阅读说明：本技术 一种重组人白介素-6的制备方法 (Preparation method of recombinant human interleukin-6 ) 是由 任军 王耀辉 贾凌云 于 2021-02-01 设计创作，主要内容包括：本发明公开了一种重组人白介素-6的制备方法,属于生物工程领域。包括如下步骤：(1)原核表达方式制备重组人白介素-6包涵体；(2)采用含尿素的洗涤缓冲液A中清洗重组人白介素-6包涵体,再用洗涤缓冲液B洗涤重组人白介素-6包涵体,离心收集固体；(3)初步纯化后的重组人白介素-6包涵体经含有β-巯基乙醇的变性液中至完全溶解,缓慢加入复性液使重组人白介素-6包涵体复性,再进行透析和超滤浓缩；(4)使用填充Anti-IL-6高亲和吸附剂的亲和吸附柱对复性的重组人白介素-6包涵体进行亲和纯化,即得。本发明操作简单,处理样本大,步骤少,复性率高。本发明所涉及的重组人白介素-6的回收率为21％,具有较高的回收率。(The invention discloses a preparation method of recombinant human interleukin-6, belonging to the field of bioengineering. The method comprises the following steps: (1) preparing a recombinant human interleukin-6 inclusion body in a prokaryotic expression mode; (2) washing the recombinant human interleukin-6 inclusion body in a washing buffer solution A containing urea, washing the recombinant human interleukin-6 inclusion body with a washing buffer solution B, and centrifugally collecting solids; (3) the recombinant human interleukin-6 inclusion body after preliminary purification is dissolved completely in a denaturating liquid containing beta-mercaptoethanol, renaturation liquid is slowly added to ensure that the recombinant human interleukin-6 inclusion body is renatured, and then dialysis and ultrafiltration concentration are carried out; (4) and (3) carrying out affinity purification on the renatured recombinant human interleukin-6 inclusion body by using an affinity adsorption column filled with Anti-IL-6 high-affinity adsorbent to obtain the recombinant human interleukin-6 inclusion body. The method has the advantages of simple operation, large sample processing, few steps and high renaturation rate. The recovery rate of the recombinant human interleukin-6 related by the invention is 21 percent, and the recovery rate is higher.)

1. A preparation method of recombinant human interleukin-6 is characterized by comprising the following steps:

(1) preparing a recombinant human interleukin-6 inclusion body in a prokaryotic expression mode;

(2) washing the recombinant human interleukin-6 inclusion body by using a washing buffer solution A containing urea, washing the recombinant human interleukin-6 inclusion body by using a washing buffer solution B to remove the urea, centrifugally collecting solids, and taking the obtained solids as the primarily purified recombinant human interleukin-6 inclusion body;

(3) adding the recombinant human interleukin-6 inclusion body after preliminary purification into a denatured liquid containing beta-mercaptoethanol until the recombinant human interleukin-6 inclusion body is completely dissolved, slowly adding a renaturation liquid to make the recombinant human interleukin-6 stand at a low temperature for renaturation, and then dialyzing and ultrafiltering for concentration to obtain renatured recombinant human interleukin-6;

(4) and carrying out affinity purification on the renatured recombinant human interleukin-6 by using an affinity adsorption column filled with Anti-IL-6 high-affinity adsorption medium to obtain the recombinant human interleukin-6.

2. The preparation method according to claim 1, wherein the specific method for preparing the recombinant human interleukin-6 inclusion body in the prokaryotic expression mode in the step (1) comprises the following steps: introducing the recombinant human interleukin-6 sequence into a pET-21a or pET-24a expression vector to prepare recombinant plasmid, transforming the recombinant plasmid into E.coli BL21 or E.coli Shuffle T7, expanding expression, collecting thalli for efficiently expressing the recombinant human interleukin-6, inducing the thalli by an IPTG inducer, collecting the thalli, crushing the thalli until bacterial liquid is relatively transparent and is not viscous any more, and collecting precipitates to obtain the recombinant human interleukin-6 inclusion body.

3. The process according to claim 1, wherein the washing buffer a in step (2) is 10 to 50mM Tris-HCl, 1 to 4M urea, 80 to 200mM NaCl, pH 8 to 9; the washing buffer B was 10-50mM Tris-HCl, pH 8-9.

4. The method according to claim 1, wherein the denaturant containing β -mercaptoethanol in step (3) is a mixture of 6-8M guanidine hydrochloride, 100-.

5. The method according to claim 1, wherein the renaturation solution in the step (3) is 0.5-1.5M guanidine hydrochloride, 50-150mM Tris-HCl, 8-12mM EDTA, 0.25-0.5mM GSSG (oxidized form), 2.5-5mM GSH (reduced form), 300-700mM arginine, 2-5% glycerol, pH 8-9; the speed of adding the renaturation liquid is 0.1-0.5 mL/min; standing at 4-6 deg.C for 20-40 h.

6. The method according to claim 1, wherein the dialysis solution used in the step (3) is 10 to 50mM Tris solution.

7. The method according to claim 1, wherein the affinity purification in step (4) comprises: enabling the renatured recombinant human interleukin-6 solution obtained in the step (3) to flow through an affinity adsorption column which takes Anti-IL-6 antibody with high affinity as affinity ligand and CL-6B agarose gel microspheres as a solid-supported medium at a rate of 0.2-1mL/min, and balancing the affinity adsorption column by using a PBS solution; eluting the adsorption column by using Gly-HCl solution with the pH value of 1.5-2, and replacing the eluted recombinant human interleukin-6 protein into 10-50mM Tris-HCl solution with the pH value of 8-9 by using an ultrafiltration tube in an ultrafiltration liquid exchange mode to obtain the purified recombinant human interleukin-6.

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a preparation method of recombinant human interleukin-6.

Background

In 1985, the human interleukin-6 gene was first cloned as B cell stimulating factor 2(BSF-2), B cell stimulating factor 2 induces B cells to produce immunoglobulin (Ig), and interleukin-6 was shown to be a typical cytokine.

Interleukin-6 (Tsiagarakis S, Kontogogs G, Glannou P, et al. Interleukin-6, a growth promoting cytokine, is present in human pituitary adenomas: an immunocytochemical study [ J ]. Clinical Endocrinology,1992,37.) is a single-chain glycoprotein used to perform signal-induced B-cell differentiation and differential stimulation or inhibition of cell growth, of different cellular origin, usually secreted by activated macrophages, lymphocytes and epithelial cells, containing 183 amino acid residues, and has a molecular mass of 22-30kDa for interleukin-6 of different cellular origin due to the different degrees of glycosylation and phosphorylation.

Interleukin-6 has a wide range of biological functions, interleukin-6 has pleiotropic effects in inflammation, immune response, hematopoiesis, etc., it can induce the synthesis of acute phase proteins such as hepatic C-reactive protein (CRP), complement C3, fibrinogen, thrombopoietin, serum amyloid A and hepcidin, etc., and inhibit the production of albumin. High concentrations of amyloid protein form complications amyloidosis over time. Interleukin-6 has a concentration dependence in hepatocyte regeneration, and high levels of Interleukin-6 may inhibit hepatocyte regeneration and even exacerbate liver injury, with hepcidin production also contributing to inflammation-associated hypoferremia and anemia. During hematopoiesis, interleukin-6 promotes differentiation of hematopoietic stem cells and maturation of megakaryocytes, resulting in the release of platelets. Interleukin-6 not only promotes fibrinogen production and platelet release, but also activates the coagulation system. Interleukin-6 induces Tissue Factor (TF) on the surface of monocytes, which promotes coagulation by initiating the extrinsic coagulation pathway, leading to the production of thrombin. Interleukin-6 has also been shown to activate Vascular Endothelial (VE) cells. VE-cadherin is an important molecule that promotes endothelial adhesion to adjacent cells by homogeneous intermolecular binding, while interleukin-6 causes its breakdown leading to vascular leakage. In addition, interleukin-6 increases the production of Vascular Endothelial Growth Factor (VEGF), thereby inducing phosphorylation and internalization of VE-cadherin, and thus has a strong vascular permeability effect on endothelial cells. Vascular permeability leads to interstitial edema, increases tissue pressure, and leads to inflammatory injury, either by interleukin-6 itself or by the introduction of VEGF.

Interleukin-6 also plays an important role in the adaptive immune response, and Interleukin-6 can stimulate antibody production, and promote CD4+ cells to induce differentiation into effector T cells. Dysregulation of the sustained or excessive secretion of interleukin-6 results in the development or progression of various diseases. The excessive expression of interleukin-6 is involved in the enlargement of lymph nodes of Castleman disease, and can also enhance the regeneration of blood vessels and the osteoporosis of patients with rheumatoid arthritis. Meanwhile, interleukin-6 also serves as an important proinflammatory cytokine and plays an important role in the amplification cascade of cytokine storms.

Interleukin-6 has numerous biological functions and has been found to be a reliable therapeutic target in several diseases, including sepsis, rheumatoid arthritis, etc., and studies have shown that targeted therapy against interleukin-6 is also effective in some cancer therapies.

However, in the current preparation of recombinant human interleukin-6, such as Li et al (Li, Y.Y., Chen, C.X., Von Specht, B.U., and Hahn, H.P (2002) Protein Expr. Purif.25, 437-447), recombinant human interleukin-6 is expressed as a soluble Protein, which yields soluble and active Protein, but the total yield is low, only 18 ug/L. There are also many reports that the soluble expression amount can be changed by adding molecular chaperone, and the maximum amount can reach 2.6 mg/L. Recombinant human interleukin-6, linked to a maltose binding protein and having a NusA tag, is highly soluble, but isolation of these markers to restore biological activity requires several proteolytic cleavage and purification steps, making this process time and labor consuming.

As with many other recombinant proteins, interleukin-6 may be expressed in E.coli as insoluble aggregates, inclusion bodies. While the interleukin-6 is dissolved by inclusion body and refolded to active structure to become active recombinant protein. Renaturation and purification of the protein are of particular importance. The recombinant human interleukin-6 is obtained by the steps of renaturation, anion exchange chromatography and gel screening purification of the Liuhong rock and the like (the cloning and expression of the gene of the interleukin-6 of the Liuhong rock and the initial detection of the pilot production process [ D ]. Beijing: university of Tonghe medical science of China, 1998.). On-column chromatographic renaturation (Ahmed, Nadeem, Matrix-assisted refolding and purification of plant-derived recombinant human interleukin-6 produced in Escherichia coli [ J ] Biotechnology and Applied Biochemistry,2014,61[5]:541 and 548) has also been used, but these methods have complicated steps and low recovery rates.

Therefore, in order to obtain active interleukin-6, a simple, repeatable, and easily scalable downstream process must be established to achieve large-scale production of recombinant human interleukin-6.

Disclosure of Invention

The invention provides a new preparation method of recombinant human interleukin-6, which is used for solving the technical problems, obtaining a large amount of recombinant human interleukin-6 inclusion bodies in a prokaryotic expression mode, washing the inclusion bodies, diluting and renaturing the inclusion bodies, and preparing the soluble recombinant human interleukin-6 with activity by affinity chromatography purification.

The invention adopts dilution renaturation, and can obtain the recombinant human interleukin-6 with higher purity only by one-step affinity purification. Has the characteristics of simple steps and high recovery rate.

A preparation method of recombinant human interleukin-6 comprises the following steps:

(1) preparing a recombinant human interleukin-6 inclusion body in a prokaryotic expression mode;

(2) washing the recombinant human interleukin-6 inclusion body by using a washing buffer solution A containing urea, washing the recombinant human interleukin-6 inclusion body by using a washing buffer solution B to remove the urea, centrifugally collecting solids, and taking the obtained solids as the primarily purified recombinant human interleukin-6 inclusion body;

(3) adding the recombinant human interleukin-6 inclusion body after preliminary purification into a denatured liquid containing beta-mercaptoethanol until the recombinant human interleukin-6 inclusion body is completely dissolved, slowly adding a renaturation liquid to make the recombinant human interleukin-6 stand at a low temperature for renaturation, and then dialyzing and ultrafiltering for concentration to obtain renatured recombinant human interleukin-6;

(4) and carrying out affinity purification on the renatured recombinant human interleukin-6 inclusion body by using an affinity adsorption column filled with Anti-IL-6 high-affinity adsorption medium to obtain the recombinant human interleukin-6.

Further, the specific method for preparing the recombinant human interleukin-6 inclusion body in the prokaryotic expression mode in the step (1) comprises the following steps: introducing the recombinant human interleukin-6 sequence into a pET-21a or pET-24a expression vector to prepare recombinant plasmid, transforming the recombinant plasmid into E.coli BL21 or E.coli Shuffle T7, expanding expression, collecting thalli for efficiently expressing the recombinant human interleukin-6, inducing the thalli by an IPTG inducer, collecting the thalli, crushing the thalli until bacterial liquid is relatively transparent and is not viscous any more, and collecting precipitates to obtain the recombinant human interleukin-6 inclusion body.

Further, the thallus is crushed by high-pressure homogenate or ultrasonic crushing, and the crushing buffer solution is as follows: 10-30mM Tris-HCl, 10-15ml/L Triton (pH 8-9), according to the mass ratio of 1: 6-1: 10 adding a crushing buffer solution, and stirring by magnetic force to fully suspend the thalli in the crushing buffer solution.

Further, high-pressure homogenate crushing is adopted, the pressure value shows 800bar for 700 plus materials, the circular crushing is carried out for 5-6 times until the bacterial liquid is relatively transparent and is not viscous any more, the thalli are crushed completely, the thalli are centrifuged for 10-20min at 5000rpm for 4000 plus materials at 4-8 ℃, the supernatant is discarded, and the recombinant human interleukin-6 inclusion body sediment is collected.

Wherein Triton is used as a neutral detergent, can effectively dissolve partial lipid and foreign protein, and adopts low-speed centrifugation to achieve the purpose of primary purification, wherein the final concentration of Triton is not less than 1%,

further, in step (2), the washing buffer a is 10 to 50mM Tris-HCl, 1 to 4M urea, 80 to 200mM NaCl (pH 8 to 9), and the washing buffer B is 10 to 50mM Tris-HCl (pH 8 to 9). Wherein the washing with the washing buffer B should remove the excess urea as much as possible.

The high-concentration urea can effectively dissolve the impure protein doped in the target inclusion body, the concentration of the target protein can be effectively improved by properly adjusting the concentration of the urea, the higher the concentration is, the better the impurity removal effect is, and when the concentration of the urea is more than 2M, the recombinant human interleukin-6 inclusion body begins to dissolve.

Further, the denaturant containing beta-mercaptoethanol in the step (3) is a mixed solution of 6-8M guanidine hydrochloride, 100-200mM NaCl, 1-1.5 ‰ beta-mercaptoethanol or a mixed solution of urea with concentration greater than 8M, 100-200mM NaCl, 10-15mM DTT. Compared with urea, guanidine hydrochloride has better dissolving effect.

The specific method of denaturation is to dissolve 20-100mg recombinant human interleukin-6 inclusion body in denaturation liquid, and standing at 4-8 ℃ until the inclusion body is fully dissolved.

Further, the renaturation solution in the step (3) is 0.5-1.5M guanidine hydrochloride, 50-150mM Tris-HCl, 8-12mM EDTA, 0.25-0.5mM GSSG (oxidized form), 2.5-5mM GSH (reduced form), 300-700mM arginine, 2-5% glycerol, and the pH value is 8-9; the speed of adding the renaturation liquid is 0.1-0.5 mL/min; standing at 4-6 deg.C for 20-40 h.

Specifically, 40-200ml of a renaturation solution containing 0.5-1.5M guanidine hydrochloride, 50-150mM Tris-HCl, 8-12mM EDTA, 0.25-0.5mM GSSG (oxidized form), 2.5-5mM GSH (reduced form), 300-700mM arginine and 2-5% glycerol (pH 8-9) was slowly added to the denaturation solution in which the inclusion bodies were dissolved at 0.1-0.5ml/min, and the mixture was stirred at 4-6 ℃.

The guanidine hydrochloride is added into the renaturation solution, so that the renaturation efficiency can be obviously improved, and the low-concentration guanidine hydrochloride can reduce the condition that inclusion bodies are separated out again when the renaturation solution is dripped into the denaturation solution due to rapid change of the environment.

Wherein the concentration of arginine in the renaturation solution can effectively inhibit protein aggregation and improve the renaturation efficiency. The final concentration should be above 300 mM.

Wherein when the concentration of arginine in the renaturation solution is only 200mM, the total renaturation rate of the protein is less than 5 percent.

Further, removing the excessive salt component in the solution by dialysis, wherein the dialysis external solution used in the step (3) is 10-50mM Tris solution. The dialysis solution is changed for more than three times, and the dialysis time is 12-18h each time.

Further, the affinity purification method in the step (4) comprises the following steps: enabling the renatured recombinant human interleukin-6 inclusion body solution obtained in the step (3) to flow through an affinity adsorption column which takes Anti-IL-6 antibody with high affinity as an affinity ligand and CL-6B agarose gel microspheres as a solid-supported medium at a rate of 0.2-1mL/min, and balancing the high affinity adsorption column by using a PBS solution; eluting the adsorption column by using Gly-HCl solution with the pH value of 1.5-2, and replacing the eluted recombinant human interleukin-6 protein into 10-50mM Tris-HCl solution with the pH value of 8-9 by using an ultrafiltration tube in an ultrafiltration liquid exchange mode to obtain the purified recombinant human interleukin-6.

The preparation method of the recombinant human interleukin-6 related by the invention can effectively save the cost by a prokaryotic expression mode, and simultaneously obtains the recombinant human interleukin-6 protein with higher purity and activity by the inclusion body pretreatment, the inclusion body dilution renaturation and the one-step affinity adsorption purification.

The invention can effectively achieve the effects of purification and enrichment only through affinity chromatography purification. The affinity purification method has the characteristics of simple operation, large sample processing, few steps and the like. Effectively reducing the processing steps can reduce the loss and improve the renaturation rate. The recovery rate of the recombinant human leukocyte hormone-6 related by the invention is 21%, and the recovery rate is higher.

Drawings

FIG. 1 is a SDS-PAGE electrophoretic analysis of the primary purification of inclusion bodies of recombinant human interleukin-6 in example 2; in the figure, lane 1: marker; lane 2: bacterial cells before induction in example 1; lane 3: the cells after induction in example 1; lane 4: precipitation after cell disruption in example 1; lane 5: the supernatant after cell disruption in example 1; lane 6: inclusion bodies after washing in example 2.

FIG. 2 is SDS-PAGE electrophoretic analysis before and after adsorption purification of the recombinant human interleukin-6 inclusion body in example 4; in the figure, lane 1: marker; lane 2: before affinity chromatography purification; lane 3: after affinity chromatography purification.

FIG. 3 is the non-denaturing electrophoresis chart of Anti-IL-6 nanobody activity assay in example 5; in the figure, lane 1: Anti-IL-6 nanobody; lane 2, lane 4, lane 6, lane 8, lane 10: mixing the Anti-IL-6 nano antibody with the renatured recombinant human interleukin-6 according to the mass ratio of 1:1, 1:2, 1:3 and 1: 4; lane 3, lane 5, lane 7, lane 9, lane 11: the corresponding antigen.

Detailed Description

The following non-limiting examples will allow one of ordinary skill in the art to more fully understand the present invention, but are not intended to limit the invention in any way.

Example 1

The experimental scheme is the preparation of the recombinant human interleukin-6 inclusion body, and the specific operation is as follows:

the recombinant human interleukin-6 gene sequence (shown as SEQ ID NO. 1) is constructed on a PET-21a expression vector (Biotechnology engineering Co., Ltd., Shanghai) to prepare recombinant plasmids. The constructed recombinant plasmid is transformed into E.coliBL-21 strain, and then is subjected to amplification culture and 4% (volume ratio) inoculation, and is cultured for 6h at 37 ℃ and induced for 5h at 37 ℃, and the final concentration of an inducer IPTG is 0.5 mM. And (3) centrifuging at a high speed to obtain thalli, washing the thalli for three times by using double distilled water, and selecting a crushing solution: 20mM Tris-HCl, 10ml/L Triton (pH 8.5) in a volume ratio of 1: 8 resuspending, homogenizing and crushing by adopting high pressure, displaying the pressure value of 700-800bar, circularly crushing for 5-6 times until the bacterial liquid is relatively transparent and is not viscous any more, after the bacterial cell is crushed, centrifuging for 10min at 5000rpm at 4 ℃, discarding the supernatant, and collecting the inclusion body precipitate.

Example 2

The experimental scheme is the primary purification of the recombinant human interleukin-6 inclusion body, and the specific operation is as follows:

inclusion bodies obtained in example 1 were washed with buffer (a): 20mM Tris-HCl, 2M urea, 100mM NaCl (pH 8.5) at 1: 20 (volume ratio), fully rinsing, centrifuging for 5min at 10000rpm of a centrifuge, discarding the supernatant, repeating for three times, and then washing with a buffer solution (B): 20mM Tris-HCl (pH 8.5) as a precipitate: the washing buffer (B) was 1: 20 (volume ratio), fully rinsing, centrifuging for 5min at 10000rpm of a centrifugal machine, discarding the supernatant, repeating for three times, and subpackaging for later use. The primary purification results are shown in figure 1, lane 1: marker; lane 2: before induction in example 1; lane 3: after induction in example 1; lane 4: the precipitate was broken as in example 1; lane 5: the supernatant was crushed as in example 1; lane 6: recombinant human interleukin-6 inclusion bodies after washing in example 2.

Example 3

The experimental scheme is a renaturation process of the recombinant human interleukin-6 inclusion body, and the specific operation is as follows:

50mg of the recombinant human interleukin-6 inclusion body obtained in example 2 after the primary purification was dissolved in 10ml of a denaturant containing 6M guanidine hydrochloride, 200mM NaCl and 1 ‰ beta-mercaptoethanol, and the dissolution was considered to be completed when no significant precipitate was present. 100ml of a renaturation solution containing 1M guanidine hydrochloride, 100mM Tris-HCl, 8mM EDTA, 0.25mM GSSG (oxidized form), 2.5mM GSH (reduced form), 500mM arginine and 3% glycerol (pH 8.5) was slowly added to the denaturation solution in which the preliminarily purified inclusion body of recombinant human interleukin-6 was dissolved at 0.3ml/min, and the mixture was stirred at 4 to 6 ℃ while being kept under agitation. After the renaturation solution is added, standing for 20 hours at 4 ℃. Using 10mM Tris (pH 8.5) solution as dialysis external liquid, dialyzing at 4 deg.C for more than three times, each time for 12 h.

Ultrafiltering and concentrating the dialyzed protein solution, adding appropriate amount of protein sample (no more than 12ml, no less than 1ml) into ultrafiltration tube with molecular weight cutoff of 3000Da, and centrifuging at 4000g at 4 deg.C for 30 min.

Example 4

The experimental scheme is an adsorption purification process of recombinant human interleukin-6, and the specific operation is as follows:

an affinity adsorption column which takes Anti-IL-6 antibody with high affinity as affinity ligand and CL-6B agarose gel microspheres as a solid-supported medium is adopted. The protein solution with good renaturation in example 3 slowly flows through an affinity adsorption column at 0.5ml/min, the affinity adsorption column is balanced by PBS solution at 1ml/min, the adsorption column is eluted by Gly-HCl solution with pH 2, the eluted recombinant human interleukin-6 protein is replaced into 10mM Tris-HCl solution with pH 8.5 by ultrafiltration, and the purity before and after purification is shown in figure 2, wherein a lane 1: marker; lane 2: before affinity chromatography purification; lane 3: after affinity chromatography purification. The total renaturation was calculated to be 21%.

The renaturation rate is the total amount of protein harvested after renaturation/the total amount of inclusion bodies dosed before renaturation.

Example 5

The experimental scheme is a recombinant human interleukin-6 activity detection process, and the specific operation is as follows:

and verifying the activity of the recombined human interleukin-6 after renaturation by adopting non-denaturing non-reducing electrophoresis. Mixing the Anti-IL-6 nano antibody and the renatured recombinant human interleukin-6 according to the mass ratio of 1:1, 1:2, 1:3, 1:4 and 1:5, sequentially loading the samples, setting a blank control verification result, and setting an electrophoresis result as shown in figure 3, wherein a lane 1: Anti-IL-6 nanobody; lane 2, lane 4, lane 6, lane 8, lane 10: mixing the Anti-IL-6 nano antibody with the renatured recombinant human interleukin-6 according to the mass ratio of 1:1, 1:2, 1:3, 1:4 and 1: 5; lane 3, lane 5, lane 7, lane 9, lane 11: the corresponding antigen.

According to the results, the antibody band becomes shallow gradually with the increase of the ratio of the recombinant human interleukin-6, which proves that the Anti-IL-6 antibody can be combined with the recombinant human interleukin-6, and the activity of the recombinant human interleukin-6 is proved.

SEQUENCE LISTING

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