Antigen purification and renaturation method

Abstract

The invention provides an antigen purification and renaturation method, which comprises the following steps: by improving a renaturation agent formula and a purification and renaturation process route, firstly completing chromatographic column purification after denaturation and then carrying out renaturation, and containing polyethylene glycol and cystine in an equilibrium buffer solution, a washing buffer solution, an elution buffer solution and a renaturation agent, the problem that recombinant protein is prone to aggregation and precipitation after renaturation is solved, CHAPS is further added to improve the renaturation effect for the recombinant protein with weak stability, the prepared recombinant protein is more stable, aggregation and precipitation are prevented, the yield is high, good immunocompetence is achieved, and the detection sensitivity when the recombinant protein is used for detecting antibodies is improved.

Description

Technical Field

[0001] The invention relates to the technical field of antigen preparation, and in particular to a method for purifying and renaturing an antigen. Background Art

[0002] In the preparation process of recombinant antigens, purification and renaturation are also very important steps. The use of appropriate purification and renaturation processes can not only improve the yield of antigens, but may even affect the immune activity of antigens, which has an important impact on the later application of recombinant antigens.

[0003] Allergens are also important antigens. Identifying allergens is the key to preventing and treating allergic diseases. Avoiding contact with allergens and carrying out specific immunotherapy are inseparable from allergen diagnosis. Dust mite allergens Der f 1 (Derf1 for short) and Der f 2 (Derf2 for short) are the main sensitizing components. Dust mite allergens Der f 3 (Derf3 for short) also play an important role in the sensitization process and can also mediate allergic reactions in the body. Different allergic patients may be allergic to different dust mite allergens (such as Derf1, Derf2 or Derf3). This requires determining which dust mite allergen the patient is allergic to in order to take targeted treatment, such as using different drugs for desensitization treatment, in order to truly achieve patient tolerance to specific dust mite allergens. In order to distinguish which dust mite allergen causes the patient's allergic reaction and carry out targeted treatment, it is often necessary to prepare Derf1, Derf2 and Derf3 recombinant antigens for the detection of IgE antibodies to the three allergens.

[0004] In the preparation process of Derf1, Derf2 and Derf3, the recombinant Derf1, Derf2 and Derf3 proteins expressed by Escherichia coli in the form of inclusion bodies lack biological activity. During the expression process, the proteins are released quickly and disorderly entangled to form insoluble proteins. In vitro folding and refolding are required to obtain biological activity. Existing conventional refolding methods include step-by-step dialysis, gradual dilution of high-concentration denaturants, chromatography, large-volume dilution, etc. However, in the above conventional methods, the recombinant Derf1, Derf2 and Derf3 proteins cannot be stably present after refolding and are prone to aggregation and precipitation.

[0005] Therefore, there is an urgent need to find a method for preparing Derf1, Derf2 and Derf3 recombinant proteins, with high yield, good renaturation and good immunoreactivity of the three prepared antigens, which provides a basis for the quantitative detection of Derf1, Derf2 and Derf3 antibodies. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a method for purifying and renaturing an antigen, by improving the formula of the renaturing agent and the process route of purification and renaturation, completing chromatography column purification before renaturation after denaturation, and containing polyethylene glycol and cysteine ​​in the equilibration buffer, washing buffer, elution buffer and renaturing agent, so as to solve the problem that Derf1, Derf2 and Derf3 recombinant proteins are prone to aggregation and precipitation after renaturation, wherein CHAPS is further added to Derf3 to improve the renaturation effect, and the prepared Derf1, Derf2 and Derf3 recombinant proteins are more stable, prevent aggregation and precipitation, have a high yield, and have good immune activity, thereby improving the detection sensitivity when used for detecting antibodies.

[0007] In one aspect, the present invention provides a method for purifying and renaturing an antigen, the method comprising the following steps:

[0008] (1) Collecting the fermentation medium containing the recombinant protein and adding a denaturant to denature and dissolve it;

[0009] (2) purification by chromatography column, wherein the chromatography column is first equilibrated with an equilibration buffer and then eluted with an elution buffer;

[0010] (3) Diluting with a refolding agent and dialyzing to obtain the recombinant antigen;

[0011] The equilibration buffer, elution buffer and refolding agent all contain polyethylene glycol and cystine.

[0012] In order to achieve short-term rapid expression of Derf1, Derf2 and Derf3 recombinant proteins, the present invention adopts Escherichia coli expression to prepare recombinant proteins, which can reduce the production cost of Derf1, Derf2 and Derf3 recombinant proteins. However, after the Derf1, Derf2 and Derf3 recombinant proteins are expressed in the form of inclusion bodies by Escherichia coli, they need to be broken and denatured to improve their solubility.

[0013] Previous experiments have shown that after Derf1, Derf2 and Derf3 recombinant proteins are expressed in E. coli in the form of inclusion bodies, the protein products obtained by fragmentation, denaturation and renaturation of the target proteins are prone to aggregation and precipitation, resulting in a decrease in the final protein yield and difficulty in exposing the antigenic epitope. Therefore, it is necessary to optimize the purification and renaturation process to improve the immune activity and stability of Derf1, Derf2 and Derf3 recombinant proteins.

[0014] Compared with the protein product after renaturation, the denatured Derf1, Derf2 and Derf3 recombinant proteins have better solubility and are more stable, so it is preferred to purify them first and then renature them. However, the denatured recombinant proteins occasionally have the problem of protein precipitation and aggregation, resulting in decreased immunological activity and reduced yield. Therefore, the present invention also optimizes the equilibration buffer and elution buffer in the purification process, completely solving the problem of protein precipitation and aggregation of Derf1, Derf2 and Derf3 recombinant proteins during ultrafiltration and purification, and can improve the immunological activity of the recombinant proteins.

[0015] After the denatured protein product, it is necessary to screen a suitable renaturing agent to improve the immune activity and long-term stability of the recombinant protein. The present invention optimizes the components in the renaturing agent formula, effectively improving the stability of the renatured Derf1, Derf2 and Derf3 recombinant proteins, ensuring that the protein does not precipitate and exposes more antigenic epitopes, thereby having good immune activity and improving the detection sensitivity when used to detect the corresponding antibodies.

[0016] After screening a suitable renaturing agent, the present invention further adjusts the purification and renaturing steps, performs renaturation after completing all purification steps, and simultaneously adds polyethylene glycol and cystine to the equilibration buffer, the elution buffer and the renaturing agent, thereby improving the stability of the target protein and preventing the protein from aggregating during the chromatography process. In addition, polyethylene glycol and cystine have relatively small molecular weights and will not be retained by the chromatography, and will not affect the chromatography effect.

[0017] The reason why polyethylene glycol and cystine can have such an effect may be related to the properties of polyethylene glycol and cystine themselves. Polyethylene glycol has a molecular exclusion effect, which can reduce the aggregation between protein molecules. Denatured recombinant proteins tend to aggregate and precipitate with each other. Polyethylene glycol can occupy the space in the solution, separate protein molecules from each other, and reduce the probability of collision and formation of aggregates. Cystine may help the protein maintain its activity, so that more antigenic epitopes can be exposed during the renaturation of the target protein, thereby improving the detection effect of antibodies. Therefore, adding polyethylene glycol and cystine during the chromatography process and the renaturation process can significantly improve the stability of Derf1, Derf2 and Derf3 recombinant proteins and maintain the activity of the proteins.

[0018] Furthermore, the refolding agent in step (3) includes polyethylene glycol, cystine, PBS and glycerol.

[0019] Furthermore, the denaturant in step (1) includes urea, EDTA-Na, mercaptoethanol and Tris-Cl solution.

[0020] Furthermore, the equilibration buffer and the elution buffer in step (2) respectively comprise polyethylene glycol, cystine, imidazole, PBS and glycerol.

[0021] Furthermore, the chromatography column in step (2) is a Ni-NTA agarose chromatography column; and also includes a washing buffer for washing the chromatography column, and the washing buffer includes polyethylene glycol, cystine, imidazole, PBS and glycerol.

[0022] By adding polyethylene glycol and cystine to the equilibration buffer, washing buffer and elution buffer during the chromatography process, the Derf1, Derf2 and Derf3 recombinant proteins can always be in the same ionic environment, which helps to improve protein stability and prevent the protein activity from being affected.

[0023] Furthermore, in the dialysis in step (3), the dialysis fluid used includes PBS and glycerol.

[0024] Furthermore, the dust mite antigens include any one or more of Derf1, Derf2, and Derf3; the amino acid sequences of the Derf1, Derf2, and Derf3 recombinant antigens are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

[0025] Furthermore, when the dust mite antigen is Derf3, the renaturing agent further comprises CHAPS.

[0026] Since the stability of Derf3 recombinant protein is weaker than that of Derf1 and Derf2 recombinant proteins, in order to improve the renaturation effect, it is preferred to add polyethylene glycol, cystine and CHAPS to the renaturing agent. Compared with Derf1 and Derf2 recombinant proteins, CHAPS is added. The reason may be that CHAPS has a solubilizing effect, which helps Derf3 recombinant protein to maintain a dispersed and active state in the solution and reduce nonspecific electrostatic interactions with other biological molecules. It also helps it fold and form more disulfide bonds, thereby promoting its renaturation.

[0027] On the other hand, the present invention provides a composition for preparing a reagent for improving the renaturation level of dust mite antigens, the composition comprising polyethylene glycol and cystine, the dust mite antigens comprising any one or more of Derf1, Derf2, and Derf3; the amino acid sequences of the Derf1, Derf2, and Derf3 recombinant antigens are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

[0028] The antigen renaturation level of the present invention includes improving the immune activity, stability and yield of the recombinant antigen, etc. The immune activity includes the detection sensitivity and accuracy when used to detect the corresponding antibody, etc.

[0029] In another aspect, the present invention provides a use of a composition for preparing a reagent for improving the renaturation level of dust mite antigen Derf3, wherein the composition comprises polyethylene glycol, cystine and CHAPS.

[0030] Beneficial effects achieved by the present invention:

[0031] 1. A suitable refolding agent was screened, and the purification and refolding process routes were improved. After denaturation, column purification was completed first and then refolding was performed. Polyethylene glycol and cystine were contained in the ultrafiltration equilibration liquid, equilibration buffer and refolding agent to solve the problem that Derf1, Derf2 and Derf3 recombinant proteins were prone to aggregation and precipitation after refolding. The prepared Derf1, Derf2 and Derf3 recombinant proteins were more stable, with high yield and purity, and good immunoreactivity, and the detection sensitivity was higher when used for detecting antibodies;

[0032] 2. By adding CHAPS to the Derf3 recombinant protein refolding agent, the refolding effect of the Derf3 recombinant protein is further improved, its stability is improved, aggregation and precipitation are prevented, the yield is increased, and it has good immunoreactivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the SDS-PAGE electrophoresis diagram of the Derf1 recombinant protein in Example 1;

[0034] Figure 2 The SDS-PAGE electrophoresis diagram of the Derf2 recombinant protein in Example 1;

[0035] Figure 3 This is the SDS-PAGE electrophoresis diagram of the Derf3 recombinant protein in Example 1. DETAILED DESCRIPTION

[0036] To make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the art without making creative work are within the scope of protection of the present invention. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels.

[0037] Example 1: Preparation, purification and renaturation of dust mite antigens

[0038] 1. Preparation of recombinant protein

[0039] In this example, E. coli was used to express the recombinant proteins Derf1, Derf2 and Derf3, and the codons of the nucleotide sequences encoding Derf1, Derf2 and Derf3 were optimized to become codons preferred by E. coli. The optimized nucleotide sequences encoding Derf1, Derf2 and Derf3 were obtained as shown in the sequence listing SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively. His-Tag sequences were added to the carboxyl ends of the encoding nucleotide sequences, respectively, and artificial synthesis was performed to obtain the encoding genes of the recombinant Derf1, Derf2 and Derf3 proteins.

[0040] The pET28a prokaryotic expression vector was selected as the expression vector of the recombinant Derf1, Derf2, and Derf3 proteins, and the coding gene fragments were respectively inserted into the expression vector after enzyme digestion to complete the vector construction. The pET28a vector was digested with NcoI and then flattened, and then digested with BamHI, and the synthesized nucleotide coding sequences of Derf1, Derf2, and Derf3 were digested with BamHI. The digested plasmid was separated on a 1% agarose gel, and the target fragment was cut from the gel, and the target fragment was recovered by gel recovery using the gel recovery kit of Axygen. After recovering the DNA fragment, the pET28a vector fragment was connected to the nucleotide coding sequence of Derf1, Derf2, and Derf3 respectively with T4 DNA ligase. After the ligation reaction was completed, the recombinant plasmid was transformed into Escherichia coli DH5a cells by heat shock transformation. The cells of the selected monoclonal colony were added to the PCR reaction system, and the PCR reaction was performed on a Bio-Rad PCR instrument. PCR was used to identify the correct clones and test the correctness of the sequence. The correct clones were re-inoculated into LB medium with kanamycin and the recombinant plasmids were extracted.

[0041] The BL21 (DE3) expression strain was used to express the Derf1, Derf2 and Derf3 recombinant proteins, respectively. The recombinant plasmids were added to the competent cells of E. coli BL21 (DE3), placed on ice for 30 minutes, and the water bath was heated to 42°C for standby use. After being placed on ice for 30 minutes, the centrifuge tube containing E. coli BL21 (DE3) was placed in a 42°C water bath, heat-shocked for 90 seconds, and then the centrifuge tube was quickly inserted into ice and placed for 1-2 minutes. 1 ml of LB medium was added, and the centrifuge tube was placed in a shaker, and cultured at 37°C and 160 rpm for 1 hour. E. coli BL21 (DE3) was collected by centrifugation, spread on LB solid medium with kanamycin added, and the plate was placed in a 37°C biochemical incubator for overnight culture. On the second day, a single clone was picked and PCR was used to identify whether BL21 (DE3) had been transferred into the recombinant plasmid. The correct single clones were identified by PCR, transferred to LB medium containing kanamycin, and cultured in a shaker at 37℃ 250rpm. When the OD600 of the bacterial solution reaches 0.6-0.8, the bacterial solution is taken out, 80% sterile glycerol is added to the final concentration of 10%, and it is stored at -20℃ for later use. The glycerol-preserved BL21 (DE3) strain is inoculated into LB liquid medium containing kanamycin at a volume ratio of 1:1000, and the flask is continuously shaken at 37℃, 100-200rpm to the logarithmic phase. Centrifuge at 3500rpm, 4℃, 20min, and collect the centrifugal precipitate, which is the bacterial body.

[0042] 2. Purification of recombinant protein

[0043] After the bacteria are disrupted by ultrasonic disruption, inclusion bodies are obtained, and inclusion bodies formed by Derf1, Derf2, and Derf3 proteins are prepared respectively. The inclusion bodies are washed, and then dissolved with a denaturant (8M urea, 1mM EDTA-Na, 10-15mM mercaptoethanol (preferably 10mM), 20mM Tris-HCl), 1:40 (w / v)), and 16000ml of the denaturing solution is added to 400g of the inclusion bodies to obtain an inclusion body solution. Purification was performed using a 5 mL nickel column, and the process included equilibration, loading, washing, and elution: (1) Equilibration, i.e., using 25 mL of equilibration buffer (0.2% polyethylene glycol (PEG-200), 0.2% cystine, 10 mM PBS, 10 mM imidazole, and 5% glycerol, pH 7.4) to equilibrate the 5 mL nickel column; (2) Loading: Add the sample to the 5 mL nickel column, mix and incubate at 4 °C for 2 h, and collect the flow-through by centrifugation; (3) Washing: Add 25 mL of washing buffer (0.2% polyethylene glycol, 0.2% cystine, 10 mM PBS, 20 mM imidazole, and 5% glycerol, pH 7.4) to wash, and repeat the wash three times; (4) Elution: Add 25 mL of elution buffer (0.2% polyethylene glycol, 0.2% cystine, 10 mM PBS, 250 mM imidazole, and 5% glycerol, pH 7.4) Elution, mix and incubate for 10 minutes, and collect the eluted product by centrifugation. Take the flow-through, washing supernatant, and eluted product for SDS-PAGE electrophoresis detection. The electrophoresis diagram is shown in Figure 1~Figure 3 , are SDS-PAGE electrophoresis diagrams of Derf1, Derf2 and Derf3 recombinant proteins.

[0044] 3. Renaturation of recombinant protein

[0045] The eluted solutions containing Derf1, Derf2, and Derf3 recombinant proteins were collected respectively, and refolding agents were added for refolding. The refolding agent formulas of Derf1 and Derf2 recombinant proteins were: 0.2% polyethylene glycol, 0.2% cystine, 10 mM PBS, 10 mM imidazole, 5% glycerol, pH 8.0; the refolding agent formula of Derf3 recombinant protein was: 0.2% polyethylene glycol (PEG-200), 0.2% cystine, 0.2% CHAPS (CAS No.: 75621-03-3 ), 10mM PBS, 10 mM imidazole, 5% glycerol, pH7.4, diluted 4 times with refolding agent, and then mixed at 4℃ for 4h, then dialyzed with 5% glycerol solution (pH 7.4), and then concentrated through 10KD ultrafiltration tube, and then packaged and stored. The obtained Derf1, Derf2, and Derf3 recombinant protein solutions were tested by turbidity detector, and their turbidity reached 0.17, 0.16, and 0.17NTU respectively.

[0046] Bradford assay and data analysis showed that the total soluble protein in the inclusion body solution of Derf1 recombinant protein was 158 mg, and the Derf1 protein content in the refolding agent obtained after purification was 105 mg, with a yield of 66.5%.

[0047] For the Derf2 recombinant protein, the total soluble protein in the inclusion body solution was 149 mg, and the Derf2 protein content in the refolding agent obtained after purification was 101 mg, with a yield of 69.6%.

[0048] For the Derf3 recombinant protein, the total soluble protein in the inclusion body solution was 151 mg, and the Derf3 protein content in the refolding agent obtained after purification was 96 mg, with a yield of 63.5%.

[0049] The purity of the final Derf1, Derf2, and Derf3 recombinant proteins can reach about 95%.

[0050] The immune activity of the refolded Derf1, Derf2, and Derf3 recombinant proteins was verified by ELISA. The products obtained after purification and refolding were collected and used as coating antigens. An ELISA method was established to detect the reaction between the recombinant proteins and serum containing known standard concentrations of Derf1, Derf2, and Derf3 recombinant protein positive IgE, and the positive binding rate was calculated. The ELISA test operation process is as follows: (1) Prepare the antigen coating solution: Take the recombinant protein and dissolve it in 0.1M PBS buffer, the final concentration is 1μg / ml, to prepare coating solution; (2) Antigen coating: 100μl per well, coating temperature: 2-8℃ overnight, coating time: no less than 15 hours; (3) Prepare PBST washing solution and wash the plate, 300μl per well, wash twice, and pat dry; (4) Prepare blocking solution and block, add 300μl blocking solution to each well, the blocking temperature is 37℃, the blocking time is 2 hours, wash the plate 5 times, and pat dry; (5) Serum is added at a ratio of 1:4 Proportional dilution (sample diluent dilution), add 100 μl / well, incubate at 4°C overnight; (6) Wash 5 times with PBST and pat dry; (7) Mouse anti-human IgE-HRP (secondary antibody): 1:5000 dilution, 100 μl per well, incubate at 37°C for 1 hour; (8) Wash 5 times with PBST and pat dry; (9) Add substrate color development solution, 100 μl per well, color development for 15 minutes in the dark; (10) Add stop solution, 50 μl / well, and measure OD405 value with an enzyme reader. The same serum and the same recombinant allergen were tested three times and the mean value was calculated (mean ± SD). The OD value of negative serum was detected by this ELISA method, and the standards of Derf1-IgE, Derf2-IgE and Derf3-IgE with concentrations of 0, 0.1, 0.3, 0.5, 1, 3, 10, 30 and 100 IU / mL were prepared respectively. The minimum detection limits of Derf1-IgE, Derf2-IgE and Derf3-IgE when the recombinant proteins were used to detect Derf1-IgE, Derf2-IgE and Derf3-IgE respectively were calculated by the standard curve method. The detection sensitivity of the recombinant antigen and the detection accuracy when 10 batches of detection were repeated were analyzed, and its immunoactivity was evaluated.

[0051] After testing, the Derf1 recombinant antigen prepared in this example had a minimum detection limit of 0.15 IU / mL when used to detect Derf1-IgE, and an accuracy of 98.9%; the Derf2 recombinant antigen had a minimum detection limit of 0.15 IU / mL when used to detect Derf2-IgE, and an accuracy of 98.5%; the Derf3 recombinant antigen had a minimum detection limit of 0.20 IU / mL when used to detect Derf3-IgE, and an accuracy of 99.0%.

[0052] Example 2: Effects of different refolding agent formulations on the turbidity of the refolded protein solution and the yield of the target protein

[0053] In the early stage of the study, during the purification of Derf1, Derf2, and Derf3 recombinant proteins, the equilibration buffer formula was: 10mM PBS, 10mM imidazole and 5% glycerol, pH8.0; the washing buffer formula was: 10mM PBS, 20mM imidazole and 5% glycerol, pH7.4; the elution buffer formula was: 10mM PBS, 250mM imidazole, 5% glycerol, pH7.4; no other ingredients were added, the renaturation agent formula was: 10mM PBS, 10 mM imidazole, 5% glycerol, pH7.4, and the other methods were consistent with the examples. It was found that there were suspended matter in the sample solution eluted after nickel column purification, which may be due to the phenomenon of protein aggregation and precipitation. Similar phenomena also existed in the sample solution after renaturation. This example attempts to screen the refolding agent formula (the proportion of each component is consistent with that in Example 1) in the hope of improving this phenomenon and increasing the yield of the target protein. Different refolding agents as shown in Table 1 are used respectively. The detection results of Derf1 and Derf2 recombinant proteins are relatively close. The detection results of Derf1 are shown in Table 1.

[0054] Table 1. Effects of different renaturing agent formulations on the preparation of Derf1 recombinant protein

[0055]

[0056] According to Table 1, the use of different refolding agents has a significant impact on the turbidity and yield of the obtained sample solution. Comparison of Groups 1 to 7 shows that the addition of polyethylene glycol and cystine to the refolding agent at the same time significantly improves the effect compared to the addition of other polymers or other amino acids alone, which can significantly improve the protein precipitation and aggregation, thereby improving the yield, and can also improve the immunological activity of the target protein, reduce the minimum detection limit of the corresponding Derf1-IgE, and improve the detection sensitivity.

[0057] Comparing groups 7 to 10, it can be seen that the effects of adding different combinations are different. When CHAPS, polyethylene glycol and cystine are added at the same time, although the turbidity can be further reduced, the immunoreactivity of the recombinant protein prepared thereby decreases (the detection sensitivity decreases), and the yield also decreases. It is possible that the addition of CHAPS has an adverse effect on the purification of Derf1.

[0058] Therefore, the most preferred is the refolding agent formula of group 7, which maintains the turbidity and yield in a better range, and has a higher detection sensitivity for Derf1-IgE and better immunoreactivity.

[0059] This example also compares the effects of different renaturing agents on Derf3 recombinant protein. The test results are shown in Table 2.

[0060] Table 2. Effects of different renaturing agent formulations on the preparation of Derf3 recombinant protein

[0061]

[0062] It can be seen from Table 2 that different refolding agent formulas have a significant impact on the turbidity and yield of the Derf3 recombinant protein solution. Comparison of Groups 1 to 6 shows that the addition of polyethylene glycol and cystine to the refolding agent significantly improves the effect compared to the addition of other polymers or other amino acids, and can significantly improve the protein precipitation and aggregation.

[0063] At the same time, on this basis, CHAPS is further added, that is, Group 6. When CHAPS, polyethylene glycol and cystine are added at the same time, the turbidity of the Derf3 recombinant protein solution can be significantly reduced, the yield can be maximized, and the immunological activity of the target protein can be improved. The minimum detection limit of Derf3-IgE is lower, and the detection sensitivity is improved. It shows that there are differences in the effects of different renaturation agent formulas on recombinant proteins with different renaturation. The addition of CHAPS makes the detection sensitivity of Derf3 recombinant protein to Derf3-IgE higher. The reason may be that different renaturation agent formulas have different folding effects during the renaturation process of the target protein. CHAPS can expose more antigenic epitopes of the Derf3 recombinant protein, thereby improving the detection effect. Therefore, the most preferred is the renaturation agent formula of Group 6.

[0064] Example 3: Effect of the order of purification and renaturation on the turbidity of the protein solution and the yield of the target protein

[0065] This example compares the effects of the order of purification and renaturation on the turbidity and yield of Derf1, Derf2, and Derf3 recombinant protein solutions, using the following two methods: the first method: first add a renaturation agent for renaturation, and then perform nickel column purification; the second method: first perform nickel column purification and then add a renaturation agent for renaturation. During the purification of Derf1, Derf2, and Derf3 recombinant proteins, the balance buffer formula is: 10mM PBS, 10mM imidazole, and 5% glycerol, pH 7.4; the washing buffer formula is: 10mM PBS, 20mM imidazole, and 5% glycerol, pH 7.4; the elution buffer formula is: 10mM PBS, 250mM imidazole, 5% glycerol, pH 7.4; no other ingredients are added, and other methods are as in Example 1. Since the detection results of Derf1, Derf2, and Derf3 recombinant proteins have similar trends, this example uses the detection results of Derf3 recombinant protein for example, see Table 3.

[0066] Table 3. Effect of the order of purification and renaturation on the preparation of Derf3 recombinant protein

[0067]

[0068] According to Table 3, renaturation followed by purification, during the purification process, the turbidity of the sample solution increased significantly, the yield also decreased significantly, and the immune activity decreased, which was not conducive to the preparation of Derf3 recombinant protein. Therefore, the preferred operation sequence was purification followed by renaturation.

[0069] Example 4: Effect of buffer formulation on turbidity of protein solution and yield of target protein

[0070] According to the results of Example 3, it can be seen that the turbidity and yield of the sample solution in the purification process will continue to decrease. Therefore, this example considers improving the formulation of the equilibration buffer, washing buffer and elution buffer in the purification process, hoping to improve the stability of the Derf1, Derf2, and Derf3 recombinant proteins in the purification process, and further improve the turbidity and yield of the recombinant protein solution. This example attempts to add several components shown in Table 4 to the equilibration buffer, washing buffer and elution buffer formulations, respectively, with the same addition ratio as in Example 1, and other methods are the same as in Example 1. Since the detection results of Derf1, Derf2, and Derf3 recombinant proteins have similar trends, this example uses the detection results of Derf3 recombinant protein for example, see Table 4.

[0071] Table 4. Effects of different buffer components on the preparation of Derf3 recombinant protein

[0072]

[0073] According to Table 4, it can be seen that adding polyethylene glycol and cystine simultaneously to the equilibrium buffer, washing buffer and elution buffer in the purification process can significantly improve the turbidity of the obtained Derf3 recombinant protein solution, increase the yield, and will not affect the purification process. The reason may be that polyethylene glycol and cystine can make the prepared Derf3 recombinant protein always in a consistent ionic environment, which can improve its stability and is not easy to precipitate and aggregate. At the same time, it can also be seen that the effects of adding different components to the buffer are different. Further adding CHAPS on the basis of polyethylene glycol and cystine not only cannot continue to improve, but also significantly reduces the yield. The reason may be that the continued addition of CHAPS hinders the purification process. Therefore, it is most preferred to add polyethylene glycol and cystine simultaneously to the equilibrium buffer, washing buffer and elution buffer in the purification process, which can significantly improve the stability of the recombinant protein, increase the yield, and ensure immune activity. The minimum detection limit for detecting Derf3-IgE can reach 0.20 IU / mL.

[0074] At the same time, this example also verifies that the addition of polyethylene glycol and cystine to the equilibration buffer, washing buffer and elution buffer in the purification process also has obvious beneficial effects on the preparation of Derf1 and Derf2, and the turbidity can be reduced to about 0.17 NTU, the yield can reach more than 66%, and the minimum detection limit for detecting Derf1-IgE or Derf2-IgE can reach 0.15 IU / mL.

[0075] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A method for purifying and renaturing an antigen, characterized in that: The following steps are involved: (1) Collecting the fermentation medium containing the recombinant protein and adding a denaturant to denature and dissolve it; (2) Purification by chromatography column, wherein the chromatography column is first equilibrated with an equilibration buffer and then eluted with an elution buffer; (3) diluting with a refolding agent and dialyzing to obtain a recombinant antigen; the equilibration buffer, elution buffer and refolding agent all contain polyethylene glycol and cystine.

2. The method according to claim 1, characterized in that The refolding agent described in step (3) includes polyethylene glycol, cystine, PBS, imidazole and glycerol.

3. The method according to claim 2, characterized in that The denaturing agent in step (1) includes urea, EDTA-Na, mercaptoethanol and Tris-HCl solution.

4. The method according to claim 3, characterized in that The equilibration buffer and elution buffer in step (2) respectively comprise polyethylene glycol, cystine, PBS, imidazole and glycerol.

5. The method according to claim 4, characterized in that The chromatography column in step (2) is a Ni-NTA agarose chromatography column; it also includes a washing buffer for washing the chromatography column, and the washing buffer includes polyethylene glycol, cystine, imidazole, PBS and glycerol.

6. The method according to claim 5, characterized in that In the dialysis of step (3), the dialysis fluid used includes PBS and glycerol.

7. The method according to claim 1, characterized in that The recombinant antigens include any one or more of Derf1, Derf2, and Derf3; the amino acid sequences of the Derf1, Derf2, and Derf3 recombinant antigens are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

8. The method according to claim 7, characterized in that When the antigen is Derf3, the renaturing agent further comprises CHAPS.

9. Use of a composition for preparing an agent for improving the renaturation level of dust mite antigens, characterized in that: The composition includes polyethylene glycol and cystine, and the dust mite antigen includes any one or more of Derf1, Derf2, and Derf3; the amino acid sequences of the Derf1, Derf2, and Derf3 recombinant antigens are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

10. Use of a composition for preparing a reagent for improving the renaturation level of dust mite antigen Derf3, characterized in that: The composition includes polyethylene glycol, cystine and CHAPS.

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