CN112358540A - Production method of recombinant IL2 in pichia pastoris - Google Patents

Abstract

The invention discloses a production method of recombinant IL2 in pichia pastoris, which comprises the following steps: step S1: constructing a recombinant plasmid; step S2: preparing competent cells; step S3: linearization of the plasmid; step S4: performing electric conversion; step S5: carrying out PCR identification on colonies of the positive clones and carrying out small-amount transfer on the clones; step S6: induction and small expression test of positive clone; step S7: amplifying and purifying; the codon optimization is carried out on the gene of IL2 in the gene synthesis process, and the later expression small test is carried out on the screening of optimal cloning and the optimization of optimal expression time, so the expression quantity of the protein is greatly improved, and the seamless cloning technology is used in the vector construction, so that the tag free protein is expressed in the pPICZ alpha A vector by the recombinant IL2, and the hydrophobic interaction chromatography is used in the later period, thereby shortening the purification process, operating at low temperature and greatly ensuring the protein activity.

Description

Production method of recombinant IL2 in pichia pastoris

Technical Field

The invention relates to the technical field of biology, and in particular relates to a production method of recombinant IL2 in Pichia pastoris.

Background

Interleukins are a class of cytokines that are produced by and act on a wide variety of cells. Since it is produced by leukocytes and acts among leukocytes, it is known that it is still used. The first is the cytokine produced by leukocyte and having regulation function among leukocytes, and the second is the cytokine which has basically definite molecular structure and biological function, has important regulation function and is uniformly named, and belongs to the same category as the blood cell growth factor. The two components are mutually coordinated and interacted to jointly complete the functions of hematopoiesis and immunoregulation, interleukin plays an important role in transmitting information, activating and regulating immune cells, mediating T, B cell activation, proliferation and differentiation and in inflammatory response, interleukin abbreviated as IL is expressed and regulated in functional relation immune response, and the regulation is participated by a plurality of factors from lymphocytes or macrophages and the like. Lymphocyte-derived lymphokines are called monokine from macrophages, the biological activities of all factors are different (such as macrophage activation, T cell proliferation promotion and the like), the physical and chemical properties of the factors are not clear, and the recombinant IL2 is obtained by successfully expressing and purifying pichia pastoris in the research, so that the foundation is laid for the subsequent research of the protein.

Disclosure of Invention

The invention aims to provide a method for producing recombinant IL2 in Pichia pastoris.

The technical problems to be solved by the invention are as follows:

the prior recombinant IL2 has low protein expression amount, long purification time and general purification effect in the preparation process.

The purpose of the invention can be realized by the following technical scheme:

a method for producing recombinant IL2 in Pichia pastoris, comprising the following steps:

step S1: constructing a recombinant plasmid;

step S2: preparing competent cells;

step S3: linearization of the plasmid;

step S4: performing electric conversion;

step S5: carrying out PCR identification on colonies of the positive clones and carrying out small-amount transfer on the clones;

step S6: induction and small expression test of positive clone;

step S7: and (5) amplifying and purifying.

Further, the specific steps of step S1 are as follows:

step S11: subcloning the optimized recombinant IL2 codon into pPICZ alpha A by a gene synthesis technology to construct a recombinant IL2 plasmid;

the gene sequence of the recombinant IL2 plasmid is:

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKRAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLCLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTVD*。

further, the specific steps of step S2 are as follows:

step S21: activating a pichia pastoris strain by using a YPD culture medium containing 50 mu g/mLCm, streaking on a YPD solid culture medium containing 50 mu g/mLCm, screening three monoclonal colonies, respectively inoculating the three monoclonal colonies into 3 50mL centrifuge tubes containing 50 mu g/mLCm YPD liquid culture medium, and carrying out overnight culture under the conditions that the temperature is 30 ℃ and the rotating speed of a shaking table is 220r/min to obtain a cloned thallus;

step S22: putting the best growing thallus in the cloned thallus obtained in the step S21 into a uniformly sterilized 1.5mL centrifuge tube, adding 500 mu L of sterilized glycerol with the volume concentration of 50%, uniformly mixing, and keeping the temperature at-20 ℃ for later use to prepare a first cloned bacterium solution;

step S23: inoculating 50 μ L of the first clone bacterial liquid prepared in step S22 into YPD liquid culture medium containing 50 μ g/mLCm, and culturing overnight at 30 deg.C until OD600 is 1.3-1.5 to obtain second clone bacterial liquid;

step S24: pre-cooling the second clone bacterial liquid prepared in the step S23 to 4 ℃ by a centrifuge, centrifuging for 5min under the condition that the rotating speed is 3000r/min, removing supernatant, and re-suspending and precipitating by 50mL of sterilized water pre-cooled to 4 ℃ to obtain a first suspension;

step S25: pre-cooling the first suspension prepared in the step S24 to 4 ℃ by a centrifuge, centrifuging for 5min under the condition that the rotating speed is 3000r/min, removing supernatant, and re-suspending and precipitating by 25mL of sterilized water pre-cooled to 4 ℃ to obtain a second suspension;

step S26: pre-cooling the second suspension prepared in the step S25 to 4 ℃ by a centrifuge, centrifuging for 5min under the condition that the rotating speed is 3000r/min, removing supernatant, and re-suspending and precipitating by using 25mL of sterilized 1M sorbitol pre-cooled to 4 ℃ to obtain a third suspension;

step S27: and (4) precooling the centrifuge to 4 ℃, centrifuging the third suspension prepared in the step S26 for 5min at the rotating speed of 3000r/min, removing the supernatant, carrying out heavy suspension precipitation by using 500 mu L of 1M sorbitol sterilized by precooling to 4 ℃ to obtain a fourth suspension, subpackaging the fourth suspension into 1.5mL of centrifuge tubes sterilized by precooling to 4 ℃, carrying out 100 mu L of each tube, and carrying out cryopreservation at the temperature of-80 ℃ to prepare the competent cells.

Further, the specific steps of step S3 are as follows:

step S31: carrying out warm bath on the recombinant IL2 plasmid prepared in the step S11 for 1-2h by using restriction enzyme SacI in a 1.5mL centrifuge tube at the temperature of 37 ℃ to prepare a primary enzyme digestion solution;

step S32: carrying out agarose gel electrophoresis detection on the initial enzyme digestion solution prepared in the step S31, and carrying out continuous incubation on the initial enzyme digestion solution which is not completely digested with restriction enzyme Sac I until complete enzyme digestion is carried out to prepare an enzyme digestion complete solution;

step S33: adding 500 mu L of IPA into the enzyme digestion complete solution prepared in the step S32, uniformly mixing, adding 100 mu LpH value of 5.2 sodium acetate solution containing 3M NaAc-HAc, uniformly mixing, using a centrifugal machine with precooling to 4 ℃, centrifuging for 5min under the condition that the rotating speed is 12000r/min, and removing the supernatant to obtain a first precipitate;

step S34: re-suspending the precipitate obtained in the step S33 with 75% volume fraction ethanol, centrifuging for 2min with a centrifuge precooled to 4 ℃ at the rotation speed of 12000r/min, removing the supernatant with a pipettor, and drying the ethanol and the second precipitate;

step S35: the second precipitate obtained in the precipitation step S34 was resuspended and dissolved in 10. mu.L of ddH2O to obtain a linearized plasmid solution, which was placed on ice for use.

Further, the specific steps of step S4 are as follows:

step S41: placing the competent cells prepared in the step S27 on ice, and thawing for 2 min;

step S42: adding 5 mu L of the linearized plasmid solution prepared in the step S35 into the melted competent cells, uniformly mixing, adding the mixture into an electric rotor which is precooled to 4 ℃, and placing the electric rotor on ice for 5 min;

step S43: the electric revolving cup is electrically shocked for 10msec under the conditions that the voltage is 1.5kV, the capacitance is 25 mu F and the resistance is 400 omega, so as to obtain electric revolving liquid;

step S44: uniformly mixing the electrotransformation liquid obtained in the step S43 with 500 mu L of precooled 1M sorbitol at 4 ℃, adding the mixture into a sterilized 1.5mL centrifuge tube, standing for 20min, adding 500 mu L of YPD liquid medium, and culturing for 2h at the temperature of 30 ℃ to obtain a culture bacterium liquid;

step S45: 100. mu.L of the culture broth obtained in step S44 was spread on a YPD solid culture plate and cultured at 30 ℃ for 2 to 3 days to prepare colonies.

Further, the specific steps of step S5 are as follows:

step S51: taking a sterilized 24-well shake culture plate, and adding sterile BMGY medium containing 50 mu g/mL Cm into 20 wells of the plate;

step S52: taking 22 PCR reaction tubes, and preparing a PCR reaction system in each tube;

step S53: selecting 20 monoclonal colonies from the colonies prepared in the step S45 by using a sterilization toothpick, placing one end which is stained with the colonies into a PCR reaction tube for rinsing once, then placing the end into a prepared bacteria shaking plate for rinsing once, and respectively adding 1 mu L of double distilled water and 1 mu L of positive bacteria liquid into the two remaining PCR reaction tubes;

step S54: the PCR reaction tube is covered by a tube cover and is placed into a PCR instrument for PCR reaction;

step S55: culturing the shake bacteria plate at 30 deg.C and 220r/min until OD600 is 2-6.

Further, the composition of the sterile BMGY medium described in step S6 was 1% Yeast Extract, 2% Peptone, 1.34% YNB, 100mM Phosphonate Buffer, 1% Glycerol, 4X 10-5% Biotin, pH 6.0.

Further, the specific steps of step S6 are as follows:

step S61: adding 500 mu L of culture in a culture hole in the shake bacteria plate which is cultured in the step S55 into a sterilized 1.5mL centrifuge tube, adding 500 mu L of glycerol with the volume fraction of 50%, and preserving at the temperature of-20 ℃;

step S62: placing the bacterium shaking plate in the step S61 in a centrifuge which is precooled to 4 ℃, centrifuging for 10min at the rotation speed of 1500r/min, removing the supernatant in each hole, adding 4mL of BMMY liquid oxygen-cultured group containing 50 mug/mLCm into each hole, inducing for 72h at the temperature of 30 ℃ and the rotation speed of 220r/min, adding 20-40 muL of methanol into each hole every 24h during the induction period, centrifuging for 5min at the rotation speed of 1500r/min after the induction is finished, and removing the supernatant;

step S63: after the sediment obtained in the step S62 is resuspended by a lysine Buffer1mL, the sediment is crushed by ultrasonic waves under the conditions of phi 3, 25 percent, 2S on/8S off and 10min, and then centrifuged for 2min under the condition of the rotating speed of 12000r/min, the crushed sediment is centrifuged to a new centrifugal tube of 1.5mL, 10 mu L of Ni-NTA is added, the mixture is incubated for 1h under the condition of the temperature of 4 ℃, centrifuged for 5min under the condition of the rotating speed of 3000r/min, 980 mu L of supernatant is removed, 20 mu L of 5 multiplied Loading Buffer is added and mixed evenly, and then the mixture is boiled and bathed for 10min to prepare the crushed supernatant of the Buffer;

step S64: the buffer-disrupted supernatant obtained in step S63 was examined by SDS-PAGE and Western Blot.

Further, the specific steps of step S7 are as follows:

step S71: and selecting a monoclonal colony with optimal expression for volume amplification culture, and performing ion exchange chromatography by using Q Sepharose Fast Flow gel to prepare the final protein.

The invention has the beneficial effects that: according to the invention, the codon optimization is carried out on the gene of IL2 in the gene synthesis process, and the optimal clone screening and optimal expression time optimization are carried out on the expression bench scale in the later period, so that the expression quantity of the protein is greatly improved, a seamless cloning technology is used in the vector construction, the tag free protein is expressed in the pPICZ alpha A vector by the recombinant IL2, the hydrophobic interaction chromatography is used in the later period, the purification process is shortened, the low-temperature operation is carried out, the protein activity is greatly ensured, and the final protein has the purity of 100% through HPLC detection.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic diagram of the HPLC technique for detecting the final protein purity.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A method for producing recombinant IL2 in Pichia pastoris, comprising the following steps:

step S1: constructing a recombinant plasmid;

step S11: subcloning the optimized recombinant IL2 codon into pPICZ alpha A by a gene synthesis technology to construct a recombinant IL2 plasmid;

the gene sequence of the recombinant IL2 plasmid is: MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKRAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLCLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTVD ^ l;

step S2: preparing competent cells;

step S21: activating a pichia pastoris strain by using a YPD culture medium containing 50 mu g/mLCm, streaking on a YPD solid culture medium containing 50 mu g/mLCm, screening three monoclonal colonies, respectively inoculating the three monoclonal colonies into 3 50mL centrifuge tubes containing 50 mu g/mLCm YPD liquid culture medium, and carrying out overnight culture under the conditions that the temperature is 30 ℃ and the rotating speed of a shaking table is 220r/min to obtain a cloned thallus;

step S22: putting the best growing thallus in the cloned thallus obtained in the step S21 into a uniformly sterilized 1.5mL centrifuge tube, adding 500 mu L of sterilized glycerol with the volume concentration of 50%, uniformly mixing, and keeping the temperature at-20 ℃ for later use to prepare a first cloned bacterium solution;

step S23: inoculating 50 mu L of the first clone bacterial liquid prepared in the step S22 into YPD liquid culture medium containing 50 mu g/mLCm, and culturing overnight at the temperature of 30 ℃ until OD600 is 1.3 to prepare a second clone bacterial liquid;

step S24: pre-cooling the second clone bacterial liquid prepared in the step S23 to 4 ℃ by a centrifuge, centrifuging for 5min under the condition that the rotating speed is 3000r/min, removing supernatant, and re-suspending and precipitating by 50mL of sterilized water pre-cooled to 4 ℃ to obtain a first suspension;

step S25: pre-cooling the first suspension prepared in the step S24 to 4 ℃ by a centrifuge, centrifuging for 5min under the condition that the rotating speed is 3000r/min, removing supernatant, and re-suspending and precipitating by 25mL of sterilized water pre-cooled to 4 ℃ to obtain a second suspension;

step S26: pre-cooling the second suspension prepared in the step S25 to 4 ℃ by a centrifuge, centrifuging for 5min under the condition that the rotating speed is 3000r/min, removing supernatant, and re-suspending and precipitating by using 25mL of sterilized 1M sorbitol pre-cooled to 4 ℃ to obtain a third suspension;

step S27: pre-cooling the third suspension prepared in the step S26 to 4 ℃ by using a centrifuge, centrifuging the third suspension for 5min at the rotating speed of 3000r/min, removing the supernatant, re-suspending and precipitating the third suspension by using 500 mu L of 1M sorbitol sterilized at the pre-cooling temperature of 4 ℃ to obtain a fourth suspension, subpackaging the fourth suspension into 1.5mL of centrifuge tubes sterilized at the pre-cooling temperature of 4 ℃, freezing and storing the fourth suspension at the temperature of-80 ℃ to prepare competent cells, wherein 100 mu L of each centrifuge tube is prepared;

step S3: linearization of the plasmid;

the specific steps of step S3 are as follows:

step S31: carrying out warm bath on the recombinant IL2 plasmid prepared in the step S11 for 1h by using restriction enzyme SacI in a 1.5mL centrifuge tube at the temperature of 37 ℃ to prepare a primary enzyme digestion solution;

step S32: carrying out agarose gel electrophoresis detection on the initial enzyme digestion solution prepared in the step S31, and carrying out continuous incubation on the initial enzyme digestion solution which is not completely digested with restriction enzyme Sac I until complete enzyme digestion is carried out to prepare an enzyme digestion complete solution;

step S33: adding 500 mu L of IPA into the enzyme digestion complete solution prepared in the step S32, uniformly mixing, adding 100 mu LpH value of 5.2 sodium acetate solution containing 3M NaAc-HAc, uniformly mixing, using a centrifugal machine with precooling to 4 ℃, centrifuging for 5min under the condition that the rotating speed is 12000r/min, and removing the supernatant to obtain a first precipitate;

step S34: re-suspending the precipitate obtained in the step S33 with 75% volume fraction ethanol, centrifuging for 2min with a centrifuge precooled to 4 ℃ at the rotation speed of 12000r/min, removing the supernatant with a pipettor, and drying the ethanol and the second precipitate;

step S35: resuspending and solubilizing the second precipitate from precipitation step S34 with 10. mu.L of ddH2O to obtain a linearized plasmid solution, which is placed on ice for use;

step S4: performing electric conversion;

the specific steps of step S4 are as follows:

step S41: placing the competent cells prepared in the step S27 on ice, and thawing for 2 min;

step S42: adding 5 mu L of the linearized plasmid solution prepared in the step S35 into the melted competent cells, uniformly mixing, adding the mixture into an electric rotor which is precooled to 4 ℃, and placing the electric rotor on ice for 5 min;

step S43: the electric revolving cup is electrically shocked for 10msec under the conditions that the voltage is 1.5kV, the capacitance is 25 mu F and the resistance is 400 omega, so as to obtain electric revolving liquid;

step S44: uniformly mixing the electrotransformation liquid obtained in the step S43 with 500 mu L of precooled 1M sorbitol at 4 ℃, adding the mixture into a sterilized 1.5mL centrifuge tube, standing for 20min, adding 500 mu L of YPD liquid medium, and culturing for 2h at the temperature of 30 ℃ to obtain a culture bacterium liquid;

step S45: spreading 100 μ L of the culture broth obtained in step S44 on YPD solid culture plate, and culturing at 30 deg.C for 2 days to obtain colony;

step S5: carrying out PCR identification on colonies of the positive clones and carrying out small-amount transfer on the clones;

the specific steps of step S5 are as follows:

step S51: taking a sterilized 24-well shake culture plate, and adding sterile BMGY medium containing 50 mu g/mL Cm into 20 wells of the plate;

step S52: taking 22 PCR reaction tubes, and preparing a PCR reaction system in each tube;

step S53: selecting 20 monoclonal colonies from the colonies prepared in the step S45 by using a sterilization toothpick, placing one end which is stained with the colonies into a PCR reaction tube for rinsing once, then placing the end into a prepared bacteria shaking plate for rinsing once, and respectively adding 1 mu L of double distilled water and 1 mu L of positive bacteria liquid into the two remaining PCR reaction tubes;

step S54: the PCR reaction tube is covered by a tube cover and is placed into a PCR instrument for PCR reaction;

step S55: culturing a shake bacteria plate at the temperature of 30 ℃ and the rotating speed of 220r/min until the OD600 is 2;

step S6: induction and small expression test of positive clone;

adding 500 mu L of culture in a culture hole in the shake bacteria plate which is cultured in the step S55 into a sterilized 1.5mL centrifuge tube, adding 500 mu L of glycerol with the volume fraction of 50%, and preserving at the temperature of-20 ℃;

step S62: placing the bacterium shaking plate in the step S61 in a centrifuge which is precooled to 4 ℃, centrifuging for 10min at the rotation speed of 1500r/min, removing the supernatant in each hole, adding 4mL of BMMY liquid oxygen-containing solution with 50 mug/mLCm into each hole, inducing for 72h at the temperature of 30 ℃ and the rotation speed of 220r/min, adding 20 muL of methanol into each hole every 24h during the induction period, centrifuging for 5min at the rotation speed of 1500r/min after the induction is finished, and removing the supernatant;

step S63: after the sediment obtained in the step S62 is resuspended by a lysine Buffer1mL, the sediment is crushed by ultrasonic waves under the conditions of phi 3, 25 percent, 2S on/8S off and 10min, and then centrifuged for 2min under the condition of the rotating speed of 12000r/min, the crushed sediment is centrifuged to a new centrifugal tube of 1.5mL, 10 mu L of Ni-NTA is added, the mixture is incubated for 1h under the condition of the temperature of 4 ℃, centrifuged for 5min under the condition of the rotating speed of 3000r/min, 980 mu L of supernatant is removed, 20 mu L of 5 multiplied Loading Buffer is added and mixed evenly, and then the mixture is boiled and bathed for 10min to prepare the crushed supernatant of the Buffer;

step S64: detecting the supernatant obtained by the buffer disruption prepared in the step S63 by SDS-PAGE electrophoresis and Western Blot;

step S7: amplifying and purifying;

step S71: and selecting a monoclonal colony with optimal expression for volume amplification culture, and performing ion exchange chromatography by using Q Sepharose Fast Flow gel to prepare the final protein.

Example 2

A method for producing recombinant IL2 in Pichia pastoris, comprising the following steps:

step S1: constructing a recombinant plasmid;

step S11: subcloning the optimized recombinant IL2 codon into pPICZ alpha A by a gene synthesis technology to construct a recombinant IL2 plasmid;

the gene sequence of the recombinant IL2 plasmid is: MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKRAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLCLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTVD ^ l;

step S2: preparing competent cells;

step S21: activating a pichia pastoris strain by using a YPD culture medium containing 50 mu g/mLCm, streaking on a YPD solid culture medium containing 50 mu g/mLCm, screening three monoclonal colonies, respectively inoculating the three monoclonal colonies into 3 50mL centrifuge tubes containing 50 mu g/mLCm YPD liquid culture medium, and carrying out overnight culture under the conditions that the temperature is 30 ℃ and the rotating speed of a shaking table is 220r/min to obtain a cloned thallus;

step S22: putting the best growing thallus in the cloned thallus obtained in the step S21 into a uniformly sterilized 1.5mL centrifuge tube, adding 500 mu L of sterilized glycerol with the volume concentration of 50%, uniformly mixing, and keeping the temperature at-20 ℃ for later use to prepare a first cloned bacterium solution;

step S23: inoculating 50 mu L of the first clone bacterial liquid prepared in the step S22 into YPD liquid culture medium containing 50 mu g/mLCm, and culturing overnight at the temperature of 30 ℃ until OD600 is 1.5 to prepare a second clone bacterial liquid;

step S24: pre-cooling the second clone bacterial liquid prepared in the step S23 to 4 ℃ by a centrifuge, centrifuging for 5min under the condition that the rotating speed is 3000r/min, removing supernatant, and re-suspending and precipitating by 50mL of sterilized water pre-cooled to 4 ℃ to obtain a first suspension;

step S25: pre-cooling the first suspension prepared in the step S24 to 4 ℃ by a centrifuge, centrifuging for 5min under the condition that the rotating speed is 3000r/min, removing supernatant, and re-suspending and precipitating by 25mL of sterilized water pre-cooled to 4 ℃ to obtain a second suspension;

step S26: pre-cooling the second suspension prepared in the step S25 to 4 ℃ by a centrifuge, centrifuging for 5min under the condition that the rotating speed is 3000r/min, removing supernatant, and re-suspending and precipitating by using 25mL of sterilized 1M sorbitol pre-cooled to 4 ℃ to obtain a third suspension;

step S27: pre-cooling the third suspension prepared in the step S26 to 4 ℃ by using a centrifuge, centrifuging the third suspension for 5min at the rotating speed of 3000r/min, removing the supernatant, re-suspending and precipitating the third suspension by using 500 mu L of 1M sorbitol sterilized at the pre-cooling temperature of 4 ℃ to obtain a fourth suspension, subpackaging the fourth suspension into 1.5mL of centrifuge tubes sterilized at the pre-cooling temperature of 4 ℃, freezing and storing the fourth suspension at the temperature of-80 ℃ to prepare competent cells, wherein 100 mu L of each centrifuge tube is prepared;

step S3: linearization of the plasmid;

the specific steps of step S3 are as follows:

step S31: carrying out warm bath on the recombinant IL2 plasmid prepared in the step S11 for 1-2h by using restriction enzyme SacI in a 1.5mL centrifuge tube at the temperature of 37 ℃ to prepare a primary enzyme digestion solution;

step S32: carrying out agarose gel electrophoresis detection on the initial enzyme digestion solution prepared in the step S31, and carrying out continuous incubation on the initial enzyme digestion solution which is not completely digested with restriction enzyme Sac I until complete enzyme digestion is carried out to prepare an enzyme digestion complete solution;

step S33: adding 500 mu L of IPA into the enzyme digestion complete solution prepared in the step S32, uniformly mixing, adding 100 mu LpH value of 5.2 sodium acetate solution containing 3M NaAc-HAc, uniformly mixing, using a centrifugal machine with precooling to 4 ℃, centrifuging for 5min under the condition that the rotating speed is 12000r/min, and removing the supernatant to obtain a first precipitate;

step S34: re-suspending the precipitate obtained in the step S33 with 75% volume fraction ethanol, centrifuging for 2min with a centrifuge precooled to 4 ℃ at the rotation speed of 12000r/min, removing the supernatant with a pipettor, and drying the ethanol and the second precipitate;

step S35: resuspending and solubilizing the second precipitate from precipitation step S34 with 10. mu.L of ddH2O to obtain a linearized plasmid solution, which is placed on ice for use;

step S4: performing electric conversion;

the specific steps of step S4 are as follows:

step S41: placing the competent cells prepared in the step S27 on ice, and thawing for 2 min;

step S42: adding 5 mu L of the linearized plasmid solution prepared in the step S35 into the melted competent cells, uniformly mixing, adding the mixture into an electric rotor which is precooled to 4 ℃, and placing the electric rotor on ice for 5 min;

step S43: the electric revolving cup is electrically shocked for 10msec under the conditions that the voltage is 1.5kV, the capacitance is 25 mu F and the resistance is 400 omega, so as to obtain electric revolving liquid;

step S44: uniformly mixing the electrotransformation liquid obtained in the step S43 with 500 mu L of precooled 1M sorbitol at 4 ℃, adding the mixture into a sterilized 1.5mL centrifuge tube, standing for 20min, adding 500 mu L of YPD liquid medium, and culturing for 2h at the temperature of 30 ℃ to obtain a culture bacterium liquid;

step S45: spreading 100 μ L of the culture broth obtained in step S44 on YPD solid culture plate, and culturing at 30 deg.C for 3 days to obtain colony;

step S5: carrying out PCR identification on colonies of the positive clones and carrying out small-amount transfer on the clones;

the specific steps of step S5 are as follows:

step S51: taking a sterilized 24-well shake culture plate, and adding sterile BMGY medium containing 50 mu g/mL Cm into 20 wells of the plate;

step S52: taking 22 PCR reaction tubes, and preparing a PCR reaction system in each tube;

step S53: selecting 20 monoclonal colonies from the colonies prepared in the step S45 by using a sterilization toothpick, placing one end which is stained with the colonies into a PCR reaction tube for rinsing once, then placing the end into a prepared bacteria shaking plate for rinsing once, and respectively adding 1 mu L of double distilled water and 1 mu L of positive bacteria liquid into the two remaining PCR reaction tubes;

step S54: the PCR reaction tube is covered by a tube cover and is placed into a PCR instrument for PCR reaction;

step S55: culturing a shake bacteria plate at the temperature of 30 ℃ and the rotating speed of 220r/min until the OD600 is 6;

step S6: induction and small expression test of positive clone;

adding 500 mu L of culture in a culture hole in the shake bacteria plate which is cultured in the step S55 into a sterilized 1.5mL centrifuge tube, adding 500 mu L of glycerol with the volume fraction of 50%, and preserving at the temperature of-20 ℃;

step S62: placing the bacteria shaking plate in the step S61 in a centrifuge which is precooled to 4 ℃, centrifuging for 10min at the rotation speed of 1500r/min, removing the supernatant in each hole, adding 4mL of BMMY liquid oxygen-containing solution with 50 mug/mLCm into each hole, inducing for 72h at the temperature of 30 ℃ and the rotation speed of 220r/min, adding 40 muL of methanol into each hole every 24h during the induction period, centrifuging for 5min at the rotation speed of 1500r/min after the induction is finished, and removing the supernatant;

step S63: after the sediment obtained in the step S62 is resuspended by a lysine Buffer1mL, the sediment is crushed by ultrasonic waves under the conditions of phi 3, 25 percent, 2S on/8S off and 10min, and then centrifuged for 2min under the condition of the rotating speed of 12000r/min, the crushed sediment is centrifuged to a new centrifugal tube of 1.5mL, 10 mu L of Ni-NTA is added, the mixture is incubated for 1h under the condition of the temperature of 4 ℃, centrifuged for 5min under the condition of the rotating speed of 3000r/min, 980 mu L of supernatant is removed, 20 mu L of 5 multiplied Loading Buffer is added and mixed evenly, and then the mixture is boiled and bathed for 10min to prepare the crushed supernatant of the Buffer;

step S64: detecting the supernatant obtained by the buffer disruption prepared in the step S63 by SDS-PAGE electrophoresis and Western Blot;

step S7: amplifying and purifying;

step S71: and selecting a monoclonal colony with optimal expression for volume amplification culture, and performing ion exchange chromatography by using Q Sepharose Fast Flow gel to prepare the final protein.

Example 3

The final protein obtained in example 2 was subjected to purity measurement using HPLC technique, and the measurement results are shown in FIG. 1.

The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions of the specific embodiments described herein may be made by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.

Claims (9)

1. A method for producing recombinant IL2 in Pichia pastoris, comprising: the method comprises the following steps:

step S1: constructing a recombinant plasmid;

step S2: preparing competent cells;

step S3: linearization of the plasmid;

step S4: performing electric conversion;

step S5: carrying out PCR identification on colonies of the positive clones and carrying out small-amount transfer on the clones;

step S6: induction and small expression test of positive clone;

step S7: and (5) amplifying and purifying.

2. The method for producing recombinant IL2 in Pichia pastoris according to claim 1, wherein: the specific steps of step S1 are as follows:

step S11: subcloning the optimized recombinant IL2 codon into pPICZ alpha A by a gene synthesis technology to construct a recombinant IL2 plasmid;

the gene sequence of the recombinant IL2 plasmid is: MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKRAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLCLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTVD.

3. The method for producing recombinant IL2 in Pichia pastoris according to claim 1, wherein: the specific steps of step S2 are as follows:

step S21: activating a pichia pastoris strain by using a YPD culture medium containing 50 mu g/mLCm, streaking on a YPD solid culture medium containing 50 mu g/mLCm, screening three monoclonal colonies, respectively inoculating the three monoclonal colonies into 3 50mL centrifuge tubes containing 50 mu g/mLCm YPD liquid culture medium, and carrying out overnight culture under the conditions that the temperature is 30 ℃ and the rotating speed of a shaking table is 220r/min to obtain a cloned thallus;

step S22: putting the best growing thallus in the cloned thallus obtained in the step S21 into a uniformly sterilized 1.5mL centrifuge tube, adding 500 mu L of sterilized glycerol with the volume concentration of 50%, uniformly mixing, and keeping the temperature at-20 ℃ for later use to prepare a first cloned bacterium solution;

step S23: inoculating 50 μ L of the first clone bacterial liquid prepared in step S22 into YPD liquid culture medium containing 50 μ g/mLCm, and culturing overnight at 30 deg.C until OD600 is 1.3-1.5 to obtain second clone bacterial liquid;

step S24: pre-cooling the second clone bacterial liquid prepared in the step S23 to 4 ℃ by a centrifuge, centrifuging for 5min under the condition that the rotating speed is 3000r/min, removing supernatant, and re-suspending and precipitating by 50mL of sterilized water pre-cooled to 4 ℃ to obtain a first suspension;

step S25: pre-cooling the first suspension prepared in the step S24 to 4 ℃ by a centrifuge, centrifuging for 5min under the condition that the rotating speed is 3000r/min, removing supernatant, and re-suspending and precipitating by 25mL of sterilized water pre-cooled to 4 ℃ to obtain a second suspension;

step S26: pre-cooling the second suspension prepared in the step S25 to 4 ℃ by a centrifuge, centrifuging for 5min under the condition that the rotating speed is 3000r/min, removing supernatant, and re-suspending and precipitating by using 25mL of sterilized 1M sorbitol pre-cooled to 4 ℃ to obtain a third suspension;

step S27: and (4) precooling the centrifuge to 4 ℃, centrifuging the third suspension prepared in the step S26 for 5min at the rotating speed of 3000r/min, removing the supernatant, carrying out heavy suspension precipitation by using 500 mu L of 1M sorbitol sterilized by precooling to 4 ℃ to obtain a fourth suspension, subpackaging the fourth suspension into 1.5mL of centrifuge tubes sterilized by precooling to 4 ℃, carrying out 100 mu L of each tube, and carrying out cryopreservation at the temperature of-80 ℃ to prepare the competent cells.

4. The method for producing recombinant IL2 in Pichia pastoris according to claim 1, wherein: the specific steps of step S3 are as follows:

step S31: carrying out warm bath on the recombinant IL2 plasmid prepared in the step S11 for 1-2h by using restriction enzyme SacI in a 1.5mL centrifuge tube at the temperature of 37 ℃ to prepare a primary enzyme digestion solution;

step S32: carrying out agarose gel electrophoresis detection on the initial enzyme digestion solution prepared in the step S31, and carrying out continuous incubation on the initial enzyme digestion solution which is not completely digested with restriction enzyme Sac I until complete enzyme digestion is carried out to prepare an enzyme digestion complete solution;

step S33: adding 500 mu L of IPA into the enzyme digestion complete solution prepared in the step S32, uniformly mixing, adding 100 mu LpH value of 5.2 sodium acetate solution containing 3M NaAc-HAc, uniformly mixing, using a centrifugal machine with precooling to 4 ℃, centrifuging for 5min under the condition that the rotating speed is 12000r/min, and removing the supernatant to obtain a first precipitate;

step S34: re-suspending the precipitate obtained in the step S33 with 75% volume fraction ethanol, centrifuging for 2min with a centrifuge precooled to 4 ℃ at the rotation speed of 12000r/min, removing the supernatant with a pipettor, and drying the ethanol and the second precipitate;

step S35: the second precipitate obtained in the precipitation step S34 was resuspended and dissolved in 10. mu.L of ddH2O to obtain a linearized plasmid solution, which was placed on ice for use.

5. The method for producing recombinant IL2 in Pichia pastoris according to claim 1, wherein: the specific steps of step S4 are as follows:

step S41: placing the competent cells prepared in the step S27 on ice, and thawing for 2 min;

step S42: adding 5 mu L of the linearized plasmid solution prepared in the step S35 into the melted competent cells, uniformly mixing, adding the mixture into an electric rotor which is precooled to 4 ℃, and placing the electric rotor on ice for 5 min;

step S43: the electric revolving cup is electrically shocked for 10msec under the conditions that the voltage is 1.5kV, the capacitance is 25 mu F and the resistance is 400 omega, so as to obtain electric revolving liquid;

step S44: uniformly mixing the electrotransformation liquid obtained in the step S43 with 500 mu L of precooled 1M sorbitol at 4 ℃, adding the mixture into a sterilized 1.5mL centrifuge tube, standing for 20min, adding 500 mu L of YPD liquid medium, and culturing for 2h at the temperature of 30 ℃ to obtain a culture bacterium liquid;

step S45: 100. mu.L of the culture broth obtained in step S44 was spread on a YPD solid culture plate and cultured at 30 ℃ for 2 to 3 days to prepare colonies.

6. The method for producing recombinant IL2 in Pichia pastoris according to claim 1, wherein: the specific steps of step S5 are as follows:

step S51: taking a sterilized 24-well shake culture plate, and adding sterile BMGY medium containing 50 mu g/mL Cm into 20 wells of the plate;

step S52: taking 22 PCR reaction tubes, and preparing a PCR reaction system in each tube;

step S53: selecting 20 monoclonal colonies from the colonies prepared in the step S45 by using a sterilization toothpick, placing one end which is stained with the colonies into a PCR reaction tube for rinsing once, then placing the end into a prepared bacteria shaking plate for rinsing once, and respectively adding 1 mu L of double distilled water and 1 mu L of positive bacteria liquid into the two remaining PCR reaction tubes;

step S54: the PCR reaction tube is covered by a tube cover and is placed into a PCR instrument for PCR reaction;

step S55: culturing the shake bacteria plate at 30 deg.C and 220r/min until OD600 is 2-6.

7. The method for producing recombinant IL2 in Pichia pastoris according to claim 6, wherein: the composition of the sterile BMGY medium described in step S6 was 1% Yeast Extract, 2% Peptone, 1.34% YNB, 100mM Phosphonate Buffer, 1% Glycerol, 4X 10-5% Biotin, pH 6.0.

8. The method for producing recombinant IL2 in Pichia pastoris according to claim 1, wherein: the specific steps of step S6 are as follows:

step S61: adding 500 mu L of culture in a culture hole in the shake bacteria plate which is cultured in the step S55 into a sterilized 1.5mL centrifuge tube, adding 500 mu L of glycerol with the volume fraction of 50%, and preserving at the temperature of-20 ℃;

step S62: placing the bacterium shaking plate in the step S61 in a centrifuge which is precooled to 4 ℃, centrifuging for 10min at the rotation speed of 1500r/min, removing the supernatant in each hole, adding 4mL of BMMY liquid oxygen-cultured group containing 50 mug/mLCm into each hole, inducing for 72h at the temperature of 30 ℃ and the rotation speed of 220r/min, adding 20-40 muL of methanol into each hole every 24h during the induction period, centrifuging for 5min at the rotation speed of 1500r/min after the induction is finished, and removing the supernatant;

step S63: after the sediment obtained in the step S62 is resuspended by a lysine Buffer1mL, the sediment is crushed by ultrasonic waves under the conditions of phi 3, 25 percent, 2S on/8S off and 10min, and then centrifuged for 2min under the condition of the rotating speed of 12000r/min, the crushed sediment is centrifuged to a new centrifugal tube of 1.5mL, 10 mu L of Ni-NTA is added, the mixture is incubated for 1h under the condition of the temperature of 4 ℃, centrifuged for 5min under the condition of the rotating speed of 3000r/min, 980 mu L of supernatant is removed, 20 mu L of 5 multiplied Loading Buffer is added and mixed evenly, and then the mixture is boiled and bathed for 10min to prepare the crushed supernatant of the Buffer;

step S64: the buffer-disrupted supernatant obtained in step S63 was examined by SDS-PAGE and Western Blot.

9. The method for producing recombinant IL2 in Pichia pastoris according to claim 1, wherein: the specific steps of step S7 are as follows:

step S71: and selecting a monoclonal colony with optimal expression for volume amplification culture, and performing ion exchange chromatography by using Q Sepharose Fast Flow gel to prepare the final protein.

Images