CN114957440A - Recombinant HMGB 1A Box protein and obtaining method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a recombinant HMGB 1A Box protein, and an acquisition method and application thereof. The invention provides a purified recombinant protein of HMGB 1A Box derived from escherichia coli, which can target and inhibit a nucleoprotein (HMGB 1) with the ability of recruiting and activating immune cells and has the function of targeting and inhibiting HMGB 1. Can realize effective inhibition of inflammation of substantia nigra of brain and reduce the continuous damage of dopaminergic neurons caused by excessive inflammation. Has high application value in preparing the medicine for treating the relevant brain inflammation of the Parkinson disease.

Description

A kind of recombinant HMGB1 A Box protein and its obtaining method and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a recombinant HMGB1 A Box protein and an acquisition method and application thereof.

Background technique

Parkinson's disease (PD) is a common neurodegenerative disease, mainly in the elderly. The main pathological change in Parkinson's disease is the degeneration and death of dopaminergic (DA) neurons in the substantia nigra, resulting in a significant decrease in striatal DA content. Data show that with age, there is a progressive decrease in dopaminergic neurons in the substantia nigra of normal adults. However, the exact cause of this pathological change is still unclear. Genetic factors, environmental factors, aging, and oxidative stress may all be involved in the degeneration and death of PD dopaminergic neurons. At present, there is no effective cure for early-stage Parkinson's disease, and there is also a lack of available therapeutic drugs for advanced-stage Parkinson's disease. Clinically, levodopa is mainly used to supplement dopamine deficiency caused by the loss of dopaminergic neurons, dopamine receptor agonists enhance the function of dopamine receptors, and B-type monoamine oxidase inhibitors reduce the oxidative damage of neurons. In recent years, authoritative studies have continuously confirmed that microglia, peripherally derived T cells and even monocytes play a very important role in promoting the continuous progression of Parkinson's disease. However, the above widely used treatment options only target the neurons themselves and cannot treat the neuroinflammation caused by immune cells in the substantia nigra of the brains of patients with Parkinson's disease.

High mobility group B1 (HMGB1) is a nuclear protein with the ability to recruit and activate immune cells, widely distributed in mammalian cells. With the discovery of its late pro-inflammatory effect, HMGB1 has become one of the hotspots in critical care medicine research in recent years. It plays an important role in the pathogenesis of sepsis, tumors, arthritis and other diseases, and HMGB1 may become a new target for the treatment of various diseases. However, there is no relevant research report on its use in the treatment of Parkinson's disease. Therefore, whether HMGB1 can be used in the treatment of Parkinson's disease is still a rather difficult and worthy question.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a recombinant HMGB1 A Box protein and its obtaining method and application. Recombinant HMGB1 A Box protein is derived from purified E. coli and has the function of targeted inhibition of HMGB1. It can effectively inhibit the inflammation of the substantia nigra in the brain and reduce the continuous damage of dopaminergic neurons caused by excessive inflammation. For the treatment of related brain inflammation in Parkinson's disease.

To achieve the above object, the present invention adopts the following technical solutions:

The present invention provides a HMGB1 A Box recombinant protein, and the amino acid sequence of the HMGB1 A Box recombinant protein is shown in SEQ ID NO: 2.

The present invention also provides a method for preparing the HMGB1 A Box recombinant protein, comprising: constructing the expression plasmid of the HMGB1 A Box recombinant protein into an Escherichia coli expression vector to express the target protein.

Further, the preparation method specifically includes:

(1) Construct the expression plasmid of the HMGB1 A Box recombinant protein; transform it into BL21 E. coli, and perform positive screening on LB solid-state culture plates containing antibiotics to obtain pET21a ⁺ E. coli positive colonies;

(2) Amplify the positive colonies obtained in step (1) to the exponential amplification stage, add IPTG, shake the bacteria, centrifuge, discard the supernatant, and retain the bacterial precipitate;

(3) adding the non-denaturing lysate to the bacterial precipitate in step (2), resuspending and mixing, and ultrasonically disrupting and lysing in an ice bath to obtain a bacterial lysate;

(4) Centrifuge the bacterial lysate obtained in step (3) to collect the lysate supernatant;

(5) Mix the lysate supernatant obtained in step (4) with the His-tag affinity resin and put it into the affinity chromatography column, open the bottom of the chromatography column, the liquid flows out, and the resin is retained;

(6) Wash the column with the washing solution, and then elute with the eluent to obtain the recombinant protein;

(7) After removing the endotoxin of the recombinant protein in step (6), put it in a dialysis bag, remove imidazole by dialysis, and filter with a 0.22 μm filter to obtain the HMGB1 A Box recombinant protein.

The nucleotide sequence of the expression plasmid described in step (1) is shown in SEQ ID NO: 1.

In step (2), the exponential phase is the OD value of the bacterial solution of 0.5-0.6; the final concentration of IPTG added is 1 mM, and the time for shaking the bacteria is 4-6 hours; the centrifugation is 3000 rcf for 10 min.

The composition of the non-denaturing lysing solution in step (3) is: each liter contains 6.90 g NaH ₂ PO ₄ ·H ₂ O, 17.54 g NaCl, and sodium hydroxide adjusts the pH to 8.0; the non-denaturing lysing solution and bacteria The volume ratio of the precipitate was 5:1.

The parameters of centrifugation in step (4) are 4°C, 10000rcf, and centrifugation for 30 minutes;

The volume ratio of the lysate supernatant to the His-tag affinity resin in step (5) is 10:1.

The composition of the washing solution in step (6) is: each liter contains 6.90 g NaH ₂ PO ₄ ·H ₂ O, 17.54 g NaCl, 0.136 g imidazole, and sodium hydroxide adjusts the pH value to 8.0;

The composition of the eluent described in step (6) is: each liter contains 6.90 g NaH ₂ PO ₄ ·H ₂ O, 17.54 g NaCl, 3.40 g imidazole, and sodium hydroxide adjusts the pH to 8.0.

The invention also provides the application of the recombinant HMGB1 A Box protein in the preparation of medicines for treating/preventing Parkinson's disease.

The present invention also provides a kit comprising recombinant HMGB1 A Box protein.

The present invention also provides the application of the above-mentioned kit in the preparation of medicines for treating/preventing Parkinson's disease.

Compared with the prior art, the beneficial effects of the present invention are:

The invention provides a purified Escherichia coli-derived HMGB1 A Box recombinant protein, which has simple preparation steps, low cost and high purification degree. It can target and inhibit HMGB1, a nuclear protein with the ability to recruit immune cells and activate immune cells. It has the biological activity function of targeting and inhibiting HMGB1. It has a recovery effect on the number of TH ⁺ dopaminergic neurons, and has a negative effect on the infiltration of CD3 ⁺ T cells. The activation of gliocytes and the infiltration of macrophages have obvious inhibitory effects. It can effectively inhibit the inflammation of the substantia nigra in the brain and reduce the continuous damage of dopaminergic neurons caused by excessive inflammation. It has high application value in the preparation of related brain inflammatory drugs for treating Parkinson's disease.

Description of drawings

Fig. 1 is the identification of pET21a-a box plasmid digestion;

Fig. 2 is eluate protein SDS-PAGE electrophoresis Coomassie brilliant blue staining diagram;

Figure 3 is the western blot detection chart;

Fig. 4 is a flow cytometry detection result of MHC II molecule expression;

Fig. 5 is the result of enzyme-linked immunosorbent assay detection of inflammatory factor TNF-α in the supernatant;

Fig. 6 is the TH immunohistochemical staining diagram of substantia nigra region;

Fig. 7 is the immunofluorescence staining of CD3 in substantia nigra region;

Fig. 8 is the immunofluorescence staining image of substantia nigra region Iba1;

Fig. 9 is the immunofluorescence staining of GFAP in substantia nigra region;

Fig. 10 is an immunofluorescence staining image of CD45 and F4/80 in the substantia nigra region.

Detailed ways

The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and embodiments. The following examples are only used to illustrate the technical solutions of the present invention more clearly, and cannot be used to limit the protection scope of the present invention. In the following examples, the experimental methods without specific conditions are selected according to the conventional methods and conditions in the art, or according to the product description. The reagents and raw materials that do not specify specific components in the following examples are commercially available.

The pET21a plasmid of the present invention is from Shanghai Sangon, His-tag affinity resin, 5×SDS-PAGE protein loading buffer and TH antibody are all from Shanghai Biyuntian Biotechnology Co., Ltd., CD3, CD45, F4/80 are all from In Biolegend, Iba1 is from Wako Chemicals, GFAP is from Novusbio, protein marker is from Thermo Fisher, SABC-POD kit and DAB color development kit are all from Wuhan Boster Biological Engineering Co., Ltd.

Example 1: Preparation of HMGB1 A Box recombinant protein

(1) The pET21a-a box plasmid with the HMGB1 A Box sequence of the 6×His tag was transformed into BL21 Escherichia coli, and positive screening was performed on LB solid-state culture plates containing ampicillin sodium (100ug/mL), and the transfected cells were obtained. Successfully transfected pET21a ⁺ Escherichia coli positive colonies; Figure 1 is the identification of pET21a-a box plasmid digestion; the nucleotide sequence of pET21a-a box plasmid is shown in SEQ ID NO: 1.

(2) Inoculate the positive colonies obtained in step (1) into 10 mL of LB liquid medium containing 100 ug/mL ampicillin sodium, and amplify for 12 hours on a shaking table at 37 degrees Celsius and 250 rpm;

(3) The bacterial solution amplified in step (2) was transferred to LB medium containing 100ug/mL ampicillin sodium at a volume ratio of 1:100, and expanded on a shaking table at 37 degrees Celsius and 250 rpm. When the OD value of the bacterial solution is increased to 0.5-0.6, add IPTG (isopropyl-β-D-thiogalactoside) to the final concentration of 1 mM in the bacterial solution, and continue to shake the bacteria for 4-6 hours;

(4) Collect the bacterial liquid obtained in step (3), centrifuge at 3000 rcf for 10 min, discard the supernatant, and retain the bacterial precipitate;

(5) Use non-denaturing lysis solution (per liter formula: 6.90 g NaH ₂ PO ₄ ·H ₂ O, 17.54 g NaCl, sodium hydroxide to adjust the pH value to 8.0) and add it to step (4) in a volume ratio of 5:1 Bacterial precipitation, fully resuspended and lysed, using 200-300w ultrasonic to break and lyse 6 times, each time for 10 seconds, the whole process of ice bath;

(6) Centrifuge the bacterial lysate obtained in step (5) at 4 degrees Celsius and 10000rcf for 30 minutes, and collect the lysate supernatant;

(7) Mix the supernatant obtained in step (6) with the His-tag affinity resin at a volume ratio of 10:1 in a 4 degree Celsius environment for 1 hour, and then load it into an empty affinity chromatography of the corresponding volume specification In the column, open the bottom of the chromatography column, gravity makes the liquid flow out naturally, and the resin is retained;

(8) Add washing solution (per liter formula: 6.90 g NaH ₂ PO ₄ ·H ₂ O, 17.54 g NaCl, 0.136 g imidazole, sodium hydroxide to adjust pH value to 8.0), wash 5-6 times , the non-specifically bound proteins on the resin were thoroughly washed away; then the eluent (per liter formulation: 6.90 g NaH ₂ PO ₄ ·H ₂ O, 17.54 g NaCl, 3.40 g imidazole, hydrated with hydroxide) was added to the column. Adjust the pH to 8.0 with sodium), elute 6-10 times, fully elute the target protein, and collect the eluent with the highest concentration in the first three times;

(9) Use DNA-EZ Reagents P liquid-phase endotoxin scavenger to remove the endotoxin in the eluate obtained in step (8), and then use the solid-phase or liquid-phase endotoxin removal reagent to remove the endotoxin more than twice, and remove the endotoxin. Reduced to below 0.1EU/mL, in line with the current Chinese National Pharmacopoeia requirements for intravenous recombinant protein drugs;

(10) Put the endotoxin-removed recombinant protein in step (9) into an 8kDa dialysis bag, seal it, soak it in sterile 0.9% NaCl solution or PBS solution at 4 degrees Celsius, and stir slowly for 12 to 18 hours. Dialysis to remove imidazole from the eluate;

(11) In an ultra-clean workbench, use a 0.22 μm filter to filter the imidazole-removed recombinant protein solution in step (10) to obtain HMGB1 A Box recombinant protein. Store the HMGB1 A Box recombinant protein in a sterile container, reserve a small amount for testing and label the protein concentration. After testing, the obtained protein concentration is 1-3 mg/mL, and the rest is stored in an ultra-low temperature refrigerator at -80 degrees Celsius for long-term storage. The HMGB1 A Box sequence is from GenBank: BC064790.1, and the 438-698 nucleotide sequence encoding the Box A domain is selected from the 438-1085 nucleotide sequence encoding the protein; the amino acid sequence encoding the HMGB1 A Box recombinant protein is shown in SEQ ID NO: 2.

Example 2

(1) Take 40 microliters of the finished protein obtained in Example 1, add 10 microliters of 5×SDS-PAGE protein loading buffer, and cook in a boiling water bath for 5-10 minutes to obtain a protein sample;

(2) Prepare a 15% acrylamide SDS-PAGE gel as a separating gel, a 5% acrylamide SDS-PAGE gel as a stacking gel, and add the protein sample and protein marker ( Thermo Fisher Scientific 26616), constant voltage electrophoresis (100 volts for stacking gel, 150 volts for separating gel) in running buffer (3g Tris-Base, 14.4g glycine, 1g SDS, _H2O to 1L) until Finish;

(3) Soak the SDS-PAGE gel in step (2) in Coomassie brilliant blue staining solution (G250 100mg, 95% ethanol 50ml to dissolve, add 85% phosphoric acid 100ml, H ₂ O to make up to 1L, filter to remove Undissolved material) was stained at room temperature for 3 hours, then soaked in decolorizing solution (100ml acetic acid, 50ml ethanol, H2O to 1L), shake slowly on a shaker to decolorize until the background was transparent, and take pictures under natural light; Figure 2 is the eluate protein SDS - PAGE electrophoresis Coomassie brilliant blue staining diagram; it can be seen from Figure 2 that the protein bands are largely enriched at the target molecular weight position, and the possibility of heterobands is excluded.

(4) After electrophoresis, take out the plate and soak it in electrophoresis transfer buffer (3g Tris-Base, 14.4g glycine, 200ml methanol, and make the volume to 1L with H ₂ O), take out the complete gel, and place the gel on a sheet. Cut out the filter paper (Whatman, 3MM CHR) soaked in electrophoresis transfer buffer to remove air bubbles, cover with a PVDF membrane soaked in methanol after activation to remove air bubbles, and then cover with a piece of electrophoresis transfer buffer soaked. Filter paper, install the device between the transfer clips, insert it into the electrophoresis transfer device, and transfer it with a constant current (300mA) in the electrophoresis transfer buffer for 30min;

(5) Take out the PVDF membrane in step (4), and seal it in a 5% bovine serum albumin solution on a shaker for 1 hour;

(6) The blocked PVDF membrane was shaken for 10 hours at 4°C in an antibody bath containing 6×His monoclonal antibody (abcam), TBST buffer (50ml 1M, pH7.5 Tris-HCl, 8g NaCl, 0.2g KCl 0.5 ml Tween 20, H ₂ O to 1 L) and washed 3 times for 15 minutes each time;

(7) Soak the PVDF membrane in step (6) in an antibody bath at room temperature with the HRP-conjugated secondary antibody (anti-species antibody from the previous step) for 1 hour, and wash it three times in TBST buffer for 15 minutes each time;

(8) Put the membrane in step (7) into the UV exposure machine, with the protein side facing up, drop the ECL color developing solution until it evenly covers the membrane, and perform exposure imaging. Figure 3 is the detection map of western blot; it can be seen from Figure 3 that the 6×His tag at the target molecular weight of A Box proves that the obtained protein is the target protein.

Example 3

(1) RAW264.7 mouse macrophage cells were seeded in a 24-well plate with 100,000 cells per well. The medium is complete DMEM medium containing 10% fetal bovine serum, and the medium volume per well is 500 microliters;

(2) Divide the cells in the culture plate in step (1) into HMGB1+A Box group and HMGB1 group; in the HMGB1+A Box group, add the protein obtained in Example 1 to a final concentration of 1ug/ml and PBS to promote dissolution. The inflammatory form disulfide-HMGB1 (HMGBiotech) was added to the final concentration of 500ng/ml, and the HMGB1 group only added the pro-inflammatory form of disulfide-HMGB1 dissolved in PBS to the final concentration of 500ng/ml; the cells in both groups were placed in a cell incubator for 12 hours;

(3) Collect the culture supernatant in step (2) in a 1.5ml centrifuge tube for use. Add 1 ml of PBS to each well to resuspend the cells by gentle pipetting, collect them into 1.5 ml centrifuge tubes, centrifuge for 5 min at 4 degrees Celsius, 500 rcf/min, discard the supernatant, resuspend the cells in 100 microliters of PBS solution, and add 1 microliter to each tube. 1 liter FITC-MHC II anti-mouse flow antibody (Biolegend), incubate at 4 degrees Celsius for 15 minutes in the dark;

(4) Take the supernatant from step (3) and add 1 ml of PBS to stop the staining, centrifuge at 4 degrees Celsius, 500 rcf/min for 5 min, discard the supernatant, resuspend the cells in 200 μl, and use FCM to detect the ratio of MHC II ⁺ cells. . Figure 4 is a graph showing the results of flow cytometry detection of the expression of MHC II molecules; as can be seen in Figure 4, the recombinant HMGB1 A Box protein has the biological activity of inhibiting HMGB1.

(5) Take the supernatant from step (3) at 4 degrees Celsius, 1000rcf/min, centrifuge for 10 minutes, aspirate the supernatant, and transfer it to a new centrifuge tube; use the Mouse TNF-a ELISA Kit (Linktech) to detect the supernatant according to the product instructions TNFα levels, the data obtained were statistically plotted using Prism 8.0 software. Figure 5 is the result of enzyme-linked immunosorbent assay detection of the inflammatory factor TNF-α in the supernatant; as can be seen in Figure 5, the enzyme-linked immunosorbent assay of the inflammatory factor TNF-α in the HMGB1-treated macrophage culture supernatant by HMGB1 A Box The test results confirm that it has the biological activity of inhibiting HMGB1.

Example 4

(1) Normal 10-week-old male C57BL/6J mice were divided into MPTP group, MPTP+A Box group and Control group; MPTP group was intraperitoneally injected with MPTP (MacLean) every day at a daily dose of 25 mg/kg body weight, continuous 7 days; MPTP+A Box group received daily intraperitoneal injection of MPTP at a daily dose of 25 mg/kg body weight, and daily tail vein injection of the recombinant HMGB1 ABox protein obtained in Example 1 at a dose of 4 mg/kg body weight for 7 consecutive days; The control group was injected with the same volume of normal saline as the other two groups by intraperitoneal and tail vein daily for 7 consecutive days;

(2) The mice in each group were anesthetized with isoflurane, and the heart was perfused with PBS solution to remove blood cells in the circulatory system, and 4% paraformaldehyde was continued to perfuse, so as to shorten the subsequent brain tissue fixation time;

(3) The mouse brain tissue after perfusion was completely removed, soaked in 4% paraformaldehyde for 3 days, then transferred to 20% sucrose-PBS solution for dehydration to the bottom of the tissue, and replaced with 30% sucrose-PBS solution to continue dehydration to the bottom of the tissue;

(4) The brain tissue of the substantia nigra of mice was intercepted by a stereotaxic device, and serial frozen sections were performed with a thickness of 20 microns;

(5) Put the frozen sections at the same position of each group of mice in PBS, shake the shaker slowly for 5 minutes, repeat 2 times, and wash off the embedding agent during sectioning;

(6) Add 3% hydrogen peroxide dropwise to the sliced tissue, let it stand at room temperature for 10 minutes to inactivate peroxidase, and immerse it in PBS twice for 5 minutes each time;

(7) 1% BSA prepared in PBS was added dropwise to the tissue, and after 1 hour of blocking at room temperature, the blocking solution was thrown off;

(8) Put the slices in a dark humid box, add the working concentrations of TH, CD3, Iba1, GFAP, CD45, F4/80 antibodies dropwise, and incubate at 4 degrees Celsius for 12 hours in the dark;

(9) Place the slices in PBS, shake the shaker slowly for 5 minutes, repeat 3 times, and wash away the unbound antibodies;

For the slices incubated with TH antibody, follow the instructions of the ready-to-use SABC-POD kit and DAB color development kit to perform the subsequent steps; finally, image them under a light microscope. Figure 6 is a picture of TH immunohistochemical staining in the substantia nigra area; it can be seen from Figure 6 that after the injection of HMGB1 A Box recombinant protein on the MPTP-induced mouse model, the TH immunohistochemical staining in the substantia nigra region confirms that it has no effect on TH ⁺ dopaminergic neurons Quantity recovery.

For the sections incubated with CD3, Iba1, GFAP, CD45, F4/80 antibodies, the corresponding anti-mouse or anti-rabbit fluorescent antibodies (Invitrogen) were added dropwise and incubated for 1 hour at room temperature in the dark; the sections were placed in PBS and shaken slowly on a shaker for 5 min, repeat 3 times, wash away the unbound fluorescent antibody; image and photograph the substantia nigra of each group of slices under a fluorescence microscope.

Figures 7-10 are the immunofluorescence staining images of CD3, Iba1, GFAP, CD45, and F4/80 in the substantia nigra; as shown in Figures 7-10, after the MPTP-induced mouse model was injected with HMGB1 A Box recombinant protein, respectively Immunofluorescence staining for CD3, Iba1, GFAP, CD45, F4/80 in the substantia nigra, compared with MPTP group, infiltration of CD3 ⁺ T cells, activation of astrocytes, macrophages in MPTP+A Box group Infiltration were significantly inhibited.

The specific embodiments of the present invention have been described above in detail, but they are only used as examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention.

sequence listing

<110> Jiangsu University

<120> A kind of recombinant HMGB1 A Box protein and its obtaining method and application

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agaggcaaaa tgtcctcata tgcattcttt gtgcaaactt gccgggagga gcacaagaag 60

aagcacccgg atgcttctgt cacttctcag agttctccaa gaagtgctca gagaggtgga 120

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Claims (10)

1. A HMGB1 A Box recombinant protein, wherein the amino acid sequence of the HMGB1 A Box recombinant protein is as shown in SEQ ID NO:2. 2. a preparation method of HMGB1 A Box recombinant protein, is characterized in that, comprises the steps: (1) Construct the expression plasmid of the HMGB1 A Box recombinant protein; transform it into BL21 E. coli, and perform positive screening on LB solid-state culture plates containing antibiotics to obtain pET21a ⁺ E. coli positive colonies; (2) Amplify the positive colonies obtained in step (1) to the exponential amplification stage, add IPTG, shake the bacteria, centrifuge, discard the supernatant, and retain the bacterial precipitate; (3) adding the non-denaturing lysate to the bacterial precipitate in step (2), resuspending and mixing, and ultrasonically disrupting and lysing in an ice bath to obtain a bacterial lysate; (4) Centrifuge the bacterial lysate obtained in step (3) to collect the lysate supernatant; (5) Mix the lysate supernatant obtained in step (4) with the His-tag affinity resin and put it into the affinity chromatography column, open the bottom of the chromatography column, the liquid flows out, and the resin is retained; (6) Wash the column with the washing solution, and then elute with the eluent to obtain the recombinant protein; (7) After removing the endotoxin of the recombinant protein in step (6), put it in a dialysis bag, remove imidazole by dialysis, and then filter to obtain a sterile HMGB1 A Box recombinant protein solution. 3 . The preparation method according to claim 2 , wherein the nucleotide sequence of the expression plasmid in step (1) is shown in SEQ ID NO: 1. 4 . 4 . The preparation method according to claim 2 , wherein the exponential phase in step (2) is the OD value of the bacterial solution of 0.5 to 0.6; the final concentration of adding IPTG is 1 mM, and the time of shaking the bacteria is 4 . -6 hours; the centrifugation was 3000rcf for 10min. 5 . The preparation method according to claim 2 , wherein the non-denaturing lysis solution in step (3) is composed of: 6.90 g NaH ₂ PO ₄ ·H ₂ O per liter, 17.54 g NaCl, 5 . The pH value was adjusted to 8.0 with sodium hydroxide; the volume ratio of the non-denaturing lysate and the bacterial precipitate was 5:1. 6 . The preparation method according to claim 2 , wherein the centrifugation parameters in step (4) are 4° C., 10000rcf, and centrifugation time 30 minutes; the lysate supernatant in step (5) and His The volume ratio of tag affinity resin is 10:1. 7 . The preparation method according to claim 2 , wherein the composition of the washing solution in step (6) is: every liter contains 6.90 g NaH ₂ PO ₄ ·H ₂ O, 17.54 g NaCl, 0.136 g imidazole, sodium hydroxide to adjust pH to 8.0. 8 . The preparation method according to claim 2 , wherein the composition of the eluent in step (6) is: 6.90 g NaH ₂ PO ₄ ·H ₂ O per liter, 17.54 g NaCl, 3.40 g g imidazole, sodium hydroxide to adjust pH to 8.0. 9. The application of the recombinant HMGB1 A Box protein of claim 1 in the preparation of medicines for treating/preventing Parkinson's disease. 10. A kit, characterized in that the kit comprises the recombinant HMGB1 A Box protein of claim 1.

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