CN110951807A - A kind of fusion expression of cecropin antibacterial peptide and its purification method - Google Patents

Abstract

The invention discloses a fusion expression and purification method of cecropin antibacterial peptide, which comprises three steps, namely the fusion expression of the cecropin antibacterial peptide; processing the fusion expression product; and (4) purifying the fusion protein. The invention adopts a self-induction fusion expression mode to replace the IPTG induction which is commonly used at present, greatly improves the expression quantity of the fusion protein, and reduces the cost by adopting a self-induction mode because the IPTG price is overhigh; the target protein is purified by adopting a mode of direct sample loading purification and elution renaturation of the denaturation of the fusion protein, so that the purification process route is shortened by simplifying the purification steps, the time is saved, and the cost is saved; the purification efficiency and the yield are improved, the purification yield is more than 50% when the purity is higher than 90%, the method is more suitable for industrial production, and the effect is more obvious.

Description

Fusion expression and purification method of cecropin antibacterial peptide

Technical Field

The invention relates to fusion expression of an antimicrobial peptide and a purification method thereof, in particular to fusion expression of cecropin antimicrobial peptide and a purification method thereof.

Background

With the overuse and even abuse of antibiotics, a series of drug-resistant bacteria and even a great number of 'super bacteria' appear, and in this background, the development of a novel and efficient antibacterial agent to replace or partially replace antibiotics is urgently needed. The antibacterial peptide is a kind of micromolecule polypeptide which is produced by a host and can resist the invasion of external microorganisms and eliminate mutant cells in vivo. The chemical essence of the polypeptide is that the protein structure is decomposed into basic unit amino acid in vivo, so that the polypeptide has no residue problem, no immunogenicity and basically no drug resistance. The antibacterial peptide not only has broad-spectrum antibacterial activity, but also can selectively kill part of fungi, viruses, cancer cells and the like; and has no obvious toxic and side effect on normal mammalian cells. Therefore, the antibacterial peptide becomes the possibility of replacing antibiotics.

Cecropin, which was originally discovered and isolated from the young Bombyx mori, was named Cecropin and was the antimicrobial peptide that was originally discovered and isolated. Cecropin is a kind of alkaline heat-resistant short peptide, has broad-spectrum antibacterial activity, is widely present in nature, and has a very important position in numerous categories of antibacterial peptides. From the discovery of cecropin, people find antibacterial peptides in bacteria, fungi, amphibians, insects, plants, mammals, human bodies and the like, the variety of the antibacterial peptides exceeds 3400, and the antibacterial peptides are recorded in an antibacterial peptide library http:// APs. Besides being extracted from animals and plants, the antibacterial peptide can also be prepared by using a chemical synthesis technology and expressed by a genetic engineering technology. The process for extracting the antibacterial peptide from the animals and plants is complex, the purification is difficult to realize, the yield is low, and the production cost is high; the chemically synthesized antibacterial peptide also has the defects of complex process, poor activity, high cost and the like, can be researched only in a laboratory, and is not suitable for industrial production; the antibacterial peptide expressed by the genetic engineering technology has the characteristics of high expression, high activity and the like, and becomes a hotspot of antibacterial peptide production research. The Escherichia coli expression system is one of the most widely used systems in the gene engineering expression technology, has the advantages of clear genetic background, high expression level of target protein, short period, low cost and the like, and has a very important position in the gene expression technology. Coli fusion expression cecropin antibacterial peptide, finds a method capable of improving the expression quantity and reducing the cost through research, and lays a foundation for industrial production.

Disclosure of Invention

The invention aims to provide fusion expression of cecropin and a purification method thereof, which can completely solve the defects of the prior art.

The purpose of the invention is realized by the following technical scheme:

a fusion expression method of cecropin antibacterial peptide comprises the following steps:

1) preparing a culture medium formula:

① preparing 1% ZY solution 100ml by weighing 2g tryptone, 1g yeast extract and ddH₂Dissolving O and fixing the volume to 100 ml;

② preparing 50 XM solution 100ml, weighing NH₄Cl 13.4g、Na₂SO₄3.55g、Na₂HPO₄17.75g、KH₂PO₄17g plus ddH₂Dissolving O, adjusting pH to 6.7, diluting to 100ml, sterilizing, and storing at 4 deg.C;

③ preparing 50X 5052 solution 100ml by weighing glycerol 25g, glucose 2.5g, lactose 10g and ddH₂Dissolving O, diluting to 100ml, sterilizing, and storing at 4 deg.C;

④ preparing trace metal solution 50ml by weighing CaCl₂0.222g、MnCl₂0.198g、ZnSO₄0.288g、NiCl₂0.047g、NaMoO₄0.0484g、H₃BO₄0.012g plus ddH₂Dissolving O, diluting to 50ml, sterilizing, and storing at 4 deg.C;

⑤ preparing ZYM-5052 solution 250ml by taking 5ml of 50 XM solution, 5ml of 50 XM 5052 solution, and 5ml of 0.1MMgSO₄The solution, 10ul of trace metal solution, 125ml of 1% ZY solution and sterile water are added to the solution to be constant volume of 250ml, and the solution is stored at 4 ℃;

2) preparing engineering bacteria: taking an expression vector PET32a to be transformed into a competent cell of escherichia coli BL21, coating, carrying out inverted culture at 37 ℃ overnight, selecting a single colony to be inoculated into a fresh LB liquid culture medium, wherein the LB liquid contains ampicillin solution with the final concentration of 0.1mg/ml, and then placing the LB liquid culture medium in an environment with the rotation speed of 220r/min and the temperature of 37 ℃ for culture overnight to prepare engineering bacteria;

3) fusion expression of cecropin: inoculating the engineering bacteria into fresh ZYM-5052 culture medium at 2% concentration, wherein the ZYM-5052 culture medium contains ampicillin solution with final concentration of 0.1mg/ml, culturing at 25 deg.C at rotation speed of 150r/min for 10 hr, and stopping culturing.

The purification method of the cecropin antibacterial peptide expressed by fusion comprises the following steps:

1) treatment of fusion expression product:

①, subpackaging the engineering bacteria liquid in a centrifuge tube, centrifuging at 4 ℃, removing the supernatant to leave inclusion bodies, adding PBS buffer solution with pH7.4 to clean the inclusion bodies, centrifuging the cleaned inclusion bodies at 4 ℃, removing the supernatant, adding PBS buffer solution with pH7.4, stirring to resuspend the bacteria, then carrying out ultrasonic crushing in an ice bath with the crushing power of 200W, crushing for 3S, cooling for 3S, and carrying out centrifugation at 4 ℃ after the crushing is finished;

② adding the inclusion bodies after crushing and centrifugation into a washing buffer solution I, wherein the washing buffer solution I contains 5mmol/L imidazole, 2mol/L urea and 20mmol/LPB buffer solution with the pH value of 7.2, then stirring and washing, centrifuging at 4 ℃, discarding supernatant, adding a washing buffer solution II, wherein the washing buffer solution II contains 0.1mol/L sodium chloride, 5mmol/L imidazole, 2mol/L urea, 0.01% TritonX-100 and 0.1mol/L Tris-HCl with the pH value of 8.0, then stirring and washing again, centrifuging at 4 ℃, discarding supernatant, and finally adding a PB buffer solution with the pH value of 7.2 to resuspend the inclusion bodies;

③ adding the inclusion body washed in method ② into a denaturant, wherein the denaturant comprises 0.3mol/L sodium chloride, 8mol/L urea and 20mmol/L Tris-HCl, and the pH value is 8.0, and then stirring until the inclusion body is dissolved;

2) and (3) purification: use of five column volumes ddH₂O rinsing of Ni²⁺Chelating the column, washing off ethanol in the column, adding 50mmol/L nickel sulfate solution to the five columns, and hanging Ni on the filler in the chromatographic column²⁺Reuse of ddH₂Washing the nickel sulfate solution which is not hung on the column by using O, balancing the column after the redundant nickel sulfate solution is completely washed, centrifuging the dissolved inclusion body solution at 4 ℃, and taking the supernatant to pass through the column; after the fusion protein sample is loaded on the column, five column volumes of denaturants are used for washing out unbound protein, after the base line is balanced, imidazole solutions with gradient concentrations are respectively used for eluting the target protein, and the fusion protein samples of each outflow peak are collected.

Compared with the prior art, the invention has the beneficial effects that:

1. the method adopts a self-induction fusion expression mode to replace the IPTG induction which is commonly used at present, greatly improves the expression quantity of the fusion protein, and reduces the cost by adopting a self-induction mode due to overhigh price of the IPTG;

2. the target protein is purified by adopting a mode of direct sample loading purification and elution renaturation of the denaturation of the fusion protein, so that the purification process route is shortened by simplifying the purification steps, the time is saved, and the cost is saved;

3. the invention improves the purification efficiency and the yield, and the purification yield is more than 50% when the purity is higher than 90%, thus being more suitable for industrial production and having more obvious effect.

Drawings

FIG. 1 is a graph of the fusion expression result of engineering bacteria at 25 ℃ and 150 r/min;

FIG. 2 is a graph showing the results of direct loading and purification on a column after denaturation of the fusion protein;

FIG. 3 is a diagram showing the results of fusion expression of engineering bacteria at 37 ℃ and 220 r/min;

FIG. 4 is a graph showing the results of sample purification after renaturation of the fusion protein.

Detailed Description

The invention will be further described with reference to specific embodiments and the accompanying drawings.

The invention discloses a cecropin antibacterial peptide fusion expression and purification method, comprising three steps.

The first step is as follows: fusion expression of the cecropin antibacterial peptide;

the second step is that: processing the fusion expression product;

the third step: and (4) purifying the fusion protein.

The process of the step one is as follows:

the formula of the culture medium is as follows:

1% ZY (100 ml): weighing 2g tryptone, 1g yeast extract and ddH₂O was dissolved and the volume was made 100 ml.

50 XM (100 ml): weighing NH₄Cl 13.4g、Na₂SO₄3.55g、Na₂HPO₄17.75g、KH₂PO₄17g plus ddH₂Dissolving O, adjusting pH to 6.7, diluting to 100ml, sterilizing, and storing at 4 deg.C.

50X 5052(100 ml): weighing 25g of glycerol, 2.5g of glucose, 10g of lactose and ddH₂Dissolving O, diluting to 100ml, sterilizing, and storing at 4 deg.C.

Trace metals (50 ml): weighing CaCl₂0.222g、MnCl₂0.198g、ZnSO₄0.288g、NiCl₂0.047g、NaMoO₄0.0484g、H₃BO₄0.012g plus ddH₂Dissolving O, diluting to 50ml, sterilizing, and storing at 4 deg.C.

ZYM-5052(250ml)：5ml(50×M)、5ml(50×5052)、5ml(0.1M MgSO₄) 10ul trace metal, 125ml of 1% ZY, and sterile water to a constant volume of 250ml, and storing at 4 ℃.

Taking an expression vector PET32a to be transformed into an escherichia coli (BL21) competent cell, coating, carrying out inverted culture at 37 ℃ overnight, picking out a single colony, inoculating the single colony into a fresh LB liquid culture medium containing ampicillin solution (the final concentration is 0.1mg/ml), and carrying out culture at 37 ℃ overnight to prepare the engineering bacteria.

Inoculating the engineering bacteria at 2% concentration into ZYM-5052 culture medium containing fresh ampicillin solution (final concentration of 0.1mg/ml), culturing at 25 deg.C for 10 hr at 150r/min, and stopping culturing.

The process of the second step is as follows:

respectively filling the engineering bacteria strain liquid into a centrifuge tube, centrifuging at 4 ℃, removing supernatant, adding PBS (pH7.4) according to the requirement, and cleaning the inclusion body; and centrifuging the washed inclusion bodies at 4 ℃, removing the supernatant, adding a PBS (phosphate buffer solution) (PH7.4), stirring and resuspending the thalli, carrying out ultrasonic crushing in an ice bath with the crushing power of 200W for 3S, cooling for 3S, and crushing for 30 min. After completion of the disruption, centrifugation was carried out at 4 ℃.

Crushing the inclusion body, adding a washing buffer solution I (containing 5mmol/L imidazole, 2mol/L urea and 20mmol/LPB buffer solution, and having a pH value of 7.2), stirring and washing, centrifuging at 4 ℃, and removing a supernatant; adding washing buffer solution II (containing 0.1mol/L sodium chloride, 5mmol/L imidazole, 2mol/L urea, 0.01% Triton X-100, 0.1mol/L Tris-HCl, pH8.0), stirring, washing again, centrifuging at 4 deg.C, and discarding the supernatant; finally, PB buffer (pH7.2) was added to resuspend the inclusion bodies.

The inclusion bodies after washing are added into a denaturant (containing 0.3mol/L sodium chloride, 8mol/L urea, 20mmol/L LTris-HCl and pH8.0) and stirred until the inclusion bodies are dissolved.

The process of the third step is as follows:

the principle is as follows: the fusion protein is separated and purified by utilizing the characteristic that thioredoxin can be combined with specific ligand and using an Affinity Chromatography (AC) method. Since thioredoxin contains (His)₆Tagged tag with Ni²⁺Specific binding when a thioredoxin-containing fusion protein sample enters Ni²⁺When the column is used, the target protein is adsorbed on Ni^{2 +}And other hybrid proteins flow out of the column along with the buffer solution, and the fusion protein is eluted by using specific eluent and is subjected to protein renaturation to finally obtain the relatively pure fusion protein.

Equipment and reagents: AKTA protein purification System, Freeze centrifuge, Ni²⁺Chelating column, 50mmol/L nickel sulfate solution, and 10, 50, 100, 200, 300mmol/L imidazole solution.

Using about five column volumes ddH₂O rinsing of Ni²⁺Chelating the column, washing off ethanol in the column, adding 50mmol/L nickel sulfate solution to the five columns, and hanging Ni on the filler in the chromatographic column²⁺Reuse of ddH₂Washing the nickel sulfate solution which is not hung, balancing the column after the redundant nickel sulfate solution is completely washed, and dissolvingThe inclusion body solution after centrifugation at 4 ℃ and supernatant through the column. After the sample is loaded on the column, about five column volumes of denaturants are used for washing out the unbound protein, after the base line is balanced, imidazole solutions with gradient concentrations are respectively used for eluting the target protein, and samples of each outflow peak are collected.

According to the invention, the following are specific experimental cases:

case one:

according to the first step, the activated engineering bacteria are inoculated into 400ml of fresh ZYM-5052 culture medium containing ampicillin solution (the final concentration is 0.1mg/ml) according to the concentration of 2%, the culture is stopped at 150r/min and the temperature is 25 ℃ for 8, 10, 12 and 14 hours, the wet bacteria are collected to be about 1g, the expression quantity of the fusion protein is detected by 15% SDS-PAGE gel electrophoresis, the result shows that the expression quantity of the fusion protein accounts for 30-50% of the total protein of the escherichia coli, and the expression quantity is higher than the average level reported at present as shown in figure 1.

Centrifuging the bacterial liquid at 4 ℃ and 8000r/min for 10min, removing the supernatant, adding PBS buffer (PH7.4) according to the proportion of 1g to 10ml, and cleaning the inclusion bodies; centrifuging the cleaned inclusion bodies at 4 ℃ at 8000r/min for 10min, removing the supernatant, adding PBS buffer (PH7.4) according to the proportion of 1g to 10ml, stirring for 30min to resuspend the thalli, then subpackaging the inclusion bodies into about 10 ml/tube (containing about 1g of wet thalli), carrying out ultrasonic crushing in an ice bath, wherein the crushing power is 200W, crushing for 3S, cooling for 3S, and crushing for 30 min. After the crushing is finished, centrifuging for 10min at 4 ℃ and 10000r/min, collecting precipitate and supernatant, and carrying out 15% SDS-PAGE gel electrophoresis detection, wherein the result shows that the protein expressed according to the method is an inclusion body and needs to be subjected to denaturation and renaturation treatment.

Adding washing buffer solution I (containing 5mmol/L imidazole, 2mol/L urea and 20mmol/L PB buffer solution, pH7.2) into 1g:10ml, stirring for 30min to clean inclusion body, centrifuging at 4 deg.C and 11000r/min for 40min, and discarding supernatant; adding washing buffer solution II (containing 0.1mol/L sodium chloride, 5mmol/L imidazole, 2mol/L urea, 0.01% Triton X-100, 0.1mol/L Tris-HCl and pH8.0) into 1 g/10 ml, stirring for 30min, cleaning inclusion body again, centrifuging at 4 deg.C and 11000r/min for 40min, and discarding the supernatant; and finally, according to the weight ratio of 1g: add PB buffer (pH7.2) to 10ml to resuspend the inclusion bodies, centrifuge at 8000r/min for 10min at 4 ℃ and discard the supernatant.

Adding the washed inclusion bodies into a denaturant (containing 0.3mol/L sodium chloride, 8mol/L urea, 20mmol/L Tris-HCl and pH8.0) according to the ratio of 1:10, stirring until the inclusion bodies are dissolved, and detecting the content of the fusion protein to be about 25.2mg/L by using a Coomassie brilliant blue method.

According to the third step, after the surplus nickel sulfate solution is washed, a denaturant (containing 0.3mol/L sodium chloride, 8mol/L urea, 20mmol/L Tris-HCl and pH8.0) is used for balancing the column, then the dissolved inclusion body solution is centrifuged for 40min at the temperature of 4 ℃ and 11000r/min, and the supernatant is taken and passed through the column. After all solutions were loaded, the column was flushed with approximately five column volumes of denaturant until baseline equilibration, using a gradient of concentration: 10. eluting target protein with 50, 100 and 200mmol/L imidazole solution, collecting each effluent peak sample, and detecting and purifying by 15% SDS-PAGE gel electrophoresis, as shown in FIG. 2, the content of the tube with the highest concentration detected by Coomassie Brilliant blue method is about: 13.8mg/L, yield 59.5%.

Case two:

according to the first step, the activated engineering bacteria are inoculated into 400ml of fresh ZYM-5052 culture medium containing ampicillin solution (the final concentration is 0.1mg/ml) according to the concentration of 2%, the culture is carried out for 8, 10, 12 and 14 hours at 37 ℃, the culture is stopped, the collected wet bacteria are about 2.4g, the expression quantity of the fusion protein is detected by 15% SDS-PAGE gel electrophoresis, as shown in figure 3, the result shows that the expression quantity of the fusion protein accounts for 30-50% of the total protein of escherichia coli, and the expression quantity is higher than the average level reported at present.

Centrifuging the bacterial liquid at 4 ℃ and 8000r/min for 10min, removing the supernatant, adding PBS buffer (PH7.4) according to the proportion of 1g to 10ml, and cleaning the inclusion bodies; centrifuging the cleaned inclusion bodies at 4 ℃ at 8000r/min for 10min, removing the supernatant, adding PBS buffer (PH7.4) according to the proportion of 1g to 10ml, stirring for 30min to resuspend the thalli, then subpackaging the inclusion bodies into about 10 ml/tube (containing about 1g of wet thalli), carrying out ultrasonic crushing in an ice bath, wherein the crushing power is 200W, crushing for 3S, cooling for 3S, and crushing for 30 min. After the crushing is finished, centrifuging for 10min at 4 ℃ and 10000r/min, collecting precipitate and supernatant, and carrying out 15% SDS-PAGE gel electrophoresis detection, wherein the result shows that the protein expressed according to the method is an inclusion body and needs to be subjected to denaturation and renaturation treatment.

Adding 1g of 10ml of PBS buffer solution with pH of 7.4, stirring for 30min to clean the inclusion body, centrifuging for 10min at 4 ℃ and 8000r/min, and removing the supernatant; the inclusion bodies are cleaned again by repeating the steps, centrifuged for 10min at 8000r/min and the supernatant is discarded at 4 ℃.

Adding the cleaned inclusion bodies into a denaturant solution of 8mol/L according to a ratio of 1:10, stirring until the inclusion bodies are dissolved, and detecting the content of the fusion protein to be about 25.0mg/L by using a Coomassie brilliant blue method.

Pumping the dissolved inclusion bodies into 2mol/L urea solution slowly according to 0.5ml/min, stirring for 14-16h to renature the protein, replacing 2mol/L urea in the solution with 20mmol/LPH7.2 PB buffer solution by using ultrafiltration, and concentrating the volume of the solution.

According to the third step, after the surplus nickel sulfate solution is washed, the column is balanced by using 20mmol/L PB buffer solution with the pH value of 7.2, after the column is balanced, the renatured protein solution is centrifuged for 10min at the temperature of 4 ℃ and the speed of 11000r/min, and the supernatant is taken to pass through the column. After the protein solution has completely passed through the column, the column is washed with about five column volumes of 20mmol/L PB buffer solution at pH7.2, and after baseline equilibration, gradient concentrations are used: 20. eluting target protein with 40, 80, 100, 200mmol/L imidazole solution, collecting each effluent peak sample, performing 15% SDS-PAGE gel electrophoresis detection and purification, and as shown in FIG. 4, detecting the content of the tube with the highest concentration by Coomassie Brilliant blue method is about: 12.3mg/L, and the yield is 48.9%.

Therefore, the invention has the following advantages and beneficial effects:

1. the invention adopts a self-induction fusion expression mode to replace the IPTG induction which is commonly used at present, greatly improves the expression quantity of the fusion protein, and reduces the cost by adopting a self-induction mode because the IPTG price is overhigh;

2. the invention adopts the mode of direct sample loading purification and elution renaturation of the denaturation of the fusion protein to purify the target protein, simplifies the purification steps, shortens the purification process route, saves time and saves cost;

3. the invention improves the purification efficiency and the yield, and the purification yield is more than 50% when the purity is higher than 90%, thus being more suitable for industrial production and having more obvious effect.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (2)

1. A fusion expression method of cecropin antibacterial peptide is characterized by comprising the following steps:

1) preparing a culture medium formula:

① preparing 1% ZY solution 100ml by weighing 2g tryptone, 1g yeast extract and ddH₂Dissolving O and fixing the volume to 100 ml;

② preparing 50 XM solution 100ml, weighing NH₄Cl 13.4g、Na₂SO₄3.55g、Na₂HPO₄17.75g、KH₂PO₄17g plus ddH₂Dissolving O, adjusting pH to 6.7, diluting to 100ml, sterilizing, and storing at 4 deg.C;

③ preparing 50X 5052 solution 100ml by weighing glycerol 25g, glucose 2.5g, lactose 10g and ddH₂Dissolving O, diluting to 100ml, sterilizing, and storing at 4 deg.C;

④ preparing trace metal solution 50ml by weighing CaCl₂0.222g、MnCl₂0.198g、ZnSO₄0.288g、NiCl₂0.047g、NaMoO₄0.0484g、H₃BO₄0.012g plus ddH₂Dissolving O, diluting to 50ml, sterilizing, and storing at 4 deg.C;

⑤ preparing ZYM-5052 solution 250ml by taking 5ml of 50 × M solution, 5ml of 50 × 5052 solution, and 5ml of 0.1M MgSO 2₄The solution, 10ul of trace metal solution, 125ml of 1% ZY solution and sterile water are added to the solution to be constant volume of 250ml, and the solution is stored at 4 ℃;

2) preparing engineering bacteria: taking an expression vector PET32a to be transformed into a competent cell of escherichia coli BL21, coating, carrying out inverted culture at 37 ℃ overnight, selecting a single colony to be inoculated into a fresh LB liquid culture medium, wherein the LB liquid contains ampicillin solution with the final concentration of 0.1mg/ml, and then placing the LB liquid culture medium in an environment with the rotation speed of 220r/min and the temperature of 37 ℃ for culture overnight to prepare engineering bacteria;

3) fusion expression of cecropin: inoculating the engineering bacteria into fresh ZYM-5052 culture medium at 2% concentration, wherein the ZYM-5052 culture medium contains ampicillin solution with final concentration of 0.1mg/ml, culturing at 25 deg.C at rotation speed of 150r/min for 10 hr, and stopping culturing.

2. The method for purifying cecropin according to claim 1, comprising the steps of:

1) treating cecropin antibacterial peptide expressed by fusion in the method:

①, subpackaging the engineering bacteria liquid in a centrifuge tube, centrifuging at 4 ℃, removing the supernatant to leave inclusion bodies, adding PBS buffer solution with pH7.4 to clean the inclusion bodies, centrifuging the cleaned inclusion bodies at 4 ℃, removing the supernatant, adding PBS buffer solution with pH7.4, stirring to resuspend the bacteria, then carrying out ultrasonic crushing in an ice bath with the crushing power of 200W, crushing for 3S, cooling for 3S, and carrying out centrifugation at 4 ℃ after the crushing is finished;

② adding the inclusion bodies after crushing and centrifugation into a washing buffer solution I, wherein the washing buffer solution I contains 5mmol/L imidazole, 2mol/L urea and 20mmol/L LPB buffer solution with the pH value of 7.2, then stirring and washing, centrifuging at 4 ℃, discarding supernatant, adding a washing buffer solution II, wherein the washing buffer solution II contains 0.1mol/L sodium chloride, 5mmol/L imidazole, 2mol/L urea, 0.01% Triton X-100 and 0.1mol/L Tris-HCl with the pH value of 8.0, then stirring and washing again, centrifuging at 4 ℃, discarding supernatant, and finally adding a PB buffer solution with the pH value of 7.2 to resuspend the inclusion bodies;

③ adding the inclusion body washed in method ② into a denaturant, wherein the denaturant comprises 0.3mol/L sodium chloride, 8mol/L urea and 20mmol/L Tris-HCl, and the pH value is 8.0, and then stirring until the inclusion body is dissolved;

2) and (3) purification: use of five column volumes ddH₂O rinsing of Ni²⁺Chelating the column, washing off ethanol in the column, adding 50mmol/L nickel sulfate solution to the five columns, and hanging Ni on the filler in the chromatographic column²⁺Reuse of ddH₂Washing the nickel sulfate solution which is not hung on the column by using O, balancing the column after the redundant nickel sulfate solution is completely washed, centrifuging the dissolved inclusion body solution at 4 ℃, and taking the supernatant to pass through the column; after the fusion protein sample is loaded on the column, five column volumes of denaturants are used for washing out unbound protein, after the base line is balanced, imidazole solutions with gradient concentrations are respectively used for eluting the target protein, and the fusion protein samples of each outflow peak are collected.

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