 100g of lactose are weighed, added into 30mM phosphate buffer salt with the pH value of 7.5, the pH value of the compound component is adjusted to 7.5 by sodium hydroxide or hydrochloric acid, and the volume is adjusted to 1L by the phosphate buffer salt.
The cultured P obtained in example 1 was collectedET-G-CSF/BL21(DE3) Shaking the PlysS strain seed liquid, and adding 15mL of seed liquid into 9 sterile centrifuge tubes respectively by using a pipettor in a clean bench; placing the centrifugal tube into a table centrifuge, setting the rotation speed to be 4000rpm, and centrifuging for 10 min; taking the centrifuged centrifuge tube, removing the supernatant in a superclean workbench, adding 9mL of compound components of the composition into 9 aseptic centrifuge tubes by using a pipettor, and uniformly mixing to form a bacterial suspension; sucking 500 μ L of the bacterial suspension with a pipettor, subpackaging into tear-drop type freeze-drying tubes, and freeze-drying according to the following freeze-drying curveAnd (3) carrying out freeze-drying treatment, and placing the freeze-drying tube of the obtained preservation composition at 2-8 ℃ for preservation:
pre-freezing: firstly, fully cooling a composition solution to be lyophilized to-45 ℃ and maintaining for 2.5 hours;
sublimation drying: vacuumizing the freeze dryer and maintaining the vacuum pressure at 0.20 +/-0.02 mbar, heating the composition to-28 ℃ at the speed of 5 ℃/h, and maintaining the temperature for 3.5 h; heating to-18 ℃ at a speed of 5 ℃/h, and maintaining for 45 h;
and (3) resolving and drying: vacuumizing the freeze-drying machine and maintaining 0.10 +/-0.02 mbar, raising the temperature of the composition to 8 ℃, continuously drying for 3 hours, carrying out ultimate vacuum for 2 hours, pressing and plugging under the vacuum state, and discharging from the box.
Example 6
(A) Components of the composition
(II) the preparation method of the composition comprises the following steps:
preparation of the complex ingredients in the composition: weighing 100g of mannitol and 350g of sucrose, adding into 50mM phosphate buffer salt with pH value of 6.5, adjusting pH value of the compound component to 6.5 with sodium hydroxide or hydrochloric acid, and diluting to 1L with phosphate buffer salt.
The cultured P obtained in example 1 was collectedET-G-CSF/BL21(DE3) Shaking the PlysS strain seed liquid, and adding 15mL of seed liquid into 9 sterile centrifuge tubes respectively by using a pipettor in a clean bench; placing the centrifugal tube into a table centrifuge, setting the rotation speed to be 4000rpm, and centrifuging for 10 min; taking the centrifuged centrifuge tube, removing the supernatant in a superclean workbench, adding 12mL of compound components into 9 aseptic centrifuge tubes by using a pipettor, and uniformly mixing to form a bacterial suspension; sucking 500 μ L of the bacterial suspension with a pipette, subpackaging into tear-drop type freeze-drying tubes, freeze-drying according to the following freeze-drying curve, and freezing the obtained preserved compositionAnd (3) storing the main pipe at 2-8 ℃:
pre-freezing: firstly, fully cooling a composition solution to be lyophilized to-50 ℃ and maintaining for 3 hours;
sublimation drying: vacuumizing the freeze dryer and maintaining the vacuum pressure at 0.20 +/-0.02 mbar, heating the composition to-30 ℃ at the speed of 5 ℃/h, and maintaining the temperature for 5 h; heating to-20 ℃ at a speed of 5 ℃/h, and maintaining for 48 h;
and (3) resolving and drying: vacuumizing the freeze-drying machine and maintaining 0.10 +/-0.02 mbar, raising the temperature of the composition to 10 ℃, continuously drying for 4 hours, carrying out ultimate vacuum for 2 hours, pressing and plugging under the vacuum state, and discharging from the box.
Example 7
(A) Components of the composition
Recombinant escherichia coli strain seed liquid OD600＝2.0
25 percent of skimmed milk powder
Purified water-
(II) the preparation method of the composition comprises the following steps:
preparation of the complex ingredients in the composition: 250g of skimmed milk powder is weighed and added into purified water, and the volume is fixed to 1L.
The cultured P obtained in example 1 was collectedET-G-CSF/BL21(DE3) Shaking the PlysS strain seed liquid, and adding 15mL of seed liquid into 9 sterile centrifuge tubes respectively by using a pipettor in a clean bench; placing the centrifugal tube into a table centrifuge, setting the rotation speed to be 4000rpm, and centrifuging for 10 min; taking the centrifuged centrifuge tube, removing the supernatant in a superclean workbench, adding 3mL of compound components into 9 aseptic centrifuge tubes by using a pipettor, and uniformly mixing to form a bacterial suspension; sucking 500 mu L of the bacterial suspension by using a pipettor, subpackaging the bacterial suspension into a tear-drop freeze-drying tube, carrying out freeze-drying treatment according to the following freeze-drying curve, and storing the freeze-drying tube of the obtained preservation composition at 2-8 ℃:
pre-freezing: firstly, fully cooling a composition solution to be lyophilized to-45 ℃ and maintaining for 2 hours;
sublimation drying: vacuumizing the freeze dryer and maintaining the vacuum pressure at 0.20 +/-0.02 mbar, heating the composition to-25 ℃ at the speed of 5 ℃/h, and maintaining the temperature for 2 h; heating to-15 ℃ at a speed of 5 ℃/h, and maintaining for 42 h;
and (3) resolving and drying: vacuumizing the freeze-drying machine and maintaining 0.10 +/-0.02 mbar, raising the temperature of the composition to 5 ℃, continuously drying for 2h, carrying out ultimate vacuum for 2h, pressing and plugging under the vacuum state, and discharging from the box.
Example 8
(A) Components of the composition
(II) the preparation method of the composition comprises the following steps:
preparation of the complex ingredients in the composition: weighing 100g of mannitol, 100g of skim milk powder, 5g of sucrose and 100g of lactose, adding into 50mM phosphate buffer salt with pH value of 6.5, adjusting the pH value of the compound component to 6.5 with sodium hydroxide or hydrochloric acid, and adding phosphate buffer salt to a constant volume of 1L.
The cultured P obtained in example 1 was collectedET-G-CSF/BL21(DE3) Shaking the PlysS strain seed liquid, and adding 15mL of seed liquid into 9 sterile centrifuge tubes respectively by using a pipettor in a clean bench; placing the centrifugal tube into a table centrifuge, setting the rotation speed to be 4000rpm, and centrifuging for 10 min; taking the centrifuged centrifuge tube, removing the supernatant in a superclean workbench, adding 12mL of compound components of the composition into 9 aseptic centrifuge tubes by using a pipettor, and uniformly mixing to form a bacterial suspension; sucking 500 mu L of the bacterial suspension by using a pipettor, subpackaging the bacterial suspension into a tear-drop freeze-drying tube, carrying out freeze-drying treatment according to the following freeze-drying curve, and storing the freeze-drying tube of the obtained preservation composition at 2-8 ℃:
pre-freezing: firstly, fully cooling a composition solution to be lyophilized to-50 ℃ and maintaining for 3 hours;
sublimation drying: vacuumizing the freeze dryer and maintaining the vacuum pressure at 0.20 +/-0.02 mbar, heating the composition to-30 ℃ at the speed of 5 ℃/h, and maintaining the temperature for 5 h; heating to-20 ℃ at a speed of 5 ℃/h, and maintaining for 48 h;
and (3) resolving and drying: vacuumizing the freeze-drying machine and maintaining 0.10 +/-0.02 mbar, raising the temperature of the composition to 10 ℃, continuously drying for 4 hours, carrying out ultimate vacuum for 2 hours, pressing and plugging under the vacuum state, and discharging from the box.
Example 9
(A) Components of the composition
(II) the preparation method of the composition comprises the following steps:
preparation of the complex ingredients in the composition: weighing 100g of mannitol and 350g of sucrose, adding into 50mM phosphate buffer salt with pH value of 6.5, adjusting pH value of the compound component to 6.5 with sodium hydroxide or hydrochloric acid, and diluting to 1L with phosphate buffer salt.
The cultured P obtained in example 2 was collectedET-G-CSF/BL21(DE3) Shaking the PlysS strain seed liquid, and adding 15mL of seed liquid into 9 sterile centrifuge tubes respectively by using a pipettor in a clean bench; placing the centrifugal tube into a table centrifuge, setting the rotation speed to be 4000rpm, and centrifuging for 10 min; taking the centrifuged centrifuge tube, removing the supernatant in a superclean workbench, adding 12mL of compound components into 9 aseptic centrifuge tubes by using a pipettor, and uniformly mixing to form a bacterial suspension; sucking 500 mu L of the bacterial suspension by using a pipettor, subpackaging the bacterial suspension into a tear-drop freeze-drying tube, carrying out freeze-drying treatment according to the following freeze-drying curve, and storing the freeze-drying tube of the obtained preservation composition at 2-8 ℃:
pre-freezing: firstly, fully cooling a composition solution to be lyophilized to-50 ℃ and maintaining for 3 hours;
sublimation drying: vacuumizing the freeze dryer and maintaining the vacuum pressure at 0.20 +/-0.02 mbar, heating the composition to-30 ℃ at the speed of 5 ℃/h, and maintaining the temperature for 5 h; heating to-20 ℃ at a speed of 5 ℃/h, and maintaining for 48 h;
and (3) resolving and drying: vacuumizing the freeze-drying machine and maintaining 0.10 +/-0.02 mbar, raising the temperature of the composition to 10 ℃, continuously drying for 4 hours, carrying out ultimate vacuum for 2 hours, pressing and plugging under the vacuum state, and discharging from the box.
Example 10
(A) Components of the composition
(II) the preparation method of the composition comprises the following steps:
preparation of the complex ingredients in the composition: weighing 100g of mannitol and 350g of sucrose, adding into 50mM phosphate buffer salt with pH value of 6.5, adjusting pH value of the compound component to 6.5 with sodium hydroxide or hydrochloric acid, and diluting to 1L with phosphate buffer salt.
The cultured P obtained in example 3 was collectedET-G-CSF/BL21(DE3) Shaking the PlysS strain seed liquid, and adding 15mL of seed liquid into 9 sterile centrifuge tubes respectively by using a pipettor in a clean bench; placing the centrifugal tube into a table centrifuge, setting the rotation speed to be 4000rpm, and centrifuging for 10 min; taking the centrifuged centrifuge tube, removing the supernatant in a superclean workbench, adding 12mL of compound components into 9 aseptic centrifuge tubes by using a pipettor, and uniformly mixing to form a bacterial suspension; sucking 500 mu L of the bacterial suspension by using a pipettor, subpackaging the bacterial suspension into a tear-drop freeze-drying tube, carrying out freeze-drying treatment according to the following freeze-drying curve, and storing the freeze-drying tube of the obtained preservation composition at 2-8 ℃:
pre-freezing: firstly, fully cooling a composition solution to be lyophilized to-50 ℃ and maintaining for 3 hours;
sublimation drying: vacuumizing the freeze dryer and maintaining the vacuum pressure at 0.20 +/-0.02 mbar, heating the composition to-30 ℃ at the speed of 5 ℃/h, and maintaining the temperature for 5 h; heating to-20 ℃ at a speed of 5 ℃/h, and maintaining for 48 h;
and (3) resolving and drying: vacuumizing the freeze-drying machine and maintaining 0.10 +/-0.02 mbar, raising the temperature of the composition to 10 ℃, continuously drying for 4 hours, carrying out ultimate vacuum for 2 hours, pressing and plugging under the vacuum state, and discharging from the box.
Example 11
(A) Components of the composition
(II) the preparation method of the composition comprises the following steps:
preparation of the complex ingredients in the composition: 100g of glycerol and 350g of sucrose are weighed, added into 50mM phosphate buffer salt with the pH value of 6.5, the pH value of the compound component is adjusted to 6.5 by sodium hydroxide or hydrochloric acid, and the volume is adjusted to 1L by the phosphate buffer salt.
The cultured P obtained in example 1 was collectedET-G-CSF/BL21(DE3) Shaking the PlysS strain seed liquid, and adding 15mL of seed liquid into 9 sterile centrifuge tubes respectively by using a pipettor in a clean bench; placing the centrifugal tube into a table centrifuge, setting the rotation speed to be 4000rpm, and centrifuging for 10 min; taking the centrifuged centrifuge tube, removing the supernatant in a superclean workbench, adding 12mL of compound components into 9 aseptic centrifuge tubes by using a pipettor, and uniformly mixing to form a bacterial suspension; sucking 500 mu L of the bacterial suspension by using a pipettor, subpackaging the bacterial suspension into a tear-drop freeze-drying tube, carrying out freeze-drying treatment according to the following freeze-drying curve, and storing the freeze-drying tube of the obtained preservation composition at 2-8 ℃:
pre-freezing: firstly, fully cooling a composition solution to be lyophilized to-50 ℃ and maintaining for 3 hours;
sublimation drying: vacuumizing the freeze dryer and maintaining the vacuum pressure at 0.20 +/-0.02 mbar, heating the composition to-30 ℃ at the speed of 5 ℃/h, and maintaining the temperature for 5 h; heating to-20 ℃ at a speed of 5 ℃/h, and maintaining for 48 h;
and (3) resolving and drying: vacuumizing the freeze-drying machine and maintaining 0.10 +/-0.02 mbar, raising the temperature of the composition to 10 ℃, continuously drying for 4 hours, carrying out ultimate vacuum for 2 hours, pressing and plugging under the vacuum state, and discharging from the box.
Example 12
(A) Components of the composition
(II) the preparation method of the composition comprises the following steps:
preparation of the complex ingredients in the composition: weighing 500g of mannitol and 350g of sucrose, adding into 50mM phosphate buffer salt with pH value of 6.5, adjusting pH value of the compound component to 6.5 with sodium hydroxide or hydrochloric acid, and diluting to 1L with phosphate buffer salt.
The cultured P obtained in example 1 was collectedET-G-CSF/BL21(DE3)Plyshaking the sS strain seed liquid, and adding 15mL of seed liquid into 9 sterile centrifuge tubes respectively by using a liquid transfer machine in a super clean workbench; placing the centrifugal tube into a table centrifuge, setting the rotation speed to be 4000rpm, and centrifuging for 10 min; taking the centrifuged centrifuge tube, removing the supernatant in a superclean workbench, adding 12mL of compound components into 9 aseptic centrifuge tubes by using a pipettor, and uniformly mixing to form a bacterial suspension; sucking 500 mu L of the bacterial suspension by using a pipettor, subpackaging the bacterial suspension into a tear-drop freeze-drying tube, carrying out freeze-drying treatment according to the following freeze-drying curve, and storing the freeze-drying tube of the obtained preservation composition at 2-8 ℃:
pre-freezing: firstly, fully cooling a composition solution to be lyophilized to-50 ℃ and maintaining for 3 hours;
sublimation drying: vacuumizing the freeze dryer and maintaining the vacuum pressure at 0.20 +/-0.02 mbar, heating the composition to-30 ℃ at the speed of 5 ℃/h, and maintaining the temperature for 5 h; heating to-20 ℃ at a speed of 5 ℃/h, and maintaining for 48 h;
and (3) resolving and drying: vacuumizing the freeze-drying machine and maintaining 0.10 +/-0.02 mbar, raising the temperature of the composition to 10 ℃, continuously drying for 4 hours, carrying out ultimate vacuum for 2 hours, pressing and plugging under the vacuum state, and discharging from the box.
Example 13
(A) Components of the composition
(II) the preparation method of the composition comprises the following steps:
preparation of the complex ingredients in the composition: weighing 100g of mannitol and 350g of sucrose, adding into 50mM phosphate buffer salt with pH value of 6.5, adjusting pH value of the compound component to 6.5 with sodium hydroxide or hydrochloric acid, and diluting to 1L with phosphate buffer salt.
The cultured P obtained in example 1 was collectedET-G-CSF/BL21(DE3) Shaking the PlysS strain seed liquid, and adding 15mL of seed liquid into 9 sterile centrifuge tubes respectively by using a pipettor in a clean bench; placing the centrifugal tube into a table centrifuge, setting the rotation speed to be 4000rpm, and centrifuging for 10 min; taking the centrifuged centrifugal tube, putting the centrifuged centrifugal tube in a superclean workbench, discarding the supernatant,adding 12mL of compound components into 9 aseptic centrifuge tubes respectively by using a pipettor, and uniformly mixing to form bacterial suspension; sucking 500 mu L of the bacterial suspension by using a pipettor, subpackaging the bacterial suspension into a tear-drop freeze-drying tube, carrying out freeze-drying treatment according to the following freeze-drying curve, and storing the freeze-drying tube of the obtained preservation composition at 2-8 ℃:
pre-freezing: firstly, fully cooling a composition solution to be lyophilized to-40 ℃ and maintaining for 3 hours;
sublimation drying: vacuumizing the freeze dryer and maintaining the vacuum pressure at 0.20 +/-0.02 mbar, heating the composition to-30 ℃ at the speed of 10 ℃/h, and maintaining the temperature for 5 h; heating to-20 ℃ at a speed of 2 ℃/h, and maintaining for 50 h;
and (3) resolving and drying: vacuumizing the freeze-drying machine and maintaining 0.10 +/-0.02 mbar, raising the temperature of the composition to 15 ℃, continuously drying for 4 hours, carrying out ultimate vacuum for 2 hours, pressing and plugging under the vacuum state, and discharging from the box.
Comparative example 1
(A) Components of the composition
(II) the preparation method of the composition comprises the following steps:
preparation of the complex ingredients in the composition: weighing 100g of mannitol and 350g of sucrose, adding into 50mM phosphate buffer salt with pH value of 6.5, adjusting pH value of the compound component to 6.5 with sodium hydroxide or hydrochloric acid, and diluting to 1L with phosphate buffer salt.
The cultured P obtained in example 1 was collectedET-G-CSF/BL21(DE3) Shaking the PlysS strain seed liquid, and adding 15mL of seed liquid into 9 sterile centrifuge tubes respectively by using a pipettor in a clean bench; placing the centrifugal tube into a table centrifuge, setting the rotation speed to be 4000rpm, and centrifuging for 10 min; taking the centrifuged centrifuge tube, placing in an ultra-clean workbench, discarding the supernatant, adding 12mL of compound components into 9 aseptic centrifuge tubes respectively by using a pipette, adding 15mL of 40% glycerol, mixing to form a bacterial suspension, pre-freezing for 4h in a refrigerator at-20 ℃, and then storing at-80 ℃ in an ultra-low temperature refrigerator.
Comparative example 2
Composition for preserving glycerin pipe
Recombinant escherichia coli strain seed liquid OD600＝3.0
40% of glycerol
(II) the preparation method comprises the following steps:
the amount of P obtained in example 1 was 2mL, which was 40% glycerol by mass volumeET-G-CSF/BL21(DE3) 3mL of PlysS strain seed liquid is uniformly mixed on a vortex oscillator, pre-frozen in a refrigerator at the temperature of-20 ℃ for 4h, and then stored in an ultra-low temperature refrigerator at the temperature of-80 ℃.
Verification examples
1. Appearance, viable count, viability and plasmid copy number
The samples obtained in the examples and the comparative examples are put under the same conditions (2-8 ℃) for storage stability examination, and the detection items are appearance, viable count, viability and plasmid copy number, and the specific operation steps are as follows:
counting viable bacteria: adding a proper amount of sterile water into a sample for resuspension, carrying out 10-fold gradient dilution by using sterile water, wherein the dilution times are 10-1 and 10-2 … … 10-9 in sequence, taking 50ul of fermentation liquor of 10-5, 10-6 and 10-9, coating the fermentation liquor on a flat plate, and repeating the steps for 1 time, wherein the marks are 1-1, 1-2, 2-1, 2-2, 3-1 and 3-2 … … respectively. And (3) putting the flat plate upside down into a water-proof constant-temperature incubator, culturing for 15h at the set temperature of 37.0 ℃, and counting bacterial colonies on the flat plate respectively after the culture is finished to obtain the number of viable bacteria.
Plasmid copy number: constructing a plasmid standard substance by adopting a gene synthesis method, and respectively carrying out internal gene GapA gene pair by utilizing a fluorescent dye embedding method; and preparing a standard curve of the target gene rhG-CSF gene. And (4) quantifying the endogenous gene and the target gene in the sample by using a standard curve.
And (3) measuring moisture: the measurement is carried out according to the first method (Fisher's method) of 0832 in general rules of Chinese pharmacopoeia.
TABLE 1 results of the strain preservation stability test (2-8 ℃ C.)
Remarking: the "-" in the table indicates no detection.
From the experimental results of table 1, it can be seen that: compared with the comparative example, the freeze-drying appearance of the strain preservation method is better, the survival rate of 3 years and 5 years is higher, and the strain preservation method is expected to realize stable preservation for a longer time; the water change of the strain after long-time preservation is small, which shows that the method has good sealing property; the copy number of the plasmid of the strain has no obvious change after long-term preservation, and the genetic stability is good.
2. Determination of thallus growth density and expression quantity after rejuvenation of recombinant escherichia coli strain
The samples obtained in the examples and comparative examples, i.e., the lyophilized compositions preserved for 5 years, were compared with those preserved for 0 day, and the growth density and expression level of the rejuvenated cells were determined by the following procedure.
2.1 measurement of cell growth density: measuring light absorption value at 600nm wavelength with ultraviolet spectrophotometer, and measuring light absorption value with OD600The values are represented.
2.2 determination of the expression level of the PET-rhG-CSF fusion protein: SDS-polyacrylamide gel electrophoresis, and a gel processing system is adopted to analyze the expression quantity of the target protein, and the specific operation steps are as follows:
2.2.1 shaking culture
The instrument equipment comprises: superclean workbench, autoclave and full-temperature air shaking table
Reagent: 1) strain: the lyophilized compositions obtained in the respective examples and comparative examples;
2) LB medium (test tube): weighing yeast extract 0.2g, tryptone 0.4g and sodium chloride 0.4g, adding water 40mL for dissolving, adjusting pH value to 7.0, evenly distributing into 4 test tubes, bundling the test tubes, and sterilizing at 121 ℃ for 30 min;
3) LB medium (shake flask): weighing yeast extract 0.5g, tryptone 1.0g and sodium chloride 1.0g, adding 100mL of water for dissolving, adjusting the pH value to 7.0, evenly distributing into 2 triangular bottles with 250mL, wherein each bottle is about 50mL, and sterilizing at 121 ℃ for 30 min;
4) 20% IPTG (isopropyl- β -D-thiogalactoside): weighing 2.0g of IPTG, adding purified water to dissolve, fixing the volume to 10mL, filtering and sterilizing by a 0.22 mu m filter membrane, subpackaging and storing at-20 ℃ for later use.
The operation is as follows:
1) taking one LB culture medium test tube in a super-clean workbench, sucking 500 mu l of sterilized water by using a pipette, dripping the sterilized water into a freeze-drying tube, slightly oscillating to dissolve solids in the freeze-drying tube into a suspension state, and completely inoculating bacterial suspension into the LB culture medium test tube; placing the freeze-dried tube in an air oscillator, culturing for 15h at 37 ℃ and the rotating speed of 150rpm to obtain activated test tube seed liquid;
2) inoculating 0.5mL of activated test tube seed liquid into an LB shake flask culture medium from a test tube after the culture is finished in a clean bench, placing an air oscillator, culturing for 4-6h at 37 ℃ and 200rpm, and adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.2mmol/L for induction for 4 h;
3) after the incubation, samples were electrophoresed.
2.2.2 reduced SDS-polyacrylamide gel electrophoresis
The instrument comprises the following steps: electrophoresis apparatus, vertical plate electrophoresis tank and glue making film
Reagent:
1) water (resistivity not less than 18.2 M.OMEGA.cm);
2) solution A1.5 mol/L Tris-hydrochloric acid buffer solution: weighing 18.15g of tris (hydroxymethyl) aminomethane, adding a proper amount of water for dissolving, adjusting the pH value to 8.8 by using hydrochloric acid, and adding water for diluting to 100 mL;
3) solution B30% acrylamide-0.8% methylenebisacrylamide solution: weighing 29.0g of acrylamide and 1.0g of methylene bisacrylamide, adding water to dissolve, diluting to 100mL, uniformly mixing, filtering by using double-layer filter paper, and storing at 4 ℃ in a dark place;
4) solution C1% sodium lauryl sulfate solution: weighing 1.0g of sodium dodecyl sulfate, adding water to dissolve and dilute the sodium dodecyl sulfate to 100mL, and mixing the sodium dodecyl sulfate and the water uniformly. Storing at room temperature;
5) solution D10% N, N, N/, N/-tetramethylethylenediamine solution: 100 mul of tetramethylethylenediamine is weighed and mixed with 900 mul of water. Preparing before use;
6) solution E10% ammonium persulfate solution: 0.1g of ammonium persulfate was weighed and dissolved in 1mL of water and mixed well. Preparing before use;
7) solution F0.5 mol/L Tris-hydrochloric acid buffer solution: weighing 6.05g of tris (hydroxymethyl) aminomethane, adding a proper amount of water for dissolving, adjusting the pH value to 6.8 by using hydrochloric acid, and adding water for diluting to 100 mL;
8) electrode buffer solution: weighing 3g of tris (hydroxymethyl) aminomethane, 14.4g of glycine and 1g of sodium dodecyl sulfate, adding a proper amount of water for dissolving, adjusting the pH value to 8.3 by using hydrochloric acid, and adding water for diluting to 1000 mL;
9) reduced test sample buffer solution: weighing 0.303g of tris (hydroxymethyl) aminomethane, 2mg of bromophenol blue, 0.8g of sodium dodecyl sulfate, 0.189mL of concentrated hydrochloric acid, 4mL of glycerol and 2mL of beta-mercaptoethanol, adding water to dissolve and dilute the solution to 12 mL;
10) coomassie brilliant blue R-250 staining solution: weighing 1g of Coomassie brilliant blue R-250, adding 200mL of methanol, 50mL of glacial acetic acid and 250mL of water, and uniformly mixing;
11) coomassie brilliant blue destaining solution: weighing 400mL of methanol, 100mL of glacial acetic acid and 500mL of water, and uniformly mixing;
12) TE buffer solution: 1.2114g of tris (hydroxymethyl) aminomethane and 0.3722g of disodium ethylene diamine tetraacetate are weighed, dissolved in a proper amount of purified water, the pH value is adjusted to 8.0, then the solution is dissolved in the purified water to 1000mL, sterilized at 121 ℃ for 15min, and stored at 4 ℃. Preparation of standard solution: taking 1 protein molecular weight standard (low), mixing uniformly according to the specification, subpackaging and storing at-20 ℃ for later use. Taking out 1 piece at the time of application, heating in boiling water bath for 3-5 minutes, centrifuging at 12000 r/min for 10 minutes, and taking supernatant and adding sample.
Preparation of a test solution: taking 1mL of the cultured bacterium solution, centrifuging for 10 minutes at 12000 rpm, discarding the supernatant, adding 300 mu L of TE buffer solution for suspension, adding 100 mu L of reduced test sample buffer solution, and mixing uniformly. Heating in boiling water bath for 3-5 min, centrifuging at 12000 rpm for 10min, and sampling the supernatant.
The determination method comprises the following steps:
(1) preparing a separation gel solution (gel concentration is 15%): measuring 4mLA liquid, 8mLB liquid, 1.6mLC liquid, 0.1mLD liquid, 0.1mLE liquid and 2.28mL of water, pouring glue to the position 4/5 of the short glass after uniformly mixing, sealing the short glass by using water slowly, and curing the short glass.
(2) Preparing a concentrated gel solution (the gel concentration is 4.5%): after the separation gel is solidified, the water layer is removed by using filter paper, 1.35mLB liquid, 0.9mLC liquid, 0.07mLD liquid, 0.07mLE liquid, 2.25mLF liquid and 4.33mL of water are measured, the mixture is uniformly mixed and immediately filled with gel to the top of short glass, and a sample comb is inserted to wait for solidification.
(3) Sample adding: carefully pulling out the sample comb after the concentrated gel solution is polymerized, adding the standard solution and the test solution into the sample comb at a concentration of 10 mu L/hole, adding the test solution into the sample comb at the same time, and recording the sample adding sequence.
(4) Electrophoresis: and switching on a power supply, starting electrophoresis under the condition of constant current of 10mA, and regulating the current to 20mA after the sample solution enters the separation gel until the electrophoresis is finished.
(5) Dyeing: the gel after electrophoresis was immersed in a staining solution and stained for 30 minutes on a horizontal shaker with shaking.
(6) And (3) decoloring: and immersing the dyed gel into a decoloring solution, oscillating and decoloring on a horizontal shaking table, and repeatedly replacing the decoloring solution until the strips are clear.
And (4) analyzing results: the gel was scanned and stored in a computer, and the expression level of the target protein was analyzed by software.
TABLE 2 Experimental results after rejuvenation of recombinant E.coli strains in examples and comparative examples
From the experimental results of table 2, it can be seen that: compared with the comparative example, under the same inoculation amount, culture temperature and rotating speed, the strain preservation method has the advantages that the growth state of the activated seed test tube is good, the activated seed test tube can be rapidly rejuvenated under the proper temperature and culture medium environment, and higher growth density can be obtained in the same culture time; the shake flask induction expression shows that the expression quantity of the method is in a higher level, which is obviously higher than that of the comparative example.
Sequence listing
<110> Shandong New times pharmaceutical Co., Ltd
<120> preservation composition of recombinant escherichia coli strain and preparation method thereof
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20 25 30
Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu
35 40 45
Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
50 55 60
Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His
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Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile
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Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
100 105 110
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala
115 120 125
Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala
130 135 140
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser
145 150 155 160
Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
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Gln Glu Lys Leu Cys Ala Met Tyr Lys Leu Cys Ala Pro Glu Glu Leu
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Val Leu Phe Leu Glu Ser Leu Gly Ile Pro Leu Glu Pro Leu Ser Ser
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Cys Pro Ser Gln Leu Gly Met Leu Ala Gly Cys Leu Ser Gln Leu His
65 70 75 80
Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ser
85 90 95
Phe Leu Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Lys Cys
100 105 110
Asp Phe Ala Thr Thr Ile Trp Gln Cys Met Glu Glu Leu Gly Met Ala
115 120 125
Leu Val Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala
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Gly Cys Arg Arg Ala Gly Gly Val Leu Val Ala Ser Leu Val Gln Ser
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Claims (10)
1. A lyophilized composition of a recombinant Escherichia coli species, said composition comprising a recombinant Escherichia coli species and a complex composition, said complex composition comprising a lyoprotectant, and phosphate buffer salt or purified water.
2. The composition of claim 1, wherein the recombinant escherichia coli strain is a pegylated recombinant human granulocyte colony stimulating factor engineered bacterium, a recombinant insulin glargine engineered bacterium, or a TNHH engineered bacterium; preferably, the recombinant escherichia coli strain is a pegylated recombinant human granulocyte colony stimulating factor engineering strain.
3. The composition of claim 1, wherein the lyoprotectant is selected from one or more of skimmed milk powder, glycine and mannitol.
4. The composition of claim 1, wherein the lyoprotectant is present in the complex component at a concentration of 5 to 25% by mass, based on 5 to 25g lyoprotectant per 100mL of solvent.
5. The composition of claim 1, wherein the complex component of the composition further comprises a polysaccharide, preferably the polysaccharide is selected from one or more of lactose, sucrose and trehalose.
6. The composition of claim 5, wherein the mass volume concentration of glycans in the complex component is 0-35%, i.e. 0-35 g glycans per 100mL solvent.
7. The composition of claim 1, wherein the recombinant Escherichia coli strain is prepared at a bacterial liquid density OD600And when the pH value reaches 1.0-1.5, adjusting the pH value of the bacterial liquid to 5.0-6.0 by using a phosphate buffer solution or a citric acid buffer solution, and continuing shaking culture.
8. A method of preparing the composition of claim 1, wherein the method comprises: mixing the recombinant Escherichia coli strain with the compound components in the composition, packaging with freeze-drying tube, and freeze-drying.
9. The method according to claim 8, comprising the steps of:
a. the recombinant Escherichia coli strain is amplified and cultured in a seed culture medium step by step, and the density OD of the strain liquid is600When the pH value reaches 1.0-1.5, using phosphate buffer solution or citric acid buffer solution to adjust the pH value of the bacterial liquid to 5.0-6.0, and continuing shaking the flask to culture until the OD of the bacterial liquid6002-3, obtaining a seed solution of the recombinant escherichia coli strain;
b. preparation of the complex ingredients in the composition: the prescription amount of the freeze-drying protective agent is mixed with phosphate buffer salt or purified water, and the mixture is obtained after constant volume;
c. and (b) taking the seed liquid obtained in the step a, centrifuging, removing supernatant, adding a compound component, suspending by using a vortex oscillator until the thalli are completely and uniformly suspended, subpackaging with a freeze-drying tube, and carrying out freeze-drying treatment to obtain the freeze-dried composition of the recombinant escherichia coli strain.
10. The method according to claim 8, wherein the lyophilization profile of the lyophilization process in the method is as follows:
pre-freezing: firstly, fully cooling a composition solution to be lyophilized to-45 to-50 ℃ and maintaining for 2 to 3 hours;
sublimation drying: vacuumizing the freeze dryer and maintaining the vacuum pressure in the freeze dryer at 0.20 +/-0.02 mbar, heating the composition to-25 to-30 ℃ at the speed of 5 ℃/h, and maintaining the temperature for 2 to 5 h; heating to-15 to-20 ℃ at a speed of 5 ℃/h, and maintaining for 42 to 48 h;
and (3) resolving and drying: vacuumizing the freeze dryer and maintaining 0.10 +/-0.02 mbar, raising the temperature of the composition to 5-10 ℃, continuously drying for 2-4 h, then carrying out ultimate vacuum for 2h, carrying out plug pressing in a vacuum state, and taking out of the box.
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