Recombinant sST2 protein as well as preparation method and application thereof

Abstract

The invention provides a recombinant sST2 protein. The amino acid sequence of the recombinant sST2 protein is as shown in SEQ ID NO. 3 or SEQ ID NO. 5. Wherein the nucleotide sequence for coding the amino acid shown as SEQ ID NO.3 is shown as SEQ ID NO.4, and the nucleotide sequence for coding the amino acid shown as SEQ ID NO.5 is shown as SEQ ID NO.6. The invention also discloses a preparation method of the amino acid sequence. The recombinant sST2 protein prepared by the invention has good performance, the modified recombinant protein has higher titer and is superior to the recombinant protein of the original sequence, the purity of the recombinant protein is greater than or equal to 95%, and the activity is greater than or equal to 95%; the protein stability is better, and the protein can be stored for one year at-20 DEG C before remelting and can be stored for more than 3 months at-20 DEG C after remelting; the recombinant protein is close to natural protein and has good intercommunity with clinical samples. The recombinant protein prepared by the invention can be used for preparing a calibrator and a quality control product in the target spot detection kit, and also can be used for related tests such as drug targeted analysis and drug research and development.

Description

Technical Field

[0001] The invention belongs to the technical field of recombinant protein preparation, and specifically relates to a recombinant sST2 protein and a preparation method and application thereof. Background Art

[0002] Growth-stimulated expressed gene 2 protein (ST2) consists of 328 amino acids. ST2 is a member of the interleukin 1 receptor family, formally known as interleukin 1 receptor-like 1 (IL1RL-1), with 2 major isoforms: transmembrane or cellular (ST2L) and soluble or circulating (sST2) forms.

[0003] ST2 is a receptor for IL-33, an IL-1-like cytokine that is secreted by living cells following cell injury. IL-33 exerts its cellular function by binding to a receptor complex composed of ST2L and IL-1R accessory proteins. The IL-33 / ST2 system is upregulated in cardiomyocytes and fibroblasts in response to mechanical stimulation or injury. The interaction between IL-33 and ST2L has been shown to be cardioprotective, reducing myocardial fibrosis, preventing cardiomyocyte hypertrophy, reducing apoptosis, and improving myocardial function in experimental models. The beneficial effects of IL-33 are specifically mediated through the ST2L receptor. sST2 strongly binds to IL-33, thereby interrupting the IL-33 / ST2L interaction and thereby abolishing the anti-remodeling effect; therefore, sST2 is considered a decoy receptor. In recent years, knowledge about the role of ST2 in the pathophysiology of cardiovascular disease has been extensively expanded, with a strong link to myocardial dysfunction, fibrosis, and remodeling.

[0004] Based on this, there is an urgent need to provide a recombinant protein with immune activity, higher potency, stable performance, and good interoperability with clinical samples, so that it can be widely used in target detection kits, drug target analysis, drug development and other related experiments. Summary of the invention

[0005] In view of this, the present invention is based on the original sequence of sST2 protein, and on this basis, in order to improve the performance of the recombinant protein, two sequences are site-directed mutated, and a gene encoding the polypeptide sequence DYKDDDDK is added to its N-terminus, and finally two recombinant proteins with sST2 immunoactivity are obtained. The prepared recombinant proteins have good performance, higher titer, better stability, and the recombinant proteins are close to natural proteins and have good interoperability with clinical samples.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: One of the purposes of the present invention is to provide a recombinant sST2 protein, the amino acid sequence of the recombinant sST2 protein is shown as SEQ ID NO.3 or SEQ ID NO.5.

[0007] The second object of the present invention is to provide a nucleotide sequence encoding the above-mentioned recombinant sST2 protein, wherein the nucleotide sequence is shown in SEQ ID NO.4 or SEQ ID NO.6; Among them, the nucleotide sequence encoding the amino acid shown in SEQ ID NO.3 is shown in SEQ ID NO.4, and the nucleotide sequence encoding the amino acid shown in SEQ ID NO.5 is shown in SEQ ID NO.6.

[0008] The third object of the present invention is to provide an expression vector containing the above nucleotides.

[0009] A fourth object of the present invention is to provide a host cell containing the above expression vector.

[0010] A fifth object of the present invention is to provide a method for preparing the above-mentioned recombinant sST2 protein, comprising the following steps: S1. Insert the nucleotide sequence shown in SEQ ID NO.4 or SEQ ID NO.6 into a vector to obtain an expression vector, and transform it into competent cells. After culturing, extract the endotoxin-free plasmid; S2. The endotoxin-free plasmid is transfected into the host cells, and after cultivation, the cells are subjected to cell lysis, affinity chromatography, washing and elution, ion exchange chromatography, dialysis and concentration to obtain the recombinant sST2 protein.

[0011] In some specific embodiments, preferably, in step S1, the vector is pcDNA3.1 and the competent cell is DH5α.

[0012] In some specific embodiments, preferably, the matrix in the affinity chromatography is SIGMA Anti-DYKDDDDK affinity purification gel; The buffer used in the affinity chromatography process is as follows: Balance buffer: 50mM Tris-HCl, 300mM NaCl, pH 7.5.

[0013] Washing buffer: 50mM Tris-HCl, 300mM NaCl, pH 7.5.

[0014] Elution Buffer: 20 mM HEPES pH 8.0, 300 mM NaCl, 0.1 mg / mL synthetic peptide DYKDDDDK, 0.1* cOmpleteTM Protease Inhibitor Cocktail, 0.1 mM AEBSF.

[0015] In some specific embodiments, preferably, the buffer used in the ion exchange chromatography process is as follows: Balance buffer: 20mM HEPES, 50mM NaCl, pH 8.0.

[0016] Binding Buffer: 20mM HEPES, 50mM NaCl, pH 8.0.

[0017] Elution Buffer A: 20 mM HEPES, 150 mM NaCl, 10% glycerol, pH 8.0.

[0018] Elution Buffer B: 20 ​​mM HEPES, 300 mM NaCl, 10% glycerol, pH 8.0.

[0019] Elution Buffer C: 20 mM HEPES, 500 mM NaCl, 10% glycerol, pH 8.0.

[0020] In some specific embodiments, preferably, the buffer formula used in the dialysis process is as follows: 20 mM Tris-HCl, 200 mM NaCl, 50% Glycerol, PH7.5, 1 mM TCEP.

[0021] The application of the above-mentioned recombinant sST2 protein in target detection kits, drug targeted analysis, and drug research and development.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The recombinant sST2 protein provided by the present invention has a purity of ≥95% and an activity of ≥95%. The quality control product prepared by the recombinant sST2 protein can be stable for 6-9 days at 37°C and for 6-12 months at -20°C, which is much higher than the original protein. The prepared control product has a CV within and between tubes of no more than ±10%, and has good uniformity. The intercommunication with the natural protein is good, and the correlation R 2 ≥0.90. The prepared recombinant sST2 protein can be well applied in target detection kits, drug targeted analysis, drug research and development and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the plasmid map of recombinant sST2 protein.

[0024] Figure 2 This is the WB detection result of recombinant sST2 protein.

[0025] Figure 3 This is the SDS-PAGE electrophoresis of recombinant sST2 protein.

[0026] Figure 4 This is a diagram for the interchangeability analysis of recombinant sST2 protein. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0028] Example 1 The present invention provides a recombinant sST2 protein, and the preparation process thereof is as follows: S1. Obtaining the coding sequence of recombinant sST2 protein The gene encoding the active sequence (Lys19-Phe328) of the sST2 protein (Gene ID: Q01638-2) was screened, subjected to site-directed mutagenesis, and a gene encoding the polypeptide sequence DYKDDDDK was added to its N-terminus for affinity purification after the expression of the recombinant sST2 protein.

[0029] Original amino acid sequence of sST2 protein SEQ ID NO.1:MGFWILAILTILMYSTAAKFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQLLKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLMYSTVSGSEKNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAH KSFLVIDNVMTEDAGDYTCKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEVEIGKNANLTCSACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNPSKECF.

[0030] The nucleotide sequence encoding the original sST2 protein is SEQ ID NO.2:ATGGGCTTCTGGATCCTGGCCATCCTGACAATCCTCATGTACAGCACAGCCGCCAAGTTCAGCAAACAGAGCTGGGGCCTGGAAAACGAGGCCCTGATTGTGCGGTGCCCTAGACAGGGCAAGCCTAGCTACACCGTGGACTGGTACTACTCTCAGACCAACAAGTCTATCCCCACACAGGAGAGAAACAGAGTGTTCGCCTCTGGCCAGCTGCTGAAGTTCCTGCCTGCTGCTGTGGCCGATAGCGGCATCTACACCTGCATCGTCCGGAGCCCTACATTTAACCGGACCGGCTACGCCAATGTGACCATCTATAAGAAGCAAAGCGACTGCAACGTTCCTGATTACCTGATGTATTCTACAGTGTCTGGAAGCGAGAAGAATAGCAAAATCTACTGCCCCACCATCGACCTGTACAACTGGACCGCTCCTCTGGAATGGTTCAAGAACTGCCAGGCCCTCCAGGGATCTAGATACAGAGCCCATAAGTCCTTCCTGGTGATCGACAACGTGATGACCGAGGACGCCGGCGACTACACCTGTAAATTCATCCACAACGAGAACGGCGCCAACTACAGCGTGACCGCTACAAGAAGCTTTACCGTCAAGGACGAGCAGGGATTCAGCCTGTTCCCCGTGATCGGCGCACCAGCCCAGAACGAGATCAAGGAAGTGGAAATCGGCAAAAATGCCAACCTGACCTGTAGCGCCTGTTTTGGCAAGGGCACCCAGTTCCTGGCTGCCGTGCTGTGGCAGCTGAATGGCACCAAGATCACAGATTTCGGCGAGCCAAGAATTCAGCAGGAAGAGGGCCAAAACCAGTCCTTTTCCAACGGACTGGCCTGCCTTGATATGGTGCTGCGGATCGCCGACGTGAAGGAGGAAGATCTGCTGCTGCAGTACGACTGCCTGGCCCTGAACCTGCACGGTCTGCGCAGACACACAGTGCGGCTGAGCAGAAAGAATCCTAGCAAGGAATGCTTC。.

[0031] Amino acid sequence of recombinant sST2 protein mutant 1, SEQ ID NO.3: MGFWILAILTILMYSTAAKFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQLLKFLPAAVADSGIYTCIVRSPTFNRTGYAVVTIYKKQSDCNVPDYLMYSTVSGSEKNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAHKSFLVIDNVMTEDAGDYTCKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEVEIGKNANLTCSACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNPSKECF。

[0032] The nucleotide sequence encoding the recombinant sST2 protein mutant 1 is SEQ ID NO.4:ATGGGCTTTTGGATCCTGGCCATCCTGACAATCCTGATGTACAGCACCGCCGCCAAGTTCAGCAAGCAGTCTTGGGGCCTCGAGAACGAGGCCCTGATCGTCAGATGTCCCAGACAGGGCAAGCCCAGCTACACCGTGGATTGGTACTACAGCCAGACCAACAAGAGCATCCCCACACAAGAGCGGAACCGGGTGTTCGCTTCTGGCCAGCTGCTGAAGTTTCTGCCTGCCGCCGTTGCCGACAGCGGCATCTACACATGTATTGTGCGGAGCCCCACCTTCAACAGAACCGGCTACGCCGTCGTGACCATCTACAAGAAGCAGAGCGACTGCAACGTGCCCGACTACCTGATGTATTCCACCGTGTCCGGCAGCGAGAAGAACAGCAAGATCTACTGCCCCACCATCGACCTGTACAACTGGACAGCCCCTCTGGAATGGTTCAAGAACTGTCAGGCCCTCCAGGGCAGCAGATACAGAGCCCACAAGAGCTTTCTGGTCATCGACAACGTGATGACCGAGGACGCCGGCGACTACACATGCAAGTTCATCCACAACGAGAACGGCGCCAACTACAGCGTGACCGCCACCAGATCCTTTACCGTGAAGGACGAGCAGGGCTTCTCTCTGTTCCCTGTGATTGGAGCCCCTGCTCAGAACGAGATCAAAGAGGTGGAAATCGGCAAGAACGCCAATCTGACCTGCAGCGCCTGCTTCGGCAAGGGCACACAATTTCTGGCTGCCGTGCTGTGGCAGCTGAACGGCACCAAGATCACCGATTTCGGCGAGCCCCGGATCCAGCAAGAGGAAGGCCAGAATCAGAGCTTCAGCAACGGCCTGGCCTGCCTGGATATGGTGCTGAGAATCGCCGACGTGAAAGAAGAGGACCTGCTGCTGCAGTACGACTGTCTGGCCCTGAATCTGCACGGCCTGAGAAGGCACACAGTGCGGCTGAGCAGAAAGAACCCCAGCAAAGAGTGCTTC。.

[0033] Amino acid sequence of recombinant sST2 protein mutant 2, SEQ ID NO.5: MGFWILAILTILMYSTAAKFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQLLKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLMYSTVSGSEKNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAHKSFLVIDNVMTEDAGDYTCKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEVEIGKNANLTCSACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSYSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNPSKECF。

[0034] The nucleotide sequence encoding the recombinant sST2 protein mutant 2 is SEQ ID NO.6:ATGGGCTTTTGGATCCTGGCCATCCTGACAATCCTGATGTACAGCACCGCCGCCAAGTTCAGCAAGCAGTCTTGGGGCCTCGAGAACGAGGCCCTGATCGTCAGATGTCCCAGACAGGGCAAGCCCAGCTACACCGTGGATTGGTACTACAGCCAGACCAACAAGAGCATCCCCACACAAGAGCGGAACCGGGTGTTCGCTTCTGGCCAGCTGCTGAAGTTTCTGCCTGCCGCCGTTGCCGACAGCGGCATCTACACATGTATTGTGCGGAGCCCCACCTTCAACAGAACCGGCTACGCCAACGTGACCATCTACAAGAAGCAGAGCGACTGCAACGTGCCCGACTACCTGATGTATTCCACCGTGTCCGGCAGCGAGAAGAACAGCAAGATCTACTGCCCCACCATCGACCTGTACAACTGGACAGCCCCTCTGGAATGGTTCAAGAACTGTCAGGCCCTCCAGGGCAGCAGATACAGAGCCCACAAGAGCTTTCTGGTCATCGACAACGTGATGACCGAGGACGCCGGCGACTACACATGCAAGTTCATCCACAACGAGAACGGCGCCAACTACAGCGTGACCGCCACCAGATCCTTTACCGTGAAGGACGAGCAGGGCTTCTCTCTGTTCCCTGTGATTGGAGCCCCTGCTCAGAACGAGATCAAAGAGGTGGAAATCGGCAAGAACGCCAATCTGACCTGCAGCGCCTGCTTCGGCAAGGGCACACAATTTCTGGCTGCCGTGCTGTGGCAGCTGAACGGCACCAAGATCACCGATTTCGGCGAGCCCCGGATCCAGCAAGAGGAAGGCCAGAATCAGAGCTACAGCAACGGCCTGGCCTGCCTGGATATGGTGCTGAGAATCGCCGACGTGAAAGAAGAGGACCTGCTGCTGCAGTACGACTGTCTGGCCCTGAATCTGCACGGCCTGAGAAGGCACACAGTGCGGCTGAGCAGAAAGAACCCCAGCAAAGAGTGCTTC。.

[0035] The above three nucleotide sequences (SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6) were all synthesized by a biological company.

[0036] S2. Construction of recombinant plasmid The three nucleotide sequences (SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6) obtained in step S1 were inserted into the vector pcDNA3.1 (Invitrogen) in sequence to form the recombinant expression plasmids pcDNA3.1-sST2-1, pcDNA3.1-sST2-2, and pcDNA3.1-sST2-3, respectively. The above process was completed by the biological company, and the recombinant plasmid and test report were returned. The plasmid map is shown in Figure 1 As shown; wherein, the sST2 sequence can be the original sequence, mutant 1 and mutant 2 respectively.

[0037] S3. Extraction of recombinant plasmid The recombinant plasmids pcDNA3.1-sST2-1, pcDNA3.1-sST2-2, and pcDNA3.1-sST2-3 obtained in the previous step were transformed into DH5α competent cells (Cat. No. CD001, Wuhan Abotek) to obtain single colonies. Pick a single colony and culture it in LB medium overnight, and use the endotoxin-free plasmid mini-extraction kit (Cat. No. DP118-02, Beijing Tiangen Biochemical) to extract the corresponding endotoxin-free pcDNA3.1-sST2-1, pcDNA3.1-sST2-2, and pcDNA3.1-sST2-3 recombinant plasmids. The specific steps are as follows: (1) Transformation: Take 50 μL of pre-cooled DH5ɑ competent cells, add 1 μL of endotoxin-free pcDNA3.1-sST2-1, pcDNA3.1-sST2-2, and pcDNA3.1-sST2-3 recombinant plasmids respectively, treat them in an ice bath for 30 min, then heat them at 42°C for 90 s (strictly control the time), and treat them in an ice bath for 5 min; then add 500 μL of antibiotic-free LB medium to the transformation system on a clean bench, and resuscitate them in a shaker at 37°C and 220 rpm for 45 min; centrifuge at 6000 rpm for 1 min, collect the bacteria, keep 200 μL of the bacterial solution, mix it with a pipette, and add it to an ampicillin-resistant plate, pour in 4 to 6 glass beads, gently shake the plate to evenly spread the bacterial solution, pour out the glass beads, and invert the plate in a 37°C incubator for overnight incubation.

[0038] (2) Extraction of endotoxin-free plasmids: Pick a single colony from the above plate and culture it in 10 mL of LB medium containing ampicillin resistance overnight. Centrifuge at 12,000 rpm for 10 min the next morning, and extract the recombinant plasmid according to the instructions of the endotoxin-free plasmid mini-extraction kit to obtain the corresponding endotoxin-free recombinant pcDNA3.1-sST2-1, pcDNA3.1-sST2-2, and pcDNA3.1-sST2-3 plasmids.

[0039] S3. Expression and purification of recombinant sST2 protein 1. Transient Transfection The endotoxin-free recombinant pcDNA3.1-sST2-1, pcDNA3.1-sST2-2, and pcDNA3.1-sST2-3 plasmids prepared above were respectively transformed into HEK293F cells. The specific process is as follows: (1) Before transfection, subculture and expand HEK293 cells until the cell density reaches about 3~4×10 6 viable cells / mL, viability ≥ 95%.

[0040] (2) One day before transfection, culture the cells in step (1) at a rate of 1.5-1.8×10 6 The cells were inoculated at a density of 10 cells / mL.

[0041] (3) On the day of transfection, adjust the cell density to 3.0×10 6 / mL, the volume is adjusted to 25.5mL, and the cell viability for transfection should be ≥95%. Note: Discard the remaining cells and do not reuse high-density cells for routine subculture.

[0042] (4) Dilute 30 µg of plasmid DNA with 1.5 mL of Opti MEM medium and mix by rotating and / or inverting the tube.

[0043] (5) Dilute 75 μL of PEIMAX (1 mg / mL, pH 7.1) with 1.5 mL of Opti MEM medium and mix by swirling and / or inverting the tube.

[0044] (6) Add the diluted PEIMAX to the diluted recombinant plasmid DNA, rotate and / or invert the tube or gently pipette 2 to 3 times to mix, and incubate the complex at room temperature for about 20 minutes.

[0045] (7) Slowly add the complex to the cell shake flask in step (3), gently shaking the shake flask during the addition process.

[0046] (8) Return the shake flask to the 37°C incubator and incubate under the following conditions: 125 rpm, 8% CO2, 37°C, and 95% humidity.

[0047] (9) On day 1, 16 to 22 hours after transfection, add 3% (v / v) 293-ProFeed, 3 g / L Glucose, and 1 mM VPA (sodium valproate) to the shake flask. Gently shake the shake flask during the addition process and return the shake flask to the 37°C incubator. (10) On day 3, during transient expression, the glucose concentration was maintained above 4 g / L and the cells were harvested when the cell viability was less than 70%.

[0048] 1.5 mL of feed was added for the first time 16 to 20 hours after transfection; cell viability was measured for 3 consecutive days, as shown in the following table:

[0049] 2. Western Blot (WB) Detection (1) Take 5 μL of the supernatant sample for SDS-PAGE electrophoresis. The electrophoresis adopts the constant voltage mode: 80V for 5% concentrated gel. When the marker begins to separate for about 25 minutes, adjust to 120V and stop the electrophoresis when bromophenol blue reaches the bottom of the separation gel.

[0050] (2) Transfer assembly sequence: transfer clamp black side (negative electrode) - sponge pad - 3 layers of filter paper - glue - membrane - 3 layers of filter paper - sponge pad - red side (positive electrode). Transfer time: 200mA, 90~180min.

[0051] (3) Blocking: After the transfer is completed, mark and wash away the transfer solution (TBST, 5 min, 2 times); place the cleaned membrane in a container containing 3% skim milk (prepared in TBST) and block at room temperature for 60-90 min; (4) Flag-tag primary antibody incubation: After blocking, pour out the blocking solution. Add primary antibody solution diluted 1:7000 with 3% skim milk (TBST), shake gently on a shaker, and incubate at room temperature for 2 hours or at 4°C overnight (after incubation at 4°C, incubate at room temperature for another 15-30 minutes). After primary antibody incubation, pour out the primary antibody solution; rinse the membrane 4 times with TBST, 5 minutes each time.

[0052] (5) Secondary antibody incubation: Before the primary antibody incubation is completed, dilute the enzyme-labeled secondary antibody corresponding to the primary antibody species at 1:5000 to the amount required for the experiment (TBST dilution). Place the cleaned membrane in a container containing the secondary antibody solution, shake it slowly on a shaker, and incubate it at room temperature for 60 to 80 minutes. After the secondary antibody incubation is completed, pour out the secondary antibody solution; rinse the membrane 4 times with TBST, 5 minutes each time.

[0053] (6) Exposure: Use tweezers to remove the membrane from TBST, drain it appropriately, and place it on the gel tray. Mix equal volumes of ECL Solution I and Solution II, and evenly add them to the membrane to completely cover it. Allow the substrate to react with the membrane for about 30 seconds, and place it on the chemiluminescence imaging system. Set the exposure time to 3 seconds, 10 seconds, 30 seconds, 60 seconds, and 120 seconds.

[0054] (7) Result analysis: WB results are shown in Figure 2 .Depend on Figure 2 It can be seen that the recombinant human sST2 protein is mainly expressed in the soluble supernatant.

[0055] In this example, HEK293F was used as a host cell, and the above-mentioned recombinant plasmids pcDNA3.1-sST2-1, pcDNA3.1-sST2-2, and pcDNA3.1-sST2-3 were introduced into the host cells by transient transfection to obtain cells expressing the recombinant sST2 protein. This method has the effect of a short transfection cycle and rapid acquisition of the target protein, and can be applied to the exploration and large-scale high-throughput screening of proteins in the research and development stage.

[0056] S4. Recombinant protein purification (1) Cell lysis Ultrasonic lysis: power 250 W, time 5 min, 3s / 3s (ultrasonication 3s, stop 3s), twice.

[0057] Lysis solution: 50mM Tris-HCl pH7.5, 300mM Nac, 0.1* cOmpleteTM ProteaseInhibitor Cocktail, 0.1mM AEBSF, 1% Triton X-100.

[0058] After breaking the bacteria, centrifuge at 12000 rpm and 4°C for 15 min and take the supernatant.

[0059] Generally, every two tubes of cells are resuspended with 30 mL of lysis solution and ultrasonicated. During centrifugation, the supernatant of the first centrifugation can be poured into an extra centrifuge tube, and then the supernatant can be poured into a new centrifuge tube after the second centrifugation.

[0060] (2) Affinity chromatography Matrix: SIGMA Anti-DYKDDDDK Affinity Purification Gel.

[0061] Incubation: Place on a rotating incubator, 20 rpm, 4°C and incubate for 4 hours.

[0062] A. Column flow penetration: After the incubation, balance the centrifuge tube and centrifuge at 4°C and 600 rpm for 10 min. Pour the supernatant after centrifugation into a new centrifuge tube, which is the flow-through. At the same time, keep about 5 mL of the supernatant to suspend the matrix and transfer the matrix to the purification column tube. After the flow-through is completed, collect the flow-through (FT).

[0063] B. Washing and elution a. Add 10mL Washing Buffer to the purification column tube to wash the column and remove the impurities in the matrix. Use gravity flow, and collect the effluent with a sterilized 5mL EP tube. The EP tube needs to be inserted on ice to keep it low temperature. After the flow is completed, use G250 to detect (take 100μL G250 in a 96-well plate and add 10μL of the elution solution that is dripping out). If the G250 turns blue, continue to add 5mL Washing Buffer to wash the column until the G250 does not turn blue, and the Washing Buffer pre-elution is completed. b. Add 1mL Elution Buffer to wash the column and wash off the proteins bound to the matrix. Collect the effluent with a 1.5mL endotoxin-free EP tube by gravity flow. The collection tube needs to be placed on an ice box to keep it low temperature. After the flow is completed, use G250 to detect (take 100μL G250 in a 96-well plate and add 10μL of the elution solution that is dripping out). If G250 turns blue, continue eluting until G250 does not turn blue, and end the Elution Buffer elution.

[0064] c. Prepare samples of the collected flow-through and eluate, generally 6× loading buffer, i.e. 20 μL protein + 5 μL 6× loading buffer, for SDS PAGE electrophoresis.

[0065] Among them, the buffer used in the affinity chromatography process is as follows: Balance buffer: 50mM Tris-HCl, 300mM NaCl, pH7.5.

[0066] Washing buffer: 50mM Tris-HCl, 300mM NaCl, pH7.5.

[0067] Elution Buffer: 20 mM HEPES pH 8.0, 300 mM NaCl, 0.1 mg / mL synthetic peptide DYKDDDDK, 0.1* cOmpleteTM Protease Inhibitor Cocktail, 0.1 mM AEBSF.

[0068] C. Collect the flow-through in step a and repeat step (2) twice. Combine the proteins obtained from the three affinity reactions and perform ion exchange.

[0069] (3) Ion exchange chromatography (Q column) A. Desalination: Take a suitable dialysis bag, rinse it, and add the protein collected in the previous step into the dialysis bag. Place the dialysis bag in the prepared desalting buffer (20mM HEPES, 50mM NaCl, pH8.0). Generally, 50mL of sample is dialyzed with 2L of buffer at 4℃ for 2h.

[0070] B. Equilibration of medium and incubation: Use Binding Buffer to balance the matrix, usually 6 to 10 column volumes. Mix the balanced matrix and desalted protein solution in a centrifuge tube, and place the centrifuge tube on a rotating incubator at 20 rpm and 4°C for 40 minutes.

[0071] C. Use 10~15 column volumes of Binding Buffer to add to the purification column tube to wash off the impurities with weaker binding ability on the matrix, control the flow rate to 2mL / min, and use G250 to detect after the flow (take 100μL G250 in a 96-well plate and add 10μL of the elution solution that is dripping out). If the G250 turns blue, continue eluting until the G250 does not turn blue, collect the effluent, and the collection tube needs to be inserted on ice to keep it cold. Add Elution Buffer A to wash the column, control the flow rate to 0.6mL / min, and use an endotoxin-free tube to collect the effluent. Each column volume of effluent is collected in an EP tube (the collection tube should be placed on an ice box to keep it cold). After the flow is completed, use G250 to detect (take 100μL G250 in a 96-well plate and add 10μL of the elution solution that is dripping out). If the G250 turns blue, continue eluting until the G250 does not turn blue.

[0072] Add elution buffer B to wash the column, control the flow rate to 0.6mL / min, collect the effluent with an endotoxin-free tube, collect the effluent of each column volume in an EP tube (the collection tube should be placed on an ice box to keep it low temperature), and detect it with G250 after the flow is completed (take 100μL G250 in a 96-well plate and add 10μL of the dripping elution solution). If G250 turns blue, continue eluting until G250 stops turning blue.

[0073] Add Elution Buffer C to wash the column, control the flow rate to 0.6mL / min, collect the effluent with an endotoxin-free tube, collect the effluent of each column volume in an EP tube (the collection tube should be placed on an ice box to keep it low temperature), and detect it with G250 after the flow is completed (take 100μL G250 in a 96-well plate and add 10μL of the dripping elution solution). If G250 turns blue, continue eluting until G250 stops turning blue.

[0074] The buffer used for ion exchange is as follows: Balance buffer: 20mM HEPES, 50mM NaCl, pH8.0.

[0075] Binding Buffer: 20mM HEPES, 50mM NaCl, pH8.0.

[0076] Elution Buffer A: 20 mM HEPES, 150 mM NaCl, 10% glycerol, pH 8.0.

[0077] Elution Buffer B: 20 ​​mM HEPES, 300 mM NaCl, 10% glycerol, pH 8.0.

[0078] Elution Buffer C: 20 mM HEPES, 500 mM NaCl, 10% glycerol, pH 8.0.

[0079] The eluted samples are collected and prepared, generally using 6× loading buffer, i.e., 20 μL protein + 5 μL 6× loading buffer, for SDS PAGE electrophoresis.

[0080] The results are as follows Figure 3 As shown: After Q column ion exchange chromatography, the purity of the protein is improved to a certain extent.

[0081] (4) Dialysis fluid exchange and concentration A. Collect the sample purified by ion exchange chromatography in the previous step and dialyze it into the final buffer (20mM Tris-HCl, 200mM NaCl, 50% Glycerol, PH7.5, 1mM TCEP). Generally, 10mL of sample is dialyzed with 1L buffer at 4℃ for 2h, the solution is changed once, and then dialyzed again for 2h.

[0082] B. After changing the liquid, use a 30kDa ultrafiltration tube to concentrate the protein to 4 mg / mL. The centrifugation conditions are 4°C, 3000 rpm, 15 min / time, and repeat the centrifugation until the concentration reaches 4 mg / mL.

[0083] C. After concentration, dispense into 100 μL / tube and freeze at -80℃.

[0084] After the above steps, the original sST2 protein, the recombinant sST2 protein mutant 1 and the recombinant sST2 protein mutant 2 are prepared.

[0085] Furthermore, the following experiments were conducted to understand the performance of the triple protein prepared above.

[0086] 1. Purity verification The specific purity verification method is as follows: 1.1 Gel preparation 1.1.1 Preparation of separation gel Prepare separation gel solution as needed, pour it into the mold to a certain height, add water to seal the top, and polymerize at room temperature.

[0087] 1.1.2 Preparation of stacking gel After the separation gel solution is polymerized, use filter paper to absorb the upper water layer, then pour in the concentrated gel solution and insert the sample comb to avoid bubbles.

[0088] 1.2 Preparation of test solution Prepare the test solution by mixing the test solution with reduced test buffer in a volume ratio of 3:1, heat in a water bath or 100°C block heater for 5 minutes, and cool to room temperature. The standard solution is operated in the same way.

[0089] 1.3 Electrophoresis 1.3.1 Loading: After the concentrated gel solution is polymerized, carefully pull out the sample comb and fill the electrophoresis tank with electrode buffer. When checking the purity and impurities, add no less than 10μg of the test solution and standard solution to the sample loading hole.

[0090] 1.3.2 Electrophoresis Constant voltage electrophoresis: The initial voltage is 80V, which is adjusted to 150-200V when entering the separation gel. When bromophenol blue migrates to the bottom of the gel, stop electrophoresis.

[0091] Constant current electrophoresis: Start electrophoresis at a constant current of 10 mA. After the test solution enters the separation gel, adjust the current to 20 mA until the electrophoresis is completed.

[0092] 1.4 Fixation and staining Coomassie Brilliant Blue staining method: Take out the electrophoresis gel slice, place it in the fixative for 60 minutes, take out the film and place it in excess Coomassie Brilliant Blue staining solution for 1-2 hours, discard the staining solution, place it in excess destaining solution, change the destaining solution several times as needed, destain until the gel background is transparent, and then store it in the preservation solution.

[0093] 1.5 Result determination After the gel color development treatment is completed, it is photographed or scanned, and the analysis results show that the molecular weight is 45-50KDa, and the protein purity of the recombinant proteins obtained by the above three recombinant plasmids is >95%.

[0094] 2. Concentration Verification The protein concentration of sST2 recombinant protein was determined by Coomassie brilliant blue method before lyophilization. This method is based on the fact that Coomassie brilliant blue G250 combines with the basic amino acid (arginine) and aromatic amino acid in the protein molecule in an acidic environment to form a blue complex. Within a certain range, the color depth is proportional to the protein concentration. The protein reference solution is used as the standard curve, and the protein content in the test sample is determined by colorimetry. The results showed that the protein concentrations were as follows: the recombinant protein of the original sequence was 3.7 mg / mL, the recombinant protein of mutant 1 was 3.9 mg / mL, and the recombinant protein of mutant 2 was 4.2 mg / mL.

[0095] 3. Activity Verification The binding capacity of immobilized recombinant human IL-33 at 10ug / mL was measured by functional ELISA with the following method: 3.1 Coating: Add the antigen (here is IL-33) solution to the wells of the ELISA plate, add 100 μL to each well, coat the antigen on the wall of the ELISA plate, and incubate at 37°C for 1-2 hours or at 4°C overnight.

[0096] 3.2 Washing: Use washing solution to wash away the residual antigen solution in the wells of the ELISA plate. Be careful to avoid splashing the liquid during washing. Shake dry after each wash. Repeat the washing 3 times. Finally, use a paper towel to gently absorb the residual liquid in the wells of the ELISA plate.

[0097] 3.3 Blocking: Add blocking solution, add 200 μL to each well, and incubate at 37°C for 30 minutes to block the remaining binding sites in the ELISA plate wells.

[0098] 3.4 Washing: Wash again 3 times using the same method as above.

[0099] 3.5 Sample addition: Add standard or sample to be tested (here is the diluted sST2 recombinant protein), add 100 μL to each well and incubate at 37°C for 1 hour.

[0100] 3.6 Washing: After the last wash, spin dry and gently absorb the remaining liquid in the wells of the ELISA plate with a paper towel.

[0101] 3.7 Add enzyme-labeled antibody: Add 1:5000 diluted HRP-labeled sST2 antibody, add 100ul to each well, and incubate at 37℃ for 30 minutes; 3.8 Washing: After washing, spin dry and gently absorb the residual liquid in the wells of the ELISA plate with a paper towel.

[0102] 3.9 Color development: Add color developers A and B. First add 50 μL of color developer A to each well, then add 50 μL of color developer B, mix gently, and incubate at 37°C for 10-15 minutes.

[0103] 3.10 Stop: Add 50 μL of stop solution to each well and mix gently.

[0104] 3.11 Determination: Use an enzyme-labeled instrument to measure the optical density of each well at a wavelength of 450 nm and record the results.

[0105] The result of multiplying the dilution factor by 3.12 showed that the protein concentrations were as follows: 3.4 mg / mL of the recombinant protein of the original sequence, 4.0 mg / mL of the recombinant protein of mutant 1, and 4.4 mg / mL of the recombinant protein of mutant 2.

[0106] 4. Stability and uniformity verification Evaluate the storage time of quality control products prepared from recombinant proteins at -20℃ and 37℃, and the storage time of the reconstituted protein solution at -20℃ under sterile conditions; evaluate the uniformity of quality control products within and between tubes.

[0107] 4.1 Stability of quality control products The three sST2 recombinant proteins were used to prepare the quality control products. After preparation, the measurement procedure selected by the enterprise was used: the conventional commercially available soluble growth stimulation expression gene 2 protein detection kit was used to assign values, and the assigned quality control products were tested with the soluble growth stimulation expression gene 2 protein (sST2) detection kit (fluorescence immunochromatography). Each concentration was tested three times, and the average value of the results was calculated. The quality control products after the test were packaged and stored at 37°C and -20°C respectively. The same batch of soluble growth stimulation expression gene 2 protein (sST2) detection kit (fluorescence immunochromatography) was used to test the quality control products at 37°C for 3 days, 6 days, 9 days, and 12 days (results are shown in Table 1) and at -20°C for 1 month, 3 months, 6 months, and 12 months (results are shown in Table 2). Each concentration was tested three times, and the average value was calculated. The test results were compared with the results of 0 days at room temperature during preparation, and the deviation was calculated. The deviation within ±10% indicates that the quality control product is stable. The specific results are shown in Table 1.

[0108] Table 1 Stability data analysis of sST2 quality control product at 37℃

[0109] Table 2 Stability data analysis of sST2 quality control product at -20℃

[0110] From the results in Tables 1 and 2, we can see that the quality control product prepared by recombinant protein ① is stable for 3 days at 37℃ and 3 months at -20℃; the quality control product prepared by recombinant protein ② is stable for 6 days at 37℃ and 6 months at -20℃; the quality control product prepared by recombinant protein ③ is stable for 9 days at 37℃ and 12 months at -20℃. The stability of the recombinant protein after mutation at 37℃ and -20℃ is significantly higher than that of the original protein.

[0111] 4.2 Uniformity of quality control products Select 5 tubes for each quality control product, repeat the test 5 times for each tube, calculate the intra-tube CV and inter-tube CV respectively, and the coefficient of variation (CV) should not be greater than ±10%.

[0112] 4.2.1. Homogeneity within the tube The results are shown in Table 3, from which it can be seen that the CV within each quality control tube does not exceed ±10%.

[0113] Table 3 sST2 quality control tube uniformity data analysis table

[0114] 4.2.2 Uniformity between tubes The results are shown in Table 4, from which it can be seen that the CV within each quality control tube does not exceed ±10%.

[0115] Table 4 sST2 quality control uniformity data analysis table

[0116] From the above results, it can be seen that the accused samples prepared from the three proteins have good uniformity both within the tube and between tubes.

[0117] 5. Interoperability testing Samples and quality control products close to the upper limit of the linear interval of the soluble growth stimulation expression gene 2 protein (sST2) detection kit (fluorescence immunochromatography) were selected, and low-value samples close to the lower limit of the linear interval were used for gradient dilution. The samples and quality control products obtained by gradient dilution were tested with the soluble growth stimulation expression gene 2 protein (sST2) detection kit (fluorescence immunochromatography), and the result values ​​were fitted with a straight line. The correlation R 2 ≥0.93 (see results Figure 4 ), indicating that the recombinant protein has good intercommunication with the natural protein.

[0118] In summary, the recombinant sST2 protein provided by the present invention has high titer and is close to the natural antigen, and can be used as a raw material for diagnostic reagents (preparation of quality control products and quality control products) or for screening suitable antibodies. In addition, the interaction between IL-33 and ST2L has been shown to have a cardioprotective effect. In experimental models, this interaction can reduce myocardial fibrosis, prevent myocardial cell hypertrophy, reduce cell apoptosis and improve myocardial function. The beneficial effects of IL-33 are specifically achieved through the ST2L receptor. sST2 strongly binds to IL-33, thereby interrupting the interaction between IL-33 / ST2L, thereby eliminating the anti-remodeling effect, and therefore, sST2 is regarded as a decoy receptor. In contrast, sST2 acts as a decoy receptor, antagonizing the cardioprotective effect of the IL-33 / ST2L interaction by isolating IL-33. Therefore, reducing the concentration of sST2 in the body has a protective effect on the myocardium. sST2 recombinant protein plays an important role in drug development and can be used to study drug targets, efficacy, toxicity, etc. It can also evaluate the correlation of sST2 recombinant protein concentration with cardiac damage, prognosis, cardiac function and inflammatory cell infiltration.

[0119] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0120] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, 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 recombinant sST2 protein, characterized in that: The amino acid sequence of the recombinant sST2 protein is shown in SEQ ID NO.3 or SEQ ID NO.

5.

2. A nucleotide sequence encoding the recombinant sST2 protein according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.4 or SEQ ID NO.6; Among them, the nucleotide sequence encoding the amino acid shown in SEQ ID NO.3 is shown in SEQ ID NO.4, and the nucleotide sequence encoding the amino acid shown in SEQ ID NO.5 is shown in SEQ ID NO.

6.

3. An expression vector containing the nucleotide sequence of claim 2.

4. A host cell containing the expression vector according to claim 3.

5. A method for preparing the recombinant sST2 protein according to claim 1, characterized in that: The following steps are involved: S1. Insert the nucleotide sequence shown in SEQ ID NO.4 or SEQ ID NO.6 into a vector to obtain an expression vector, and transform it into competent cells. After culturing, extract the endotoxin-free plasmid; S2. The endotoxin-free plasmid is transfected into the host cells, and after cultivation, the cells are subjected to cell lysis, affinity chromatography, washing and elution, ion exchange chromatography, dialysis and concentration to obtain the recombinant sST2 protein.

6. The method according to claim 5, characterized in that In step S1, the vector is pcDNA3.1 and the competent cell is DH5α.

7. The method according to claim 5, characterized in that The matrix in the affinity chromatography is SIGMA Anti-DYKDDDDK affinity purification gel; The buffer used in the affinity chromatography process is as follows: Balance buffer: 50mM Tris-HCl, 300mM NaCl, pH 7.5; Washing buffer: 50mM Tris-HCl, 300mM NaCl, pH 7.5; Elution Buffer: 20 mM HEPES pH 8.0, 300 mM NaCl, 0.1 mg / mL synthetic peptide DYKDDDDK, 0.1* cOmpleteTM Protease Inhibitor Cocktail, 0.1 mM AEBSF.

8. The method according to claim 5, characterized in that The buffer used in the ion exchange chromatography process is as follows: Balance buffer: 20mM HEPES, 50mM NaCl, pH 8.0; Binding Buffer: 20mM HEPES, 50mM NaCl, pH 8.0; Elution Buffer A: 20 mM HEPES, 150 mM NaCl, 10% glycerol, pH 8.0; Elution Buffer B: 20 ​​mM HEPES, 300 mM NaCl, 10% glycerol, pH 8.0; Elution Buffer C: 20 mM HEPES, 500 mM NaCl, 10% glycerol, pH 8.

0.

9. The method according to claim 5, characterized in that The buffer formula used during dialysis was as follows: 20 mM Tris–HCl, 200 mM NaCl, 50% Glycerol, pH 7.5, 1 mM TCEP.

10. Use of the recombinant sST2 protein according to claim 1 in target detection kits, drug targeted analysis, and drug development.

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