A method for purifying and refolding an antigen

Abstract

The present invention provides a method for purifying and refolding an antigen. By improving the refolding agent formulation and the process routes for purification and refolding, chromatography column purification is first completed after denaturation and then refolding is carried out. Polyethylene glycol and cystine are contained in the equilibration buffer, washing buffer, elution buffer and refolding agent, so as to solve the problem that the recombinant protein is prone to aggregation and precipitation after refolding. For recombinant proteins with relatively weak stability, CHAPS is further added to improve the refolding effect. The prepared recombinant protein is more stable, prevents aggregation and precipitation, has a high yield, has good immunological activity, and improves the detection sensitivity when used for detecting antibodies.

Description

Technical Field

[0001] The present invention relates to the technical field of antigen preparation, and particularly to a method for purifying and refolding an antigen. Background Art

[0002] In the preparation process of recombinant antigens, purification and refolding are also very important steps. By adopting appropriate purification and refolding processes, not only can the yield of antigens be improved, but it may even affect the immunological activity of antigens, which has an important impact on the later application of recombinant antigens.

[0003] Allergens are also a kind of important antigens. Identifying allergens is the key to preventing and treating allergic diseases. Avoiding contact with allergens and carrying out specific immunotherapy both rely on allergen diagnosis. The dust mite allergens Der f 1 (abbreviated as Derf1) and Der f 2 (abbreviated as Derf2) are the main sensitizing components. The dust mite allergen Der f 3 (abbreviated as Derf3) also plays an important role in the sensitization process and can also mediate the body to produce allergic reactions. Different allergy 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, so as to truly achieve the patient's tolerance to specific dust mite allergens. In order to distinguish which dust mite allergen specifically causes the allergic reaction in the patient and thus carry out targeted treatment, it is often necessary to prepare recombinant antigens of Derf1, Derf2 and Derf3 for detecting IgE antibodies of 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 rapidly released and randomly entangled to form insoluble proteins. It is necessary to undergo in vitro folding and refolding to obtain biological activity. Existing conventional refolding methods include stepwise dialysis method, gradual dilution method of high-concentration denaturant, chromatographic separation method, large-volume dilution method, etc. However, in the above conventional methods for the recombinant Derf1, Derf2 and Derf3 proteins, the proteins cannot stably exist after refolding and are prone to aggregation and precipitation.

[0005] Therefore, there is an urgent need to find a method for preparing recombinant proteins of Derf1, Derf2 and Derf3, with high yields, good refolding and good immunoreactivity of the three prepared antigens, providing a basis for quantitatively detecting 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 refolding an antigen. By improving the refolding agent formulation and the process routes of purification and refolding, chromatography column purification is first completed after denaturation and then refolding is carried out. Polyethylene glycol and cystine are contained in the equilibration buffer, washing buffer, elution buffer and refolding agent, so as to solve the problem that the recombinant proteins Derf1, Derf2 and Derf3 are prone to aggregation and precipitation after refolding. For Derf3, CHAPS is further added to improve the refolding effect. The prepared recombinant proteins Derf1, Derf2 and Derf3 are more stable, prevent aggregation and precipitation, have a high yield, and have good immunological activity, thereby improving the detection sensitivity when used for detecting antibodies.

[0007] On the one hand, the present invention provides a method for purifying and refolding an antigen, and the method comprises the following steps:

[0008] (1) Collecting a fermentation medium containing a recombinant protein, and adding a denaturing agent for denaturation and dissolution;

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

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

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

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

[0013] Previous experiments have proved that after the recombinant proteins Derf1, Derf2 and Derf3 are expressed in Escherichia coli in the form of inclusion bodies, the protein products obtained by breaking, denaturing and refolding the target proteins are prone to aggregation and precipitation, resulting in problems such as a decrease in the final protein yield and difficulty in exposing antigenic epitopes. Therefore, it is necessary to optimize the purification and refolding processes to improve the immunological activity and stability of the recombinant proteins Derf1, Derf2 and Derf3.

[0014] Compared with the refolded protein products, the denatured recombinant Derf1, Derf2, and Derf3 proteins have better solubility and are more stable. Therefore, it is preferred to perform purification first and then refolding. However, for denatured recombinant proteins, there may occasionally be problems of protein precipitation and aggregation, resulting in a decrease in immunological activity and yield. Therefore, the present invention also optimizes the equilibration buffer and elution buffer during the purification process, completely solving the problem of protein precipitation and aggregation of recombinant Derf1, Derf2, and Derf3 proteins during ultrafiltration and purification, and improving the immunological activity of the recombinant proteins.

[0015] For the denatured protein products, a suitable refolding agent needs to be screened to improve the immunological activity and long-term stability of the recombinant proteins. The present invention optimizes the components in the refolding agent formulation, effectively improving the stability of the refolded recombinant Derf1, Derf2, and Derf3 proteins, ensuring that the proteins do not precipitate, and exposing more antigenic epitopes, thus having good immunological activity and improving the detection sensitivity when detecting the corresponding antibodies.

[0016] After screening a suitable refolding agent, the present invention also adjusts the purification and refolding steps, performing refolding after completing all the purification steps. At the same time, polyethylene glycol and cystine are added to the equilibration buffer, elution buffer, and refolding agent, thereby improving the stability of the target protein, preventing protein aggregation during chromatography, and since the molecular weights of polyethylene glycol and cystine are small, they will not be retained by 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 their own properties. Polyethylene glycol has a molecular exclusion effect, which can reduce the aggregation between protein molecules. Denatured recombinant proteins are prone to aggregate and precipitate with each other, and polyethylene glycol can occupy the space in the solution, separating protein molecules from each other and reducing the probability of forming aggregates by mutual collision. Cystine may help the protein maintain its activity, enabling more antigenic epitopes to be exposed during the refolding process of the target protein, thereby improving the detection effect on antibodies. Therefore, adding polyethylene glycol and cystine during the chromatography process and refolding process can significantly improve the stability of recombinant Derf1, Derf2, and Derf3 proteins and maintain the activity of the proteins.

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

[0019] Further, the denaturing agent described in step (1) includes urea, EDTA-Na, mercaptoethanol, and Tris-Cl solution.

[0020] Further, both the equilibration buffer and elution buffer described in step (2) each include polyethylene glycol, cystine, imidazole, PBS, and glycerol.

[0021] Further, the chromatography column described in step (2) is a Ni-NTA agarose chromatography column; it further 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 recombinant proteins Derf1, Derf2, and Derf3 can always be in the same ionic environment, which helps improve protein stability and prevent the influence on protein activity.

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

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

[0025] Further, when the dust mite antigen is Derf3, the renaturant further includes CHAPS.

[0026] Since the stability of the Derf3 recombinant protein is relatively weaker than that of the Derf1 and Derf2 recombinant proteins, in order to improve the renaturation effect, it is preferable to add polyethylene glycol, cystine, and CHAPS to the renaturant. Compared with the Derf1 and Derf2 recombinant proteins, CHAPS is added. The reason may be that CHAPS has a solubilizing effect, which helps the Derf3 recombinant protein maintain a dispersed and active state in the solution, reduces the non-specific electrostatic interaction with other biomolecules, and also helps its folding to form more disulfide bonds, thereby promoting its renaturation.

[0027] On the other hand, the present invention provides the use of a composition for preparing a reagent for improving the renaturation level of a dust mite antigen. 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 recombinant antigens Derf1, Derf2, and Derf3 are shown as SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 respectively.

[0028] The renaturation level of the antigen described in the present invention includes improving the immunological activity, stability, and yield of the recombinant antigen, etc. The immunological activity includes detection sensitivity and accuracy when detecting the corresponding antibody, etc.

[0029] On the other hand, the present invention provides the use of a composition for preparing a reagent for increasing the refolding level of dust mite antigen Derf3, and the composition comprises polyethylene glycol, cystine and CHAPS.

[0030] Beneficial effects achieved by the present invention:

[0031] 1. Appropriate refolding agents are screened, and the process routes of purification and refolding are improved. After denaturation, column purification is first completed and then refolding is carried out. Polyethylene glycol and cystine are contained in the ultrafiltration equilibration solution, equilibration buffer and refolding agent, solving the problem that the recombinant proteins Derf1, Derf2 and Derf3 are prone to aggregation and precipitation after refolding. The prepared recombinant proteins Derf1, Derf2 and Derf3 are more stable, with high yield and high purity, and have good immunoreactivity, and the detection sensitivity is higher when used for detecting antibodies;

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

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

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

[0035] Figure 3 It is the SDS-PAGE electrophoresis diagram of the Derf3 recombinant protein in Example 1. Detailed implementation manners

[0036] To make the objectives, 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 with reference to the drawings of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention. Reagents or instruments not indicating the manufacturer can be all conventional products commercially available through regular channels.

[0037] Example 1: Preparation, purification and refolding of dust mite antigen

[0038] I. Preparation of recombinant protein

[0039] In this embodiment, Escherichia coli was used to express the recombinant proteins Derf1, Derf2, and Derf3. The codons of the nucleotide sequences encoding Derf1, Derf2, and Derf3 were optimized to the codons preferred by Escherichia coli. The optimized nucleotide sequences encoding Derf1, Derf2, and Derf3 obtained the nucleotide sequences shown in Sequence Listing SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 respectively, and a His-Tag sequence was added to the carboxyl terminus of their encoding nucleotide sequences, followed by artificial synthesis 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 for the recombinant Derf1, Derf2, and Derf3 proteins. Their encoding gene fragments were digested with enzymes and inserted into the expression vector respectively to complete the vector construction. The pET28a vector was digested with NcoI, filled in, and then digested with BamHI. At the same time, the synthetic nucleotide coding sequences of Derf1, Derf2, and Derf3 were digested with BamHI. The digested plasmids were separated on a 1% agarose gel, and the target fragments were cut from the gel. The gel recovery kit from Axygen Company was used to recover the target fragments from the gel. After recovering the DNA fragments, the pET28a vector fragments were ligated with the nucleotide coding sequences of Derf1, Derf2, and Derf3 respectively using T4 DNA ligase. After the ligation reaction was completed, the recombinant plasmids were transformed into Escherichia coli DH5a cells by heat shock transformation. The cells of the selected monoclonal colonies were added to the PCR reaction system, and the PCR reaction was carried out on a Bio-Rad PCR instrument. The monoclonal colonies with correct PCR identification were used to detect the correctness of the sequence. The monoclonal colonies with correct sequencing were re-inoculated into LB medium supplemented with kanamycin for culture, and the recombinant plasmids were extracted.

[0041] Use the BL21(DE3) expression strain to express Derf1, Derf2, and Derf3 recombinant proteins respectively. Add the recombinant plasmids into Escherichia coli BL21(DE3) competent cells, place them on ice for 30 minutes, and heat the water bath to 42 °C for standby. After placing on ice for 30 minutes, put the centrifuge tube containing Escherichia coli BL21(DE3) into the 42 °C water bath for heat shock for 90 sec, and then quickly insert the centrifuge tube into the ice and place it for 1 - 2 minutes. Add 1 ml of LB medium, place the centrifuge tube on a shaker, and culture at 37 °C and 160 rpm for 1 hour. Centrifuge to collect Escherichia coli BL21(DE3), spread it on the LB solid medium added with kanamycin, and place the plate in a 37 °C biochemical incubator to culture overnight. The next day, pick monoclonal colonies and use the PCR method to identify whether the recombinant plasmid has been transferred into BL21(DE3). For the monoclonal colonies with correct PCR identification, transfer them to the LB medium containing kanamycin and culture at 37 °C and 250 rpm in a shaker. When the OD600 of the bacterial solution reaches 0.6 - 0.8, take out the bacterial solution, add sterile glycerol at 80% to a final concentration of 10%, and store it at -20 °C for standby. Inoculate the glycerol-preserved BL21(DE3) strain into the LB liquid medium containing kanamycin at a volume ratio of 1:1000, and continuously shake-flask culture at 37 °C and 100 - 200 rpm until the logarithmic phase. Centrifuge at 3500 rpm, 4 °C for 20 min, and collect the centrifugation precipitate, which is the bacterial cells.

[0042] II. Purification of Recombinant Proteins

[0043] After disrupting the bacterial cells by ultrasonic fragmentation, inclusion bodies were obtained. Inclusion bodies formed by Derf1, Derf2, and Derf3 proteins were prepared separately. The inclusion bodies were washed, and then dissolved with a denaturing agent (8M urea, 1mM EDTA-Na, 10 - 15mM mercaptoethanol (preferably 10mM), 20mM Tris-HCl, 1:40 (w / v)). 16000 ml of the denaturing solution was added to 400 g of inclusion bodies to obtain an inclusion body solution. Purification was carried out using a 5 mL nickel column, and the process included equilibration, sample loading, washing of impurities, and elution: (1) Equilibration: Equilibrate a 5 mL nickel column with 25 ml of equilibration buffer (0.2% polyethylene glycol (PEG-200), 0.2% cystine, 10mM PBS, 10mM imidazole, and 5% glycerol, pH 7.4); (2) Sample loading: Add the sample to the 5 mL nickel column, mix well and incubate at 4°C for 2 h, and centrifuge to collect the flow-through; (3) Washing of impurities: Wash with 25 mL of washing buffer (0.2% polyethylene glycol, 0.2% cystine, 10mM PBS, 20mM imidazole, and 5% glycerol, pH 7.4), and repeat the washing 3 times; (4) Elution: Add 25 mL of elution buffer (0.2% polyethylene glycol, 0.2% cystine, 10mM PBS, 250mM imidazole, 5% glycerol, pH 7.4) for elution, mix well and incubate for 10 min, and centrifuge to collect the elution product. Take the flow-through, washing supernatant, and elution product for SDS-PAGE electrophoresis detection. The electrophoresis pattern is shown in Figures 1 to 3 , which are the SDS-PAGE electrophoresis patterns of Derf1, Derf2, and Derf3 recombinant proteins respectively.

[0044] III. Renaturation of Recombinant Proteins

[0045] Solutions containing Derf1, Derf2, and Derf3 recombinant proteins obtained by elution were collected separately and added with a renaturing agent for renaturation. Among them, the renaturing agent formulations for Derf1 and Derf2 recombinant proteins are both: 0.2% polyethylene glycol, 0.2% cystine, 10mM PBS, 10 mM imidazole, 5% glycerol, pH 8.0; the renaturing agent formulation for Derf3 recombinant protein is: 0.2% polyethylene glycol (PEG-200), 0.2% cystine, 0.2% CHAPS (CAS number: 75621-03-3 ), 10mM PBS, 10 mM imidazole, 5% glycerol, pH 7.4. Dilute with the renaturing agent by 4 times respectively, then mix well at 4°C for 4 h, then dialyze with a 5% glycerol solution (pH 7.4), and then concentrate through a 10KD ultrafiltration tube. After concentration, the concentrated solution was aliquoted and stored. The prepared Derf1, Derf2, and Derf3 recombinant protein solutions were detected by a turbidity detector, and their turbidities reached 0.17, 0.16, and 0.17 NTU respectively.

[0046] After detection by Bradford method and data analysis, for the Derf1 recombinant protein, the total soluble protein in the inclusion body solution was 158 mg, the content of Derf1 protein in the refolding agent obtained after purification was 105 mg, and the yield was 66.5%.

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

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

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

[0050] The immunological activities of the refolded recombinant proteins Derf1, Derf2, and Derf3 were verified by ELISA. The products obtained after purification and refolding were collected. Using these recombinant proteins as coated antigens, an ELISA method was established to detect their reaction with sera containing known standard concentrations of the recombinant proteins Derf1, Derf2, and Derf3 positive for IgE, and the positive binding rate was calculated. The ELISA detection operation process was as follows: (1) Prepare the antigen coating solution: Absorb the recombinant protein and dissolve it in 0.1 M PBS buffer to a final concentration of 1 μg / ml to make the coating solution; (2) Antigen coating: Coat 100 μl per well, coating temperature: 2 - 8°C overnight, coating time: not less than 15 hours; (3) Prepare PBST washing solution and wash the plate, 300 μl per well, wash 2 times, and pat dry; (4) Prepare the blocking solution and perform blocking, add 300 μl of blocking solution per well, blocking temperature is 37°C, blocking time is 2 hours, wash the plate 5 times, and pat dry; (5) Dilute the serum at a ratio of 1:4 (diluted with sample diluent), add 100 μl / well, and incubate overnight at 4°C; (6) Wash with PBST 5 times and pat dry; (7) Mouse anti-human IgE-HRP (secondary antibody): Dilute 1:5000, add 100 μl per well, and incubate at 37°C for 1 h; (8) Wash with PBST 5 times and pat dry; (9) Add the substrate chromogenic solution, 100 μl per well, and develop color in the dark for 15 min; (10) Add the stop solution, 50 μl / well, and measure the OD405 value with an enzyme-linked immunosorbent assay reader. The same serum was measured three times with the same recombinant allergen, and the (mean ± SD) was calculated. The OD values of negative sera were detected by this ELISA method, and 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 the recombinant proteins Derf1, Derf2, and Derf3 for detecting Derf1-IgE, Derf2-IgE, and Derf3-IgE were calculated by the standard curve method, the detection sensitivity of this recombinant antigen was analyzed, and the detection accuracy during 10 batches of repeated detections was evaluated to assess its immunological activity.

[0051] After detection, for the Derf1 recombinant antigen prepared in this example, the minimum detection limit for detecting Derf1-IgE reached 0.15 IU / mL, and the accuracy reached 98.9%; for the Derf2 recombinant antigen, the minimum detection limit for detecting Derf2-IgE reached 0.15 IU / mL, and the accuracy reached 98.5%; for the Derf3 recombinant antigen, the minimum detection limit for detecting Derf3-IgE reached 0.20 IU / mL, and the accuracy reached 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 initial stage of the research, during the purification process of Derf1, Derf2, and Derf3 recombinant proteins, the formulation of the equilibration buffer was: 10 mM PBS, 10 mM imidazole, and 5% glycerol, pH 8.0; the formulation of the washing buffer was: 10 mM PBS, 20 mM imidazole, and 5% glycerol, pH 7.4; the formulation of the elution buffer was: 10 mM PBS, 250 mM imidazole, 5% glycerol, pH 7.4; no other components were added. The formulation of the renaturant was: 10 mM PBS, 10 mM imidazole, 5% glycerol, pH 7.4. Other methods were the same as those in the examples. It was found that there were suspended substances in the sample solution eluted after nickel column purification, which might be the phenomenon of protein aggregation and precipitation. A similar phenomenon also existed in the renatured sample solution. In this example, an attempt was made to screen the renaturant formulation (the proportion of each component was the same as that in Example 1) in the hope of improving this phenomenon and also hoping to increase the yield of the target protein. Different renaturants shown in Table 1 were used respectively. The detection results of Derf1 and Derf2 recombinant proteins were relatively close, and the detection results of Derf1 are shown in Table 1.

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

[0055]

[0056] As can be seen from Table 1, using different renaturants had obvious effects on the turbidity and yield of the obtained sample solution. By comparing groups 1 - 7, it can be seen that adding polyethylene glycol and cystine to the renaturant simultaneously significantly improved the effect compared with adding other polymers or other amino acids alone. It could significantly improve the protein precipitation and aggregation situation, thereby increasing the yield, improving the immunoreactivity of the target protein, reducing the lowest detection limit of the corresponding Derf1 - IgE, and improving the detection sensitivity.

[0057] By comparing groups 7 - 10, it can be seen that adding different compositions also had different effects. When adding CHAPS, polyethylene glycol, and cystine simultaneously, although the turbidity could be further reduced, the immunoreactivity of the prepared recombinant protein decreased (the detection sensitivity decreased), and the yield also decreased. It was possible that the addition of CHAPS had an adverse effect on the purification of Derf1.

[0058] Therefore, the most preferred renaturant formulation was that of group 7, which maintained the turbidity and yield in a relatively optimal range, had a higher detection sensitivity for Derf1 - IgE, and better immunoreactivity.

[0059] This example also compared the effects of different renaturants on Derf3 recombinant protein, and the detection results are shown in Table 2.

[0060] Table 2. Effects of Different Renaturant Formulations on the Preparation of Derf3 Recombinant Protein

[0061]

[0062] As can be seen from comparing Table 2, there are obvious effects on the turbidity and yield of the Derf3 recombinant protein solution obtained with different renaturant formulations. By comparing groups 1 - 6, it can be seen that adding polyethylene glycol and cystine to the renaturant significantly improves the effect compared to adding other polymers or other amino acids, and can significantly improve the aggregation of protein precipitation.

[0063] Meanwhile, on this basis, further adding CHAPS, that is, group 6, when adding CHAPS, polyethylene glycol and cystine simultaneously, can significantly reduce the turbidity of the Derf3 recombinant protein solution, achieve the highest yield, and can also improve the immunoreactivity of the target protein, with a lower minimum detection limit for Derf3 - IgE, improving the detection sensitivity. It shows that using different renaturant formulations has different effects on the renaturation of different recombinant proteins. The addition of CHAPS makes the detection sensitivity of Derf3 recombinant protein to Derf3 - IgE higher. The reason may be that different renaturant formulations result in different folding effects during the renaturation of the target protein, and CHAPS can expose more antigenic epitopes of the Derf3 recombinant protein, thus improving the detection effect. Therefore, the most preferred is the renaturant formulation of group 6.

[0064] Example 3. Effects of the Order of Purification and Renaturation on the Turbidity of Protein Solution and the Yield of Target Protein

[0065] This example compares the effects of the order of purification and renaturation operations on the turbidity and yield of Derf1, Derf2, and Derf3 recombinant protein solutions, using the following two methods respectively: The first: After adding the renaturant for renaturation first, then perform nickel column purification; The second: First perform nickel column purification and then add the renaturant for renaturation. During the purification process of Derf1, Derf2, and Derf3 recombinant proteins, the formulation of the equilibration buffer is: 10 mM PBS, 10 mM imidazole, and 5% glycerol, pH 7.4; The formulation of the washing buffer is: 10 mM PBS, 20 mM imidazole, and 5% glycerol, pH 7.4; The formulation of the elution buffer is: 10 mM PBS, 250 mM imidazole, 5% glycerol, pH 7.4; No other components are added, and other methods are as in Example 1. Since the detection result trends of Derf1, Derf2, and Derf3 recombinant proteins are similar, the detection results of Derf3 recombinant protein are used for illustration in this example, as shown in Table 3.

[0066] Table 3. Effects of the Order of Purification and Renaturation on the Preparation of Derf3 Recombinant Protein

[0067]

[0068] According to Table 3, after renaturation and then purification, during the purification process, the turbidity of the sample solution increased significantly, the yield decreased significantly, and the immunological activity decreased, which was not conducive to the preparation of the Derf3 recombinant protein. Therefore, it is preferred to adopt the operation sequence of purification first and then renaturation.

[0069] Example 4. Influence of buffer formulation on the turbidity of the protein solution and the yield of the target protein

[0070] It can be seen from the results of Example 3 that the turbidity and yield of the sample solution in the purification process will continue to decrease. Therefore, in this example, it is considered to improve the formulations of the equilibration buffer, washing buffer, and elution buffer during the purification process, hoping to improve the stability of the Derf1, Derf2, and Derf3 recombinant proteins during the purification process, and further improve the turbidity and recovery rate of the recombinant protein solution. In this example, several components shown in Table 4 were simultaneously added to the formulations of the equilibration buffer, washing buffer, and elution buffer respectively, and the addition ratio was the same as that in Example 1, and other methods were the same as those in Example 1. Since the detection result trends of the Derf1, Derf2, and Derf3 recombinant proteins were similar, the detection results of the Derf3 recombinant protein were used for illustration in this example, as shown in Table 4.

[0071] Table 4. Influence of different buffer additives on the preparation of the Derf3 recombinant protein

[0072]

[0073] It can be seen from Table 4 that when polyethylene glycol and cystine are simultaneously added to the equilibration buffer, washing buffer, and elution buffer during the purification process, the turbidity of the obtained Derf3 recombinant protein solution can be significantly improved, the yield can be increased, and it will not affect the purification process. The reason may be that polyethylene glycol and cystine can keep the prepared Derf3 recombinant protein in a consistent ionic environment all the time, 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 addition of CHAPS hinders the purification process. Therefore, the most preferred is to simultaneously add polyethylene glycol and cystine to the equilibration buffer, washing buffer, and elution buffer during the purification process, which can significantly improve the stability of the recombinant protein, increase the yield, and ensure the immunological activity, and the lowest detection limit can reach 0.20 IU / mL when detecting Derf3-IgE.

[0074] Meanwhile, this embodiment also verified that adding polyethylene glycol and cystine simultaneously to the equilibration buffer, washing buffer, and elution buffer during the purification process also has obvious beneficial effects on the preparation of Derf1 and Derf2. The turbidity can be reduced to about 0.17 NTU, the yield can reach over 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 should be subject to the scope defined by the claims.

Claims

1. A method for purifying and renaturing dust mite antigen Derf3 to improve the detection sensitivity of Derf3 antibody, 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 renaturing agent and dialyzing to obtain a recombinant antigen; The equilibration buffer consists of 0.2% polyethylene glycol, 0.2% cystine, 10 mM PBS, 10 mM imidazole and 5% glycerol; the elution buffer consists of 0.2% polyethylene glycol, 0.2% cystine, 10 mM PBS, 250 mM imidazole and 5% glycerol; the refolding agent consists of 0.2% polyethylene glycol, 0.2% cystine, 0.2% CHAPS, 10 mM PBS, 10 mM imidazole and 5% glycerol; The chromatography column in step (2) is a Ni-NTA agarose chromatography column; and further comprises a washing buffer for washing the chromatography column, wherein the washing buffer is composed of 0.2% polyethylene glycol, 0.2% cystine, 10 mM PBS, 20 mM imidazole and 5% glycerol; The amino acid sequence of the Derf3 recombinant antigen is shown in SEQ ID NO.

3.

2. The method according to claim 1, characterized in that In the dialysis of step (3), the dialysis fluid used includes PBS and glycerol.

3. A method for purifying and renaturing dust mite antigens to improve antibody detection sensitivity, 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 renaturing agent and dialyzing to obtain a recombinant antigen; The equilibration buffer consists of 0.2% polyethylene glycol, 0.2% cystine, 10 mM PBS, 10 mM imidazole and 5% glycerol; the elution buffer consists of 0.2% polyethylene glycol, 0.2% cystine, 10 mM PBS, 250 mM imidazole and 5% glycerol; the refolding agent consists of 0.2% polyethylene glycol, 0.2% cystine, 10 mM PBS, 10 mM imidazole and 5% glycerol; The chromatography column in step (2) is a Ni-NTA agarose chromatography column; it also includes a washing buffer for washing the chromatography column, the washing buffer is composed of 0.2% polyethylene glycol, 0.2% cystine, 10mM PBS, 20mM imidazole and 5% glycerol; the dust mite antigen includes Derf1 or Derf2; the amino acid sequences of the Derf1 and Derf2 recombinant antigens are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

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

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