Method for purifying vjbr protein and use thereof

Abstract

This invention provides a method for purifying VjbR protein and its applications, relating to the field of protein purification technology. The purification method for VjbR protein provided by this invention effectively improves the purity and yield of VjbR protein by adding specific concentrations of reducing agent and surfactant to the equilibration buffer and elution buffer of Ni affinity chromatography, and adding specific concentrations of surfactant to the equilibration buffer and elution buffer of ion exchange chromatography. Furthermore, the purified VjbR protein is stable, does not easily precipitate, and can be stored for a long time, providing a foundation for the development of diagnostic technologies for brucellosis.

Description

Technical Field

[0001] This invention relates to the field of protein purification technology, and in particular to a method for purifying VjbR protein and its application. Background Technology

[0002] Brucellosis, commonly known as Brucella infection, is caused by infection with the Gram-negative, intracellular parasitic bacillus Brucella. It is a zoonotic disease that can cause abortion and fever in animals, seriously threatening livestock production and human health. The VjbR protein influences Brucella's virulence and intracellular parasitic ability by regulating the expression of various Brucella genes. Belonging to the LuxR protein family, the VjbR protein is a crucial component of the Brucella Quorum Sensing (QS) system. The QS system is a mechanism by which bacteria sense their own population density and regulate gene expression, enabling them to adjust their physiological behavior and virulence based on signals from their surrounding environment. When Brucella infects and parasitizes macrophages in host cells, the VjbR protein regulates Brucella's target genes, helping it adapt to harsh intracellular environments such as high salt, hyperosmolarity, acid, heat shock, and oxygen stress, thereby enhancing the bacteria's resistance and survival ability. In addition, the VjbR protein also participates in regulating Brucella's replication and spread within host cells, enabling it to establish chronic infection within the host.

[0003] The VjbR protein, a transcriptional activator of the QS system, influences the expression of various Brucella genes, including the type IV secretion system (T4SS) and flagellar genes. VjbR regulates the type IV secretion system by modulating the expression of the VirB operon, exhibiting a positive regulatory relationship with it. VjbR also regulates the expression of other Brucella virulence-related genes, such as flagellar genes and cell surface structure-related genes, thereby modulating Brucella virulence. Due to the specific expression of VjbR protein in Brucella, antibody detection technologies based on VjbR protein can be developed. Studies have found that serum from naturally infected cattle and sheep contains antibodies against the VjbR protein; therefore, detecting specific antibodies against the VjbR protein in serum can aid in the diagnosis of brucellosis. Therefore, the preparation of high-purity VjbR protein is crucial for the development of diagnostic methods. Currently, VjbR protein is mainly prepared using the *E. coli* expression system. This involves cloning the VjbR gene into appropriate prokaryotic expression vectors, such as pET-30a and pET-28a, and inducing expression to obtain the VjbR protein. Although prokaryotic expression methods for VjbR exist in the published literature, the reported purity of the VjbR protein is only 65.7%, and no description of a VjbR protein preparation method is found in the published patents. Because it is expressed through recombinant *E. coli*, the presence of unremoved *E. coli* host proteins in serum antibody detection can lead to significant background absorption, resulting in false positives. Furthermore, *E. coli*-expressed VjbR protein often exists in inclusion body form; purification after urea denaturation causes the protein to lose its native conformation, which also affects subsequent applications.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The primary objective of this invention is to provide a method for purifying VjbR protein.

[0006] A second objective of this invention is to provide the application of the above-described purification method in the preparation of diagnostic products for brucellosis.

[0007] To achieve the above objectives, the following technical solution is adopted:

[0008] In a first aspect, the present invention provides a method for purifying VjbR protein, comprising the following steps:

[0009] The solution containing VjbR protein was diluted with Ni affinity chromatography equilibration buffer and then subjected to Ni affinity chromatography. The VjbR protein eluent was collected and then subjected to ion exchange chromatography to obtain purified VjbR protein.

[0010] The equilibration buffer and elution buffer of the Ni affinity chromatography each contain 2-10 mM of reducing agent and 0.2% vol-1.5% vol of surfactant.

[0011] The equilibration buffer and elution buffer of the ion exchange chromatography each contain 0.2% vol to 0.8% vol of surfactant independently;

[0012] The VjbR protein carries a His tag.

[0013] As a further technical solution, in the equilibration buffer and elution buffer of the Ni affinity chromatography, the reducing agent is DTT or TCEP, preferably DTT, and the surfactant is Tween 20 or Tween 80, preferably Tween 20.

[0014] Preferably, the equilibration buffer and elution buffer for Ni affinity chromatography each contain 5 mM DTT and 0.5% vol Tween 20 independently.

[0015] As a further technical solution, the equilibration buffer for Ni affinity chromatography also contains 40–60 mM Tris-HCl and 0.1–0.2 M NaCl;

[0016] The elution buffer for Ni affinity chromatography also contains 40–60 mM Tris-HCl, 0.1–0.2 M NaCl, and 450–550 mM imidazole.

[0017] As a further technical solution, the dilution factor is 3 to 5 times;

[0018] And / or, the chromatography column for Ni affinity chromatography includes GE Ni Sepharose. TM 6FF.

[0019] As a further technical solution, the surfactant in the equilibration buffer and elution buffer of the ion exchange chromatography is Tween 20 or Tween 80, preferably Tween 20;

[0020] Preferably, the equilibration buffer and elution buffer of the ion exchange chromatography each contain 0.5% vol of Tween 20 independently.

[0021] As a further technical solution, the equilibration buffer for the ion exchange chromatography also contains 40–60 mM Tris-HCl;

[0022] The elution buffer for the ion exchange chromatography also contains 40–60 mM Tris-HCl and 0.4–0.6 M NaCl.

[0023] As a further technical solution, the chromatography column for the ion exchange chromatography includes Cytiva QHP.

[0024] As a further technical solution, the solution containing VjbR protein includes a lysate of cells expressing VjbR protein;

[0025] Preferably, the cells comprise Escherichia coli.

[0026] As a further technical solution, the method for preparing the lysate of cells expressing VjbR protein includes: fermenting engineered bacteria expressing VjbR protein, collecting the fermenting bacteria, and then sequentially performing cell disruption and solid-liquid separation to obtain the lysate of cells expressing VjbR protein.

[0027] Secondly, the present invention provides the application of the above-mentioned purification method in the preparation of diagnostic products for brucellosis.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The purification method for VjbR protein provided by this invention effectively improves the purity and yield of VjbR protein by adding specific concentrations of reducing agent and surfactant to the equilibration buffer and elution buffer of Ni affinity chromatography, and adding specific concentrations of surfactant to the equilibration buffer and elution buffer of ion exchange chromatography. Furthermore, the purified VjbR protein is stable, does not easily precipitate, and can be stored for a long time, providing a foundation for brucellosis diagnosis technology. Attached Figure Description

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 SDS-PAGE electrophoresis image of VjbR protein fermentation results;

[0032] Figure 2SDS-PAGE results for Ni affinity chromatography and ion exchange chromatography of the supernatant of disrupted bacterial cells (Left panel: Ni affinity chromatography: Lane 1 (leftmost lane) - supernatant of ultrasonically disrupted bacterial cells, Lane 2 - supernatant of ultrasonically disrupted bacterial cells passed through a 0.22 μm filter membrane, Lanes 3, 4, 5, 7, 8, 9 - samples eluted under different gradients, Lane 6 - protein marker; Right panel: Ion exchange chromatography: Lane 1 (leftmost lane) - protein marker, Lane 2 - sample diluted 4-fold by 50% gradient elution of Ni affinity chromatography, Lane 3 - flow-through sample, Lanes 4-10 - samples with segmented linear elution peaks).

[0033] Figure 3 SDS-PAGE results optimized for Ni affinity chromatography (lane 1 (leftmost lane) - supernatant from ultrasonically disrupted bacterial cells, lane 2 - supernatant from ultrasonically disrupted bacterial cells filtered through a 0.22 μm filter, lane 3 - flow-through sample, lanes 4-6 - eluted samples at different gradients, lane 7 - protein marker, lane 8 - supernatant from ultrasonically disrupted bacterial cells, lane 9 - supernatant from ultrasonically disrupted bacterial cells filtered through a 0.22 μm filter, lane 10 - flow-through sample, lanes 11-13 - eluted samples at different gradients);

[0034] Figure 4 SDS-PAGE results of ion exchange chromatography eluted samples (in the figure, lane 1 (leftmost lane) - protein marker, lanes 2-6 - different concentrations of BSA, lane 7 - linearly eluted samples, lane 8 - gradient eluted samples). Detailed Implementation

[0035] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0036] In a first aspect, the present invention provides a method for purifying VjbR protein, comprising the following steps:

[0037] The solution containing VjbR protein was diluted with Ni affinity chromatography equilibration buffer and then subjected to Ni affinity chromatography. The VjbR protein eluent was collected and then subjected to ion exchange chromatography to obtain purified VjbR protein.

[0038] The equilibration buffer and elution buffer of the Ni affinity chromatography each contain 2-10 mM of reducing agent and 0.2% vol-1.5% vol of surfactant.

[0039] The equilibration buffer and elution buffer of the ion exchange chromatography each contain 0.2% vol to 0.8% vol of surfactant independently;

[0040] The VjbR protein carries a His tag.

[0041] The purification method for VjbR protein provided by this invention effectively improves the purity and yield of VjbR protein by adding specific concentrations of reducing agent and surfactant to the equilibration buffer and elution buffer of Ni affinity chromatography, and adding specific concentrations of surfactant to the equilibration buffer and elution buffer of ion exchange chromatography. Furthermore, the purified VjbR protein is stable, does not easily precipitate, and can be stored for a long time, providing a foundation for brucellosis diagnosis technology.

[0042] In some optional embodiments, the reducing agent in the equilibration buffer and elution buffer of the Ni affinity chromatography is DTT or TCEP, preferably DTT, and the surfactant is Tween 20 or Tween 80, preferably Tween 20.

[0043] Preferably, the equilibration buffer and elution buffer for Ni affinity chromatography each contain 5 mM DTT and 0.5% vol Tween 20 independently.

[0044] The inventors discovered that adding a specific concentration of DTT to the equilibration buffer and elution buffer can effectively improve the purity of VjbR protein; adding a specific concentration of Tween 20 to the equilibration buffer and elution buffer can effectively prevent protein loss on the column due to precipitation, prevent protein aggregation, and improve the membrane recovery rate.

[0045] In some optional embodiments, the equilibration buffer for Ni affinity chromatography further contains Tris-HCl and NaCl, wherein the concentration of Tris-HCl may be, for example, but not limited to, 40 mM, 50 mM or 60 mM, and the concentration of NaCl may be, for example, but not limited to, 0.1 M, 0.15 M or 0.2 M, and the pH of the equilibration buffer is 8.

[0046] The elution buffer for Ni affinity chromatography also contains Tris-HCl, NaCl, and mM imidazole. The concentration of Tris-HCl can be, for example, but not limited to, 40mM, 50mM, or 60mM; the concentration of NaCl can be, for example, but not limited to, 0.1M, 0.15M, or 0.2M; the concentration of imidazole can be, for example, but not limited to, 450mM, 500mM, or 550mM; and the pH of the elution buffer is 8.

[0047] In some optional embodiments, the dilution factor is 3 to 5 times, preferably 4 times;

[0048] In some alternative embodiments, the Ni affinity chromatography column comprises GE Ni Sepharose. TM 6FF.

[0049] In some optional embodiments, the surfactant in the equilibration buffer and elution buffer of the ion exchange chromatography is Tween 20 or Tween 80, preferably Tween 20;

[0050] Preferably, the equilibration buffer and elution buffer of the ion exchange chromatography each contain 0.5% vol of Tween 20 independently.

[0051] The inventors discovered that by adding a specific concentration of Tween 20 to the equilibration buffer and elution buffer of ion exchange chromatography, column loss due to protein precipitation can be effectively avoided, protein aggregation can be prevented, and membrane recovery rate can be improved.

[0052] In some optional embodiments, the equilibration buffer for the ion exchange chromatography further contains Tris-HCl, the concentration of which may be, for example, but not limited to, 40 mM, 50 mM or 60 mM, and the pH of the equilibration buffer is 8.

[0053] The elution buffer for the ion exchange chromatography also contains Tris-HCl and NaCl, wherein the concentration of Tris-HCl can be, for example, but not limited to, 40 mM, 50 mM or 60 mM, and the concentration of NaCl can be, for example, but not limited to, 0.4 M, 0.5 M or 0.6 M, and the pH of the elution buffer is 8.

[0054] In some alternative embodiments, the VjbR protein eluent is diluted 4-fold with Ni affinity chromatography elution buffer and then subjected to ion exchange chromatography.

[0055] In some alternative embodiments, the ion exchange chromatography column includes Cytiva QHP.

[0056] In some alternative embodiments, the solution containing VjbR protein includes a lysate of cells expressing VjbR protein;

[0057] Preferably, the cells include, but are not limited to, Escherichia coli, or other fermenting bacteria known to those skilled in the art.

[0058] In some optional embodiments, the method for preparing the lysate of cells expressing VjbR protein includes: fermenting engineered bacteria expressing VjbR protein, collecting the fermenting bacteria, and then sequentially performing cell disruption and solid-liquid separation to obtain the lysate of cells expressing VjbR protein.

[0059] In some alternative embodiments, the solid-liquid separation includes filtration using a 0.22 μm filter membrane.

[0060] Secondly, the present invention provides the application of the above-mentioned purification method in the preparation of diagnostic products for brucellosis.

[0061] For example, the VjbR protein prepared by this invention can be used to prepare an ELISA kit for the detection of anti-VjbR protein antibodies, thereby achieving the diagnosis of brucellosis.

[0062] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0063] Example 1: Expression of VjbR protein

[0064] The organic culture medium (1L) formula is as follows: glycerol 10g; glucose 3g; sucrose 5g; ammonium sulfate 2.67g; Na2HPO4·12H2O 8.59g; yeast extract 10g; potassium dihydrogen phosphate 3.4g; sodium sulfate 1.42g; magnesium sulfate 1g; calcium chloride 0.25g, pH adjusted to 7.1.

[0065] The LB medium (1L) formula is: 10g yeast extract; 10g tryptone; 5g sodium chloride, pH adjusted to 8.0.

[0066] Escherichia coli expressing the VjbR protein were inoculated at a rate of 1% into 60 mL of LB medium containing 25 μg / mL kanamycin and cultured at 37°C and 180 rpm until OD500. 600 2-3 were used as seed culture; the obtained seed culture was inoculated at a 1% inoculum into 120 mL culture flasks of organic medium and LB medium containing 25 μg / mL kanamycin, and cultured at 37℃ and 180 rpm until OD. 600 The OD value was approximately 1.5, and the culture was carried out with a gradient cooling process until it reached 22°C, at which point the OD value was reached. 600Add IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 0.2 mM to 2-3, and induce fermentation at 180 rpm. Induce fermentation for 18 h in organic medium and 14 h in LB medium.

[0067] The fermentation broth was centrifuged to collect the bacterial cells, which were then resuspended in 10-fold volume of Tris-HCl buffer (pH 8.0, 50 mM Tris-HCl, 5 mM EDTA-2Na buffer). After overnight freeze-thaw cycles at -80°C, the cells were sonicated. The sonication conditions were: 25 mL of resuspended cells, 180 W, 30% power, 5 s sonication followed by a 10 s interval, for a total of 20 min. The supernatant was collected by centrifugation and filtered through a 0.22 μm filter to obtain the lysed bacterial supernatant. Protein analysis was then performed using gel electrophoresis. Figure 1 (In the figure, "cell disruption supernatant (8x)" refers to the supernatant of ultrasonically disrupted bacterial cells diluted 8 times with Tris-HCl buffer without passing through a 0.22 μm filter; "cell disruption supernatant passed through a 0.22 μm filter (8x)" refers to the supernatant of ultrasonically disrupted bacterial cells diluted 8 times with Tris-HCl buffer and passed through a 0.22 μm filter; "precipitate (4x)" refers to the resuspended precipitate of ultrasonically disrupted bacterial cells after centrifugation, diluted 4 times with Tris-HCl buffer.) The results showed that the soluble expression level in the organic medium was 126.44 μg / mL, while the soluble expression level in the LB medium was 71.8 μg / mL. Therefore, the organic medium exhibited significantly better culture performance compared to the LB medium.

[0068] In the following examples, the supernatant of broken bacterial cells obtained from fermentation in organic culture medium was used as the sample for purification.

[0069] Example 2: Conventional Purification Method

[0070] The supernatant of the lysed bacterial cells obtained in Example 1 was purified sequentially by Ni affinity chromatography and ion exchange chromatography. The experimental steps are as follows:

[0071] Prepare Ni affinity chromatography equilibration buffer: 50mM Tris-HCl, 0.15M NaCl, pH 8.0.

[0072] Prepare the elution buffer: 50mM Tris-HCl, 0.15M NaCl, 500mM imidazole.

[0073] Centrifuge the supernatant of the lysed bacterial cells from Example 1 at 11000 rpm for 10 min, collect the supernatant, dilute it 4-fold with equilibration buffer, and filter it through a 0.22 μm filter membrane for later use. Use GE Ni Sepharose... TMAfter equilibrating 5 column volumes of 6FF (column volume: 2 mL) with equilibration buffer, load 20 mL of sample at a flow rate of 2 mL / min and elution buffer at a flow rate of 2 mL / min. Set up 20% gradient elution for 8 CV, 50% gradient elution for 5 CV, and 100% gradient elution for 3 CV. Collect the 50% gradient elution peak.

[0074] Prepare ion exchange chromatography equilibration buffer: 50 mM Tris-HCl, pH 8.0.

[0075] Prepare the elution buffer: 50mM Tris-HCl, 0.5M NaCl, pH 8.0.

[0076] The sample collected by 50% elution of Ni affinity chromatography was diluted 4-fold with 50 mM Tris-HCl, pH 8.0, and filtered through a 0.22 μm filter membrane for later use. Cytiva Q HP (column volume: 5 mL) was equilibrated with 5 column volumes of equilibration buffer, and then 40 mL of the sample was loaded at a flow rate of 2 mL / min. The elution buffer flow rate was 2 mL / min. 0-100% linear elution was performed for 15 CV, and 100% gradient elution was performed for 5 CV. The 0-100% linear elution peak was collected to obtain the VjbR protein sample.

[0077] Protein samples purified by Ni affinity chromatography and ion exchange chromatography were subjected to gel electrophoresis, and the results are as follows: Figure 2 As shown, although the recombinant protein carries a His tag, a large amount of contaminating protein remains difficult to remove after Ni affinity chromatography. Ion exchange chromatography is also a common method for protein purification, but after another ion exchange chromatography purification, contaminating proteins that Ni affinity chromatography could not remove still exist, and the final purity after two steps of chromatography is only 62%. Changing the Ni affinity packing material did not yield better results. In addition, after two steps of chromatography, flocculent precipitate quickly appeared in the eluent, which was identified as VjbR protein by SDS-PAGE, indicating that this protein is prone to aggregation and precipitation and has poor stability.

[0078] Example 3: Optimization of Ni affinity chromatography conditions

[0079] Prepare the equilibration buffer: 50mM Tris-HCl, 0.15M NaCl, pH 8.0.

[0080] Prepare the elution buffer: 50mM Tris-HCl, 0.15M NaCl, 500mM imidazole.

[0081] Different components were added to the equilibration buffer and elution buffer according to Table 1 to serve as equilibration buffer and elution buffer.

[0082] Centrifuge the supernatant of the lysed bacterial cells from Example 1 at 11,000 rpm for 10 min, take the supernatant, dilute it 4 times with equilibration buffer and filter it through a 0.22 μm filter membrane for later use.

[0083] GE Ni Sepharose TM After equilibrating 5 column volumes (CV) of 6FF (column volume: 2 mL) with equilibration buffer, 20 mL of sample was loaded at a flow rate of 2 mL / min. The elution buffer flow rate was 2 mL / min. A 10% gradient elution was set up for 8 CV, a 60% gradient elution for 5 CV, and a 100% gradient elution for 3 CV. The 60% gradient elution peak sample was collected, filtered through a 0.22 μm filter, and stored at 4°C for later use. Electrophoresis was performed, and the effect of various buffer compositions on the yield and purity of the purified protein was calculated based on the grayscale of the target band in the electrophoresis. The purity and purification yield of the Ni affinity eluted sample, the yield of the eluted sample filtered through a 0.22 μm filter, and the overall yield are shown in Table 1.

[0084] Table 1 Optimization of Ni affinity chromatography buffer conditions

[0085]

[0086]

[0087] In conventional Ni affinity chromatography mobile phase systems, 60% eluted samples contain a large amount of disulfide-bonded contaminating proteins that cannot be removed by conventional Ni affinity chromatography mobile phase. As shown in Table 1 of Example 3, adding reducing agents, including DTT or TCEP, can effectively improve purity to over 60%, with DTT being superior. Samples collected after 60% gradient elution in Ni affinity chromatography aggregate immediately, resulting in significant losses during the 0.22 μm filter step before the next ion exchange chromatography step. Adding surfactants Tween-20 and Tween-80 to the buffer improves the yield of purified samples after filtration through a 0.22 μm filter, with Tween-20 being superior. Therefore, simultaneous addition of reducing agents and surfactants is necessary to achieve both improved purity and yield. The optimal purification effect is achieved with the addition of 5 mM DTT and 0.5% Tween-20, as shown in the results. Figure 3 As shown.

[0088] Example 4

[0089] To investigate the effect of the addition method of 5 mM DTT and 0.5% Tween-20 in Ni affinity chromatography on protein purification, the inventors made the following adjustments to the addition method of 5 mM DTT and 0.5% Tween-20 based on the above-mentioned Group 10 experiment:

[0090] Method 1: The difference from Group 10 is that when diluting the sample, a equilibration buffer (50mM Tris-HCl, 0.15M NaCl, pH 8.0) without reducing agents and surfactants is used.

[0091] Method 2: The difference from Group 10 is that 5 mM DTT and 0.5% Tween-20 are added to the equilibration buffer, while no addition is made to the elution buffer.

[0092] Method 3: The difference from Group 10 is that no DTT and 0.5% Tween-20 are added to the equilibration buffer instead of the elution buffer.

[0093] The purity, purification yield, elution yield of Ni affinity eluted samples with the above addition method, the yield of eluted samples filtered through a 0.22 μm membrane, and the total yield are shown in Table 2.

[0094] Table 2 Comparison of 5mM DTT and 0.5% Tween-20 addition methods

[0095]

[0096] In Ni affinity chromatography, the purification effect and total yield of adding 5 mM DTT and 0.5% Tween-20 at different stages varied. As shown in Table 2, when 5 mM DTT and 0.5% Tween-20 were added alone to the binding buffer (Method 1) and equilibration buffer (Method 2), the purity of the purified VjbR protein was less than 75%, and the yield after passing through a 0.22 μm filter was less than 80%. When added alone to the elution buffer (Method 3), although the yield after passing through a 0.22 μm filter could reach 95%, the purity of the eluted protein was less than 60%, which still could not achieve the effect of adding 5 mM DTT and 0.5% Tween-20 to both the equilibration buffer and the elution buffer simultaneously.

[0097] The following examples use the protein eluent obtained from group 10 as a sample for ion exchange chromatography.

[0098] Example 5: Optimization of Ion Exchange Chromatography

[0099] Prepare the equilibration buffer: 50mM Tris-HCl, pH 8.0.

[0100] Prepare the elution buffer: 50mM Tris-HCl, 0.5M NaCl, pH 8.0.

[0101] Different components were added to the equilibration buffer and elution buffer according to Table 3, with the control group having no additional components added.

[0102] The VjbR protein elution collected in Group 10 of Example 3 was diluted 4 times with the elution buffer of Group 10 of Example 3, filtered through a 0.22 μm filter membrane and used for later use.

[0103] After equilibrating Cytiva Q HP (column volume: 20 mL) for 5 column volumes with equilibration buffer, 320 mL of the sample was loaded at a flow rate of 15 mL / min. The elution buffer flow rate was also 15 mL / min. Linear elution from 0 to 100% was performed for 15 CV, and gradient elution from 100% was performed for 5 CV. The 100% elution peak was collected. The purification yield of this step was calculated based on the electrophoretic bands.

[0104] The purified protein was stored at 4℃ for 14 days and 30 days, and the concentration was measured again to investigate the stability of proteins prepared by different methods.

[0105] Table 3 Effects of Ion Exchange Chromatography Buffer Components on Yield and Stability

[0106] Group number Components purity Ion exchange yield Save 14 days of remaining amount Save 30 days of remaining amount 1 control group 90％ 51％ 44.6％ 2.8％ 2 0.2% Tween-20 95％ 81％ 97.9％ 96.2％ 3 0.5% Tween-20 96％ 83.3％ 98％ 97％ 4 0.8% Tween-20 96％ 82.1％ 98.2％ 96％ 5 1% Tween-20 95％ 65％ 75.4％ 42.6％ 6 0.5% - Tween-80 91％ 68％ 69％ 27％ 7 5% glycerin 88％ 54％ 49％ 5.6％

[0107] Adding different components to the ion-exchange mobile phase, when 0.2%-0.8% Tween-20 is added, the eluted VjbR protein sample exhibits good stability at 4°C. The preferred condition is 0.5% Tween-20, and the purification results are as follows: Figure 4 As shown, the purity of the obtained VjbR protein is greater than 95%.

[0108] Example 6

[0109] To investigate the effects of DTT and Tween-20 on protein purification efficiency in a two-step purification process, the following experiment was designed:

[0110] 1) Ni affinity chromatography:

[0111] Prepare the equilibration buffer: 50mM Tris-HCl, 0.15M NaCl, pH 8.0.

[0112] Prepare the elution buffer: 50mM Tris-HCl, 0.15M NaCl, 500mM imidazole.

[0113] Different components were added to the equilibration buffer and elution buffer according to Table 4.

[0114] Centrifuge the supernatant of the lysed bacterial cells from Example 1 at 11000 rpm for 10 min, collect the supernatant, dilute it 4-fold with equilibration buffer, and filter it through a 0.22 μm filter membrane. Use GE Ni Sepharose... TMAfter equilibrating the 6FF (column volume: 2 mL) column to 5 column volumes (CV) with equilibration buffer, load 20 mL of sample at a flow rate of 2 mL / min and elution buffer at a flow rate of 2 mL / min. Set up 10% gradient elution for 8 CV, 60% gradient elution for 5 CV, and 100% gradient elution for 3 CV. Collect the 60% gradient elution peak sample, filter it through a 0.22 μm filter membrane, and store it at 4℃ for later use.

[0115] 2) Ion exchange chromatography:

[0116] Prepare the equilibration buffer: 50mM Tris-HCl, pH 8.0.

[0117] Prepare the elution buffer: 50mM Tris-HCl, 0.5M NaCl, pH 8.0.

[0118] Different components were added to the equilibration buffer and elution buffer according to Table 4.

[0119] The VjbR protein eluent collected by Ni affinity chromatography was diluted 4-fold with Ni affinity chromatography elution buffer and filtered through a 0.22 μm filter membrane for later use. Cytiva Q HP (column volume: 20 mL) was equilibrated with 5 column volumes of equilibration buffer, and then 320 mL of the eluent was loaded at a flow rate of 15 mL / min. Elution buffer flow rate was 15 mL / min. Linear elution from 0-100% for 15 CV and gradient elution from 100% for 5 CV were performed, and the 100% elution peak was collected. The purification yield was calculated based on the electrophoretic bands. The purified protein was stored at 4 °C for 14 days, and the concentration was retested to investigate the stability of proteins prepared by different methods.

[0120] Table 4. Effects of Addition Methods on Purity and Stability

[0121]

[0122] As shown in Table 4, only by simultaneously adding 5 mM DTT and 0.5% Tween-20 in the Ni affinity chromatography step and adding 0.5% Tween-20 in the ion exchange chromatography step can high purity and good stability of VjbR protein be obtained, with a high total protein yield.

[0123] Example 7: The prepared VjbR protein was tested in Brucella-infected serum to evaluate the identification effect of the VjbR protein prepared by the method of the present invention.

[0124] The identification effect of VjbR proteins prepared by different preparation schemes (group 3 in Table 3, group 1 in Table 3, and group 2 in Table 4) and stored for 0 and 3 months was evaluated using the following method:

[0125] 1) Dilute VjbR protein to 2 μg / mL (200 ng / well) with 1×CBS coating buffer, add 100 μL to each well, and incubate overnight at 4°C; 2) Wash 5 times with 1×PBST and blot dry; 3) Add 200 μL of Jinno blocking buffer to each well and block at 37°C for 2 h; 4) Wash 5 times with 1×PBST and blot dry; 5) Add 100 μL of negative / positive serum diluted 200 times with 1×PBST to each well and incubate at 37°C for 1 h; 6) Wash 5 times with 1×PBST and blot dry; 7) Add 100 μL of goat anti-bovine secondary antibody diluted 1×PBST to each well and incubate at 37°C for 1 h; 8) Wash 5 times with 1×PBST and blot dry; 9) Add 100 μL of TMB and develop color at room temperature in the dark for 10 min; 10) Add 50 μL of stop solution to stop the reaction, and measure the OD value using a microplate reader. 450 reading.

[0126] ROC analysis was performed on the detection results of 16 positive and 16 negative serum samples according to the above method. The results are shown in Table 5. As can be seen from the table, the VjbR protein prepared by adding 5 mM DTT and 0.5% Tween-20 to the Ni affinity chromatography mobile phase and adding 0.5% Tween-20 to the ion exchange chromatography mobile phase has good stability and identification effect.

[0127] Table 5. Identification effect of VjbR protein prepared by different methods after 0 and 3 months of storage.

[0128]

[0129] In summary, the inventors have developed a method for preparing VjbR protein. After recombinant expression and fermentation in E. coli, the purified protein has a purity >95% and can be stably stored at 4°C. This method can obtain large quantities of VjbR protein with high purity and good stability, providing a foundation for the subsequent development of disease diagnostic methods targeting VjbR protein.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for purifying VjbR protein, characterized in that, Includes the following steps: The solution containing VjbR protein was diluted with Ni affinity chromatography equilibration buffer and then subjected to Ni affinity chromatography. The VjbR protein eluent was collected and then subjected to ion exchange chromatography to obtain purified VjbR protein. The equilibration buffer and elution buffer for Ni affinity chromatography each contain 2-10 mM of reducing agent and 0.2% vol-1.5% vol of surfactant. The equilibration buffer and elution buffer of the ion exchange chromatography each independently contain 0.2% vol to 0.8% vol of Tween 20 or Tween 80; The VjbR protein carries a His tag. In the equilibration buffer and elution buffer of the Ni affinity chromatography, the reducing agent is DTT or TCEP, and the surfactant is Tween 20 or Tween 80. The solution containing VjbR protein is a lysate of cells expressing VjbR protein; The cells were Escherichia coli.

2. The purification method according to claim 1, characterized in that, The equilibration buffer and elution buffer for Ni affinity chromatography contain DTT as the reducing agent and Tween 20 as the surfactant.

3. The purification method according to claim 2, characterized in that, The equilibration buffer and elution buffer for Ni affinity chromatography each contain 5 mM DTT and 0.5% vol Tween 20 independently.

4. The purification method according to claim 1, characterized in that, The equilibration buffer for Ni affinity chromatography also contains 40–60 mM Tris-HCl and 0.1–0.2 M NaCl; The elution buffer for Ni affinity chromatography also contains 40–60 mM Tris-HCl, 0.1–0.2 M NaCl, and 450–550 mM imidazole.

5. The purification method according to claim 1, characterized in that, The dilution factor is 3 to 5 times; And / or, the Ni affinity chromatography column is a GE Ni Sepharose column. TM 6FF.

6. The purification method according to claim 1, characterized in that, The equilibration buffer and elution buffer of the ion exchange chromatography each contain 0.5% vol of Tween 20.

7. The purification method according to claim 1, characterized in that, The equilibration buffer for the ion exchange chromatography also contains 40-60 mM Tris-HCl; The elution buffer for the ion exchange chromatography also contains 40–60 mM Tris-HCl and 0.4–0.6 M NaCl.

8. The purification method according to claim 1, characterized in that, The ion exchange chromatography column used was Cytiva Q HP.

9. The purification method according to claim 1, characterized in that, The method for preparing the lysate of cells expressing VjbR protein includes: fermenting engineered bacteria expressing VjbR protein, collecting the fermenting bacteria, and then sequentially performing cell disruption and solid-liquid separation to obtain the lysate of cells expressing VjbR protein.

10. The use of the purification method according to any one of claims 1-9 in the preparation of brucellosis diagnostic products.

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