A simplified macroglobulin capable of inhibiting tumor necrosis factor and its synthesis method

Abstract

The present invention relates to a simplified macroglobulin and a synthesis method capable of inhibiting tumor necrosis factor. The fusion protein gene consisting of a TNF-α targeting peptide and an RBD domain of an α2-macroglobulin is cloned into a plasmid vector to obtain a recombinant expression plasmid, and then the host cell is further transformed and an inducer is used to induce the host cell to overexpress the fusion protein, and the fusion protein is obtained after separation and purification. The TNF-α targeting peptide comprises a sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4. The RBD domain of the α2-macroglobulin comprises a sequence shown in SEQ ID NO.5 or SEQ ID NO.6. The synthesis of a simplified macroglobulin SM that can be used as a TNF-α inhibitor is achieved by a microbial method. The process is mild and efficient, low in cost, low in equipment requirements, and easy to expand production in the later stage.

Description

Technical Field

[0001] The present invention relates to a simplified macroglobulin capable of inhibiting tumor necrosis factor-α (TNF-α) and a synthesis method thereof, and also relates to the gene design of the fusion protein, the construction of a recombinant expression vector containing a fusion protein gene fragment, the fermentation expression in Escherichia coli, the separation and purification method of the fusion protein and the effect on inhibiting inflammation. Background Art

[0002] Tumor necrosis factor (TNF-α) is a proinflammatory cytokine discovered more than 40 years ago and has received widespread attention as a major regulatory factor after tissue damage. TNF-α can amplify inflammation by activating the classic NF-κB pathway and mitogen-activated protein kinase (MAPK) signaling pathway, and in most cases induces apoptosis through the kinase receptor interacting serine / threonine kinase (RIPK1) pathway. Therefore, inhibiting the action of TNF-α is an effective way to relieve inflammation after tissue damage.

[0003] Currently, there are five FDA-approved biologics used to inhibit TNF-α to promote the resolution of inflammation in rheumatoid arthritis (RA), inflammatory bowel disease (IBD), psoriasis, ankylosing spondylitis (AS), juvenile idiopathic arthritis, psoriatic arthritis, Crohn's disease, etc. All of these marketed drugs are monoclonal antibodies (Infliximab, Adalimumab, Certolizumab Pegol, Golimumab) or crystallizable IgG1 fragments (Etanercept) that can bind to soluble tumor necrosis factor receptor 2 (TNFR2). They show high affinity for TNF-α and competitively block the TNF-α-TNFR interaction, thereby exerting an effective therapeutic effect. However, these TNF-α inhibitors are also subject to some limitations, such as high cost and immunogenicity, especially long-term use will increase the risk of lymphoma and optic neuritis.

[0004] With the development of biotechnology, phage display technology and computer simulation have promoted the development of small molecule peptides and used them for targeted binding and inhibition of TNF-α. Although small molecule peptides have great prospects in inhibiting TNF-α, their short half-life and low absorption of the intestinal barrier may limit their clinical application, and no approved products have been obtained so far. Therefore, new TNF-α antagonists as alternatives to TNF-α antibodies are still urgently needed.

[0005] During the immune response, antibodies initially use the antigen epitope in the antigen binding region (Fab) fragment to bind and capture the antigen to form an antigen-antibody complex, which is then cross-linked with the Fc region by the Fc receptor (FcR) or interacts with the complement receptor (CR) to be phagocytosed by leukocytes. 2 -Macroglobulin, as a broad-spectrum protease inhibitor, can regulate protein degradation in complex biological processes. With the capture and binding of peptidases, α 2 - macroglobulin receptor binding domain (RBD) exposure, resulting in, α 2 -macroglobulin-peptidase complexes are internalized and then degraded in lysosomes. 2 -macroglobulin can also regulate the activity of various cytokines and inflammatory factors. 2 - Macroglobulin is usually extracted from blood, which has limited sources, and α 2 -Macroglobulins have complex functions and low specificity.

[0006] Therefore, affected by α 2 -Inspired by the "capture-internalization mechanism" of macroglobulin to regulate proteases and growth factors, we used genetic engineering technology to target TNF-α and α 2 - RBD domain fusion of macroglobulin to form simplified α 2 -Macroglobulin (simplified α 2 -macroglobulin, SM) as a TNF-α inhibitor. Summary of the invention

[0007] The object of the present invention is to provide a simplified macroglobulin capable of inhibiting tumor necrosis factor (TNF-α), wherein the simplified macroglobulin fuses a TNF-α targeting peptide and an α 2 -The RBD domain of macroglobulin. The simplified macroglobulin prepared by the present invention can be used as a TNF-α inhibitor in α 2 -Macroglobulin works in the "capture-internalization" mode, effectively binding to TNF-α, and then being internalized into lysosomes by cells for decomposition of the SM-TNF-α complex, thereby inhibiting the biological function of TNF-α.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] A simplified macroglobulin SM that can inhibit tumor necrosis factor, containing TNF-α targeting peptide and α 2 -A fusion protein of the RBD domain of macroglobulin, wherein the TNF-α targeting peptide comprises the sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4; α 2-The RBD domain of macroglobulin comprises the sequence shown in SEQ ID NO.5 or SEQ ID NO.6.

[0010] The second object of the present invention is to provide a method for synthesizing the simplified macroglobulin SM, which comprises: cloning the gene of the fusion protein into a plasmid vector to obtain a recombinant expression plasmid, transforming the recombinant expression plasmid into a host cell, inducing the host cell to overexpress the fusion protein with an inducer, and obtaining the fusion protein after separation and purification.

[0011] In the above technical solution, further, according to a preferred embodiment of the present invention, the amino acid sequence of the simplified macroglobulin SM is SEQ ID NO. 7, and the nucleotide sequence is SEQ ID NO. 8. The recombinant expression vector is obtained by cloning the simplified macroglobulin SM gene into the Escherichia coli expression vector pET-28a, and the recombinant expression vector contains NcoI and XhoI restriction enzyme sites, T7 strong promoter, lca lactose operon and kanamycin resistance screening gene.

[0012] The host cell expressing the simplified macroglobulin SM is a host cell containing the nucleotide sequence of SEQ ID NO. 8; or a host cell containing a recombinant expression vector. Preferably, the cell is Escherichia coli BL21 (DE3).

[0013] Preferably, the method for synthesizing the simplified macroglobulin SM of the present invention comprises using genetic engineering technology to synthesize the TNF-α targeting peptide and α 2 -The RBD domain of macroglobulin is fused to construct a recombinant expression vector, and then the recombinant expression vector is transformed into Escherichia coli BL21 (DE3) to express the fusion protein, and the simplified macroglobulin SM is purified by column chromatography. The following steps are included:

[0014] (1) Artificially synthesizing a simplified macroglobulin SM gene, the nucleotide sequence of which is shown in SEQ ID NO.8;

[0015] (2) The synthesized simplified macroglobulin SM gene was cloned into the E. coli expression vector pET-28a, and an NcoI restriction enzyme site was added to the 5' end of the fusion protein gene and an XhoI restriction enzyme site was added to the 3' end of the gene to construct a recombinant expression vector;

[0016] ⑶ The successfully constructed recombinant expression vector was transformed into Escherichia coli BL21 (DE3), and the transformants were screened using kanamycin-resistant LB solid culture medium plates;

[0017] (4) Expression of simplified macroglobulin SM protein by fermentation of E. coli BL21 (DE3);

[0018] ⑸ Simplify the separation and purification of macroglobulin SM.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention combines TNF-α targeting peptide and α 2 -The RBD domain of macroglobulin was fused at the gene level to construct a simplified macroglobulin SM that can inhibit tumor necrosis factor (TNF-α). On this basis, the present invention further uses E. coli fermentation to express the fusion protein, and uses affinity chromatography technology to purify the supernatant protein to construct a high-purity separation and purification method. The process is gentle and efficient, low cost, low equipment requirements, and easy to expand production later. The simplified macroglobulin SM developed by the invention retains both the TNF-α targeting peptide and the α 2 -The function of the RBD domain of macroglobulin shows efficient TNF-α binding and inhibition function, and has good cell compatibility, which can effectively reduce the level of cellular inflammation. It is expected to become a new TNF-α antagonist and a substitute for TNF-α antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the recombinant expression vector pET-28a-SM; the recombinant expression vector pET-28a-SM contains a T7 strong promoter, an lca lactose operator, a kanamycin resistance marker site, a simplified macroglobulin SM encoding gene and a hexahistidine tag.

[0022] Figure 2 This is the elution purification diagram of SM protein purified by affinity chromatography.

[0023] Figure 3 To simplify the polyacrylamide gel electrophoresis of macroglobulin SM; it includes a protein Maker lane and a lane for the separated and purified SM protein.

[0024] Figure 4 This is the inhibition curve of SM protein on the cytotoxicity of L929 cells induced by TNF-α / actinomycin D (AMD).

[0025] Figure 5 A quartz crystal microbalance was used to detect the binding of SM protein to TNF-α.

[0026] Figure 6 Flow cytometry was used to detect the endocytosis of macrophages to particles coated with SM protein. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific examples.

[0028] The simplified macroglobulin SM capable of inhibiting tumor necrosis factor of the present invention comprises a TNF-α targeting peptide and an α 2 -A fusion protein of the RBD domain of macroglobulin, wherein the TNF-α targeting peptide comprises the sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4; α 2 -The RBD domain of macroglobulin comprises the sequence shown in SEQ ID NO.5 or SEQ ID NO.6. TNF-α targeting peptide and α 2 -The RBD domains of macroglobulins can be connected by a flexible linker to reduce the influence between the two sequences.

[0029] The gene of the fusion protein is cloned into a plasmid vector to obtain a recombinant expression plasmid. After the recombinant expression plasmid is transformed into a host cell, an inducer is used to induce the host cell to over-express the fusion protein. After separation and purification, the fusion protein, namely simplified macroglobulin SM, can be obtained.

[0030] The simplified macroglobulin SM of the present invention retains both TNF-α targeting peptide and α 2 -The function of the RBD domain of macroglobulin is to 2 -Macroglobulin works in the "capture-internalization" mode, effectively binds to TNF-α, and then is internalized into the lysosome by the cell for the decomposition of the SM-TNF-α complex, thereby inhibiting the biological function of TNF-α, and can be used as a new type of TNF-α antagonist. This effect is caused by the simplified macroglobulin SM containing both TNF-α targeting peptide and α 2 -The structural design of the RBD domain of macroglobulin determines that the TNF-α targeting peptide is the sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4; α 2 -The RBD domain of macroglobulin is the sequence shown in SEQ ID NO.5 or SEQ ID NO.6, both of which can achieve the above effect.

[0031] According to a specific example of the present invention, the fusion protein is expressed by fermentation with E. coli, and codon optimization is performed according to the expression preference of E. coli, so that the fusion protein can be stably overexpressed in E. coli, and the amino acid sequence of the simplified macroglobulin SM is shown in SEQ ID NO. 7. The nucleotide sequence is shown in SEQ ID NO. 8.

[0032] The present invention provides a recombinant expression vector containing the simplified macroglobulin SM gene. The simplified macroglobulin SM gene is cloned into the Escherichia coli expression vector pET-28a to obtain a recombinant expression vector, which is named pET-28a-SM. The recombinant expression vector contains NcoI and XhoI restriction enzyme cutting sites, a T7 strong promoter, an lca lactose operon and a kanamycin resistance screening gene.

[0033] The present invention provides a host cell containing the simplified macroglobulin SM gene, wherein the host cell is a host cell containing the nucleotide sequence shown in SEQ ID NO.7, or a host cell containing the recombinant expression vector pET-28a-SM. Preferably, the cell is Escherichia coli BL21 (DE3).

[0034] Genetic engineering technology was used to synthesize TNF-α targeting peptide and α 2 -The RBD domain of macroglobulin was fused to construct a recombinant expression vector pET-28a-SM, and the pET-28a-SM plasmid vector was transformed into Escherichia coli BL21 (DE3) to express the simplified macroglobulin SM, and the simplified macroglobulin SM was purified by column chromatography.

[0035] According to a specific embodiment of the present invention, the above method for producing simplified macroglobulin SM may include the following steps:

[0036] ⑴ Artificial synthesis of simplified macroglobulin SM gene

[0037] ① Simplify the determination of the macroglobulin SM gene sequence.

[0038] TNF-α targeting peptides and α 2 -The amino acid sequence of the RBD domain of macroglobulin. In order to more conveniently verify the role of these two sequences and to expand the application of SM protein in the material direction, according to the characteristics of increased expression of matrix metalloproteinase (MMP) after tissue damage, a matrix metalloproteinase-cleavable polypeptide sequence (PLGLLA) and a domain containing Dopa were introduced into SM. On the basis of not changing the amino acid sequences of these protein polypeptides, the gene was codon optimized according to the preference of Escherichia coli expression, and the nucleotide sequence SEQ ID NO.8 was obtained.

[0039] ②Synthesis of SM gene.

[0040] The optimized gene sequence was submitted to GENEWISE to artificially synthesize the simplified macroglobulin SM gene.

[0041] (2) The synthesized simplified macroglobulin SM gene was cloned into the E. coli expression vector pET-28a, an NcoI restriction enzyme site was added to the 5' end of the fusion protein gene, and an XhoI restriction enzyme site was added to the 3' end of the gene to construct a recombinant expression vector pET-28a-SM.

[0042] ⑶ The successfully constructed recombinant expression vectors pET-28a-SM and pACYC-de-1-TYR were transformed into Escherichia coli BL21 (DE3), and the transformants were screened using LB solid culture medium plates with dual resistance to kanamycin and chloramphenicol.

[0043] Since the conversion of Dopa in the Dopa domain in the SM protein sequence must be achieved under the action of tyrosinase, in order to achieve the conversion of tyrosine in the protein to Dopa, the recombinant plasmid pACYC-de-1-TYR containing the tyrosinase gene and the recombinant expression vector plasmid pET-28a-SM were mixed evenly with the competent Escherichia coli BL21 (DE3), and the competent Escherichia coli BL21 (DE3) was made to absorb the recombinant expression vector plasmid pET-28a-SM and pACYC-de-1-TYR through a chemical method of ice incubation-heat shock-ice incubation. After recovery, the bacterial solution was spread on a solid culture medium plate containing kanamycin and chloramphenicol double resistance to screen the transformants.

[0044] ⑷Expression of simplified macroglobulin SM by fermentation in Escherichia coli BL21 (DE3).

[0045] E. coli BL21 (DE3) transformants were streaked and activated on LB resistance plates, and single colonies were picked for seed culture. 1 mL of seed culture was transferred to a shake flask containing 50-100 mL of LB medium to expand the culture of E. coli and ferment to express the fusion protein SM. When the concentration of the E. coli culture was OD600 = 0.6-0.8, 0.4-1.5 mM IPTG was used for induction, and the E. coli cells were collected by centrifugation at 4 ° C, 6000-10000 rpm for 20-40 min at 12-20 ° C, 200-300 rpm for 18-24 h.

[0046] ⑸ Simplify the separation and purification of macroglobulin SM.

[0047] After the E. coli fermentation is completed, the cells are collected by centrifugation and resuspended in binding buffer (20-40 mM sodium phosphate, 0.4-0.8 M sodium chloride, 20-40 mM imidazole, pH 7.0-7.4). The bacterial solution is high-pressure broken in an ice-water bath, using a pressure of 800-1000 bar and high-pressure breaking 3-5 times. After centrifugation, the supernatant is collected and filtered with a 0.22 μm membrane to remove solid particles.

[0048] The simplified macroglobulin SM has a hexa-histidine tag, which can react with Ni in the nickel column. 2+ Through coordination bonds and other bonding, imidazole in the solution can compete with Ni 2+ Binding, so high concentration imidazole solution can elute the protein bound to the nickel column. A 5mL Histrap column from GE was used to separate the SM protein supernatant by affinity chromatography. After the Histrap column was washed with water and binding buffer, the supernatant solution that passed through the membrane was loaded onto the column at a flow rate of 0.2-2mL / min to bind the protein to the column. Wash the impurities in the column with wash buffer (20-40mM sodium phosphate, 0.4-0.8M sodium chloride, 20-200mM imidazole, pH7.0-7.4) at a flow rate of 1-5mL / min. After washing, elution buffer (20-40mM sodium phosphate, 0.4-0.8M sodium chloride, 500mM imidazole, pH7.0-7.4) was used together with binding buffer to form a linear eluent to elute the simplified macroglobulin SM bound to the column. The eluted protein solution was dialyzed with PBS for desalting and then freeze-dried.

[0049] Finally, the final purified protein was subjected to polyacrylamide gel electrophoresis to analyze its purity.

[0050] Example 1: Construction of recombinant expression vector and strain

[0051] ① Construction of recombinant expression vector

[0052] Genetic engineering technology was used to synthesize TNF-α targeting peptide and α 2 -The RBD domain of macroglobulin was designed at the gene level, and a matrix metalloproteinase (MMP) cleavable polypeptide sequence (PLGLLA) and a domain containing Dopa were introduced at the same time. Three GGGGS repeat sequences were used as connecting polypeptides. The gene was codon optimized according to the preference of Escherichia coli expression without changing its amino acid sequence to obtain a nucleotide sequence. An NdeI restriction enzyme site was added to the 5' end of the gene sequence and a HindIII restriction enzyme site was added to the 3' end of the gene. The recombinant expression vector pET-28a-SM was artificially synthesized and constructed, such as Figure 1 shown.

[0053] ②Chemical transformation of Escherichia coli

[0054] The correctly constructed recombinant expression vectors pET-28a-SM and pACYC-de-1-TYR were mixed with competent Escherichia coli BL21 (DE3) in proportion and incubated on ice for 30 minutes. After heat shock at 42°C for 45 seconds, they were quickly placed on ice for 3 minutes. 450 μL LB medium was added and shaken for 2 hours. After recovery, 100 μL of bacterial solution was evenly spread on a double-resistant LB solid medium plate containing kanamycin and chloramphenicol for resistance screening. Single colonies grown on the plate were picked for colony PCR by aseptic operation, and the PCR products were subjected to agarose electrophoresis to verify the transformation results. The electrophoresis graph showed a band of about 750 bp, which was consistent with the size of the SM protein gene. The plasmids extracted at the same time were sent to Jinweizhi Company for sequencing. The sequencing results were compared with the gene sequence shown in SEQ ID NO.8. The similarity was greater than 99%, and the gene fragment was considered to be the SM protein gene. The colonies with successful colony PCR and sequencing were the target transformants. The successfully transformed E. coli strains were stored in glycerol at -80°C.

[0055] Example 2: Expression and purification of fusion protein SM

[0056] ①Expression of simplified macroglobulin SM by E. coli fermentation

[0057] The successfully transformed E. coli was streaked and activated on a double-resistant LB solid medium plate containing kanamycin and chloramphenicol. A single colony was picked in a test tube containing 5 mL of LB liquid medium containing 50 μg / mL of kanamycin and chloramphenicol, and cultured overnight at 37°C and 200 rpm. The E. coli seed solution was transferred to a 4L shake flask containing 1L of culture medium at a ratio of 1:100. The growth curve of E. coli was measured with a UV spectrophotometer. After the E. coli transfer, when the bacterial solution concentration was OD600 = 0.8, 1.0 mM isopropyl-β-d-thiogalactoside (IPTG) was used to induce E. coli to express SM protein. The E. coli cells were collected after continuing to culture at 16°C and 250 rpm for 24 hours.

[0058] ②Simplify the separation and purification of macroglobulin SM

[0059] The E. coli fermentation broth was centrifuged at 4°C and 6000rpm for 10 minutes and the culture medium was discarded. The E. coli cells were resuspended in binding buffer (20mM sodium phosphate, 0.5M sodium chloride, 40mM imidazole, pH 7.4), and the bacterial liquid was crushed under high pressure in an ice water bath at a pressure of 800bar, and the crushing cycle was repeated 5 times. The clarified bacterial liquid obtained by crushing was centrifuged at 4°C to obtain the supernatant. Solid particles were removed by filtration with a 0.22μm membrane to prevent clogging during the affinity chromatography process.

[0060] A 5mL Histrap column from GE was used to perform affinity chromatography separation on the SM protein supernatant. The Histrap column was connected to AKATpure. The Histrap column was washed with 10 times the column volume of high-purity water and binding buffer in sequence until the baseline was flat. The protein supernatant solution that passed the membrane was loaded onto the column at a flow rate of 1mL / min to allow the protein to bind to the column. After the loading was completed, wash buffer (20mM sodium phosphate, 0.5M sodium chloride, 50mM imidazole, pH7.4) was used to wash the impurities in the column at a flow rate of 2mL / min until the baseline was flat. After washing, the proteins bound to the column were eluted with a linear imidazole eluent composed of elution buffer (20mM sodium phosphate, 0.5M sodium chloride, 500mM imidazole, pH7.4) and binding buffer, and the eluate was collected when the absorption peak appeared, such as Figure 2 After elution, the Histrap column was washed with 8 column volumes of binding buffer and high-purity water in turn, and finally the Histrap column was stored in 20% ethanol. The eluate was dialyzed with PBS (dialysis bag molecular weight cutoff 8000-14000) for desalination.

[0061] The purified SM protein was dissolved in ultrapure water, added with loading buffer, boiled, and subjected to SDS-PAGE electrophoresis and purity analysis. The purity of the purified SM protein was greater than 80%, and the molecular weight of the purified SM protein was about 21KD. Figure 3 shown.

[0062] Example 3: Nitroblue tetrazolium (NBT) staining to verify Dopa in SM

[0063] Three sample solutions of water, SM protein and protein without tyrosinase co-expression (SM-WD) were dropped on polyvinylidene fluoride (PVDF) membranes. After the protein samples were bound to the membrane, the PVDF membrane was immersed in a 2M potassium glycine (pH 10) solution containing 0.5mg / mL nitro blue tetrazolium (NBT) and treated in the dark for 1h. The PVDF membrane was washed with 0.2M sodium borate (pH 8.5) and high-purity water in turn. The SM protein droplets were blue-purple, indicating that Dopa in the SM protein under the experimental conditions under which the redox reaction occurred, and nitro blue tetrazolium (NBT) was reduced to form blue-purple formazan. The water-treated area was colorless, and the protein without tyrosinase co-expression (SM-WD) showed a slightly yellow protein residue. The NBT staining experiment showed that co-expression of tyrosinase can realize the conversion of tyrosine to Dopa in SM.

[0064] Example 4: Biocompatibility test

[0065] The biocompatibility of SM protein was tested using RAW264.7 macrophages, H9C2 cardiomyocytes, and SWs chondrocytes. Briefly, RAW264.7 cells were seeded at a density of 2 × 10 4 cells / well, and the seeding density of H9C2 and SWs cells was 0.5×10 4 Cells / well were plated in 96-well plates for 12 hours, and 1 mg / mL SM protein was added and cultured for 24 hours. Afterwards, cell viability was measured by CCK8 detection and calcein / indium iodide (PI) cell live and dead staining. It was found that the activity of the three cells was similar to that of normal well plates. After cell live and dead staining, the cells all showed green color, and there were almost no red dead cells.

[0066] Example 5: Inhibition of TNF-α / actinomycin D (AMD) cytotoxicity by SM protein on L929 cells. L929 cells were grown at a density of 1×10 4 Cells / well were seeded into 96-well plates and cultured in MEM medium at 37°C and 5% CO2 for 12 h. Subsequently, SM was serially diluted and mixed with 10 ng / mL TNF-α and 1 μg / mL actinomycin D (AMD), further incubated for 18 h, and cell viability was measured by CCK8. The inhibition rate of TNF-α-mediated cytotoxicity was calculated using the following formula. (ODAMD+TNF-α+SM-ODAMD+TNF-α) / (ODAMD-ODAMD+TNF-α)×100. It can be found that with the increase of SM concentration, TNF-α-mediated cytotoxicity gradually weakened, and the half-maximal inhibitory concentration (IC50) was 4.66 μM. Figure 4 shown.

[0067] Example 6: Detection of the binding effect of SM protein on TNF-α

[0068] Quartz crystal microbalance (QCM-D) was used to qualitatively detect the interaction between SM and TNF-α. The gold sensor chip was heated with 30% HO. 2 O 2 :NH 3 -H 2 O: water = 1:1:5 solution, and N 2Blow dry. During the QCM-D measurement, the fluid flow rate was 50μL / min. The chip was first washed with PBS until the baseline was stable, and then 50μg / mL SM was loaded into the detection system. Because Dopa in SM has a strong adhesion ability, it can be adsorbed on the chip. It was found that the QCM chip frequency (F) dropped significantly, and the frequency did not change even after rinsing with PBS, indicating that SM can form a stable protein adsorption on the chip. After that, 1mg / mL BSA was loaded, and it was found that the chip frequency curve dropped, and the frequency increased after rinsing with PBS, indicating that BSA reversibly adsorbed on the chip and blocked the chip surface where SM was not adsorbed. Subsequently, 50μg / mL TNF-α was loaded, and it was found that it caused the chip frequency to drop sharply, and it was not affected by PBS washing, indicating that TNF-α was firmly captured on the chip surface. The same test was performed without SM and used as a negative control. It was found that after 1mg / mL BSA shielded nonspecific interactions, the loading of TNF-α caused a small drop in the chip frequency, which was about 1 / 4 of that with SM. These results show that SM has a strong interaction with TNF-α.

[0069] Example 7: Enhanced endocytosis of macrophages to particles coated with SM protein

[0070] Nile red was loaded in polyurethane nanoparticles (NPs) as a matrix for SM protein coating. Due to the excellent adhesion ability of Dopa in SM, particle surface modification can be achieved by simple adsorption. First, the NPs were soaked in 0.4 mg / mL BSA or SM solution overnight and then washed three times with ultrasound. The protein concentration on the NPs was then determined using a Micro BCA protein assay kit. In addition, the particle size and ζ potential of the NPs were characterized using a dynamic light scattering nanoparticle sizer. Finally, RAW264.7 cells were used to explore their endocytosis of NPs. First, RAW264.7 cells were seeded in 48-well plates at a cell density of 5×104 / well and then cultured in DMEM medium for 12 h. 400 nM receptor-associated protein (RAP) was added to some RAW264.7 cells and incubated for 1 h as a pretreatment. Then 100 μg / mL NPs were added to all wells and flow cytometry analysis was performed after incubation for 6 h. The results showed that RAW264.7 cells co-incubated with NP@SM showed stronger fluorescence intensity compared with the NP@BSA group. However, the fluorescence intensity of cells in the NP@SM group decreased when pretreated with RAP (an effective LRP1 antagonist). These results indicate that the RBD in SM can promote the endocytosis of NPs by interacting with LRP1 on the cell membrane.

[0071] Example 8: SM protein inhibits macrophage inflammation level

[0072] RAW264.7 cells were cultured at 10 × 10 4 The density of cells / well was cultured in a 24-well plate for 12 hours. Thereafter, 100 ng / mL LPS and 20 ng / mL IFN-γ were added to activate RAW264.7 cells to produce an inflammatory response. At the same time, 1 mg / mL SM was added to verify its efficacy on cellular inflammation. After 8 hours of culture, the supernatant was collected and the concentrations of inflammatory factors TNF-α, IL-6 and IL-1β were measured by ELISA. The cells were stained with 10 μM DCFH-DA in FBS-free culture medium to detect the level of cellular ROS, the fluorescence state of the cells was measured using a microscope, and the fluorescence intensity was further measured using a flow cytometer. The results showed that SM can also effectively reduce the concentrations of TNF-α, IL-6 and IL-1β of RAW264.7 under the stimulation of LPS and IFN-γ, and the cellular ROS level is also similar to that of the unstimulated control group, indicating that SM protein can effectively inhibit the level of macrophage inflammation. The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the above embodiments have described the present invention in detail, those skilled in the art can still modify the technical solutions of the above embodiments, or replace some of the technical features therein by equivalents, to achieve the final preparation of the fusion protein. It should be particularly pointed out that any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A simplified macroglobulin capable of inhibiting tumor necrosis factor, characterized in that: The simplified macroglobulin is a fusion protein containing a TNF-α targeting peptide and an RBD domain of α2-macroglobulin; the amino acid sequence of the fusion protein is SEQ ID NO.7, and the nucleotide sequence is SEQ ID NO.

8.

2. The method for synthesizing simplified macroglobulin according to claim 1, characterized in that: The recombinant expression vector is obtained by cloning the simplified macroglobulin gene into the Escherichia coli expression vector pET-28a. The recombinant expression vector contains NcoI and XOt Restriction enzyme sites, T7 strong promoter, lac The invention relates to a novel protein encoding a lactose operon and a kanamycin resistance screening gene. The host cell expressing the simplified macroglobulin is a host cell containing the nucleotide sequence of SEQ ID NO.8, or a host cell containing the recombinant expression vector. The recombinant plasmid pACYC-de-1-TYR containing the tyrosinase gene is transformed into the host cell together with the recombinant expression vector.

3. The method for synthesizing simplified macroglobulin according to claim 2, characterized in that: The host cell is Escherichia coli BL21 (DE3).

4. The method for synthesizing simplified macroglobulin according to claim 2, characterized in that: The TNF-α targeting peptide was fused with the RBD domain of α2-macroglobulin using genetic engineering technology to construct a recombinant expression vector, and then the recombinant plasmid pACYC-de-1-TYR containing the tyrosinase gene and the recombinant expression vector were transformed into Escherichia coli BL21 (DE3) to express the fusion protein, and the protein was purified by column chromatography.

5. The method for synthesizing simplified macroglobulin according to claim 2, characterized in that: The following steps are involved: (1) Artificially synthesizing a simplified macroglobulin gene, the nucleotide sequence of which is shown in SEQ ID NO.8; ⑵ The synthesized simplified macroglobulin gene was cloned into the E. coli expression vector pET-28a, and the fusion protein gene was added to the 5' end NcoI Restriction enzyme sites were added at the 3' end of the gene XOt Restriction enzyme cutting sites, construction of recombinant expression vector; ⑶ The recombinant plasmid pACYC-de-1-TYR containing the tyrosinase gene and the successfully constructed recombinant expression vector were transformed into Escherichia coli BL21 (DE3), and the transformants were screened using LB solid culture medium plates with kanamycin and chloramphenicol double resistance; (4) Expression of simplified macroglobulin by fermentation with E. coli BL21 (DE3); ⑸ Simplify the separation and purification of macroglobulin.

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