A recombinant sST2 protein and its preparation method and application

Abstract

The present invention provides a recombinant sST2 protein, the amino acid sequence of the recombinant sST2 protein is shown in SEQ ID NO.3 or SEQ ID NO.5. 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. The recombinant sST2 protein prepared by the present invention has good performance, and the modified recombinant protein has a higher potency, which is better than the recombinant protein of the original sequence. The purity of the recombinant protein is ≥95%, and the activity is ≥95%; the protein stability is better, and it can be stored for one year at ‑20°C before reconstitution, and can be stored for more than 3 months at ‑20°C after reconstitution; the recombinant protein is close to the natural protein and has good interoperability with clinical samples. The recombinant proteins prepared by the present invention can be used for the preparation of calibrators and quality control products in the target detection kit, and can also be used for related experiments such as drug targeted analysis and drug development.

Description

Technical Field

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

[0002] Growth-stimulating factor 2 (ST2) protein 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 two major isoforms: a transmembrane or cellular (ST2L) and a soluble or circulating (sST2) form.

[0003] ST2 is the receptor for IL-33, an IL-1-like cytokine secreted by living cells in response to cellular injury. IL-33 exerts its cellular functions by binding to a receptor complex composed of ST2L and the IL-1R accessory protein. 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, decreasing 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 abrogating the anti-remodeling effects; therefore, sST2 is considered a decoy receptor. Knowledge of the role of ST2 in the pathophysiology of cardiovascular disease has expanded extensively in recent years, 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 tests. Summary of the Invention

[0005] In light of this, the present invention, based on the original sequence of the sST2 protein, enhanced the performance of the recombinant proteins by site-directed mutagenesis at two locations and by adding a gene encoding the polypeptide sequence DYKDDDDK to its N-terminus. Ultimately, two recombinant proteins with sST2 immunoreactivity were obtained. The resulting recombinant proteins exhibited excellent performance, with higher potency and improved stability. They also resembled the native protein and exhibited good interoperability with clinical samples.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] One of the objectives 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.

[0008] 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;

[0009] 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.

[0010] The third object of the present invention is to provide an expression vector containing the above-mentioned nucleotides.

[0011] A fourth object of the present invention is to provide a host cell containing the above expression vector.

[0012] A fifth object of the present invention is to provide a method for preparing the above-mentioned recombinant sST2 protein, comprising the following steps:

[0013] S1. Insert the nucleotide sequence shown in SEQ ID NO.4 or SEQ ID NO.6 into a vector to obtain an expression vector, transform the vector into competent cells, and extract the endotoxin-free plasmid after culture;

[0014] S2. The endotoxin-free plasmid is transfected into the host cells, and after culture, the cells are subjected to cell lysis, affinity chromatography, washing and elution, ion exchange chromatography, dialysis and concentration to obtain the recombinant sST2 protein.

[0015] In some specific embodiments, preferably, in step S1, the vector is pcDNA3.1 and the competent cell is DH5α.

[0016] In some specific embodiments, preferably, the matrix in the affinity chromatography is SIGMA Anti-DYKDDDDK affinity purification gel;

[0017] The buffers used in affinity chromatography are as follows:

[0018] Balance buffer: 50mM Tris-HCl, 300mM NaCl, pH 7.5.

[0019] Washing buffer: 50mM Tris-HCl, 300mM NaCl, pH 7.5.

[0020] Elution Buffer: 20 mM HEPES pH 8.0, 300 mM NaCl, 0.1 mg / mL synthetic peptide DYKDDDDK, 0.1*cOmplete TM Protease Inhibitor Cocktail, 0.1mM AEBSF.

[0021] In some specific embodiments, preferably, the buffer used in the ion exchange chromatography process is as follows:

[0022] Balance buffer: 20mM HEPES, 50mM NaCl, pH 8.0.

[0023] Binding Buffer: 20mM HEPES, 50mM NaCl, pH 8.0.

[0024] Elution Buffer A: 20 mM HEPES, 150 mM NaCl, 10% glycerol, pH 8.0.

[0025] Elution Buffer B: 20 mM HEPES, 300 mM NaCl, 10% glycerol, pH 8.0.

[0026] Elution Buffer C: 20 mM HEPES, 500 mM NaCl, 10% glycerol, pH 8.0.

[0027] In some specific embodiments, preferably, the buffer solution used in the dialysis process has the following formula: 20 mM Tris-HCl, 200 mM NaCl, 50% Glycerol, pH 7.5, 1 mM TCEP.

[0028] Application of the above-mentioned recombinant sST2 protein in target detection kits, drug targeted analysis, and drug development.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The recombinant sST2 protein provided by the present invention has a purity of ≥95% and an activity of ≥95%. The quality control product prepared from it is 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 of no more than ±10% within or between tubes, and has good homogeneity. It has good interoperability with the natural protein and a 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

[0031] Figure 1 This is the plasmid map of recombinant sST2 protein.

[0032] Figure 2 This is the WB detection result of recombinant sST2 protein.

[0033] Figure 3 This is the SDS-PAGE electrophoresis diagram of recombinant sST2 protein.

[0034] Figure 4 This is a diagram of the interchangeability analysis of recombinant sST2 protein. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0036] Example 1

[0037] The present invention provides a recombinant sST2 protein, the preparation process of which is as follows:

[0038] S1. Obtaining the coding sequence of recombinant sST2 protein

[0039] 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 recombinant sST2 protein expression.

[0040] Original amino acid sequence of sST2 protein SEQ ID NO.1: MGFWILAILTILMYS TAAKFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQLLKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLMYSTVSGSEKNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAHKSFLVIDNVM TEDAGDYTCKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEVEIGKNANLTCSACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNPSKECF.

[0041] The nucleotide sequence encoding the original sST2 protein is SEQ ID NO.2:ATGGGCTTCT GGATCCTGGCCATCCTGACAATCCTCATGTACAGCACAGCCGCCAAGTTCAGCAAACAGAGCTGGGGCCTGGAAAACGAGGCCCTGATTGTGCGGTGCCCTAGACAGGGCAAGCCTAGCTACACCGTGGACTGGTACTACTCTCAGACCAACAAGTCTATCCCCACACAGGAGAGAAACAGAGTGTTCGCCTCTGGCCAGCTGCTGAAGTTCCTGCCTGCTGCTGTGGCCGATAGCGGCATCTACACCTGCATCGTCCGGAGCCCTACATTTAACCGGACCGGCTACGCCAATGTGACCATCTATAAGAAGCAAAGCGACTGCAACGTTCCTGATTACCTGATGTATTCTACAGTGTCTGGAAGCGAGAAGAATAGCAAAATCTACTGCCCCACCATCGACCTGTACAACTGGACCGCTCCTCTGGAATGGTTCAAGAACTGCCAGGCCCTCCAGGGATCTAGATACAGAGCCCATAAGTCCTTCCTGGTGATCGACAACGTGATGACCGAGGACGCCGGCGACTACACCTGTAAATTCATCCACAACGAGAACGGCGCCAACTACAGCGTGACCGCTACAAGAAGCTTTACCGTCAAGGACGAGCAGGGATTCAGCCTGTTCCCCGTGATCGGCGCACCAGCCCAGAACGAGATCAAGGAAGTGGAAATCGGCAAAAATGCCAACCTGACCTGTAGCGCCTGTTTTGGCAAGGGCACCCAGTTCCTGGCTGCCGTGCTGTGGCAGCTGAATGGCACCAAGATCACAGATTTCGGCGAGCCAAGAATTCAGCAGGAAGAGGGCCAAAACCAGTCCTTTTCCAACGGACTGGCCTGCCTTGATATGGTGCTGCGGATCGCCGACGTGAAGGAGGAAGATCTGCTGCTGCAGTACGACTGCCTGGCCCTGAACCTGCACGGTCTGCGCAGACACACAGTGCGGCTGAGCAGAAAGAATCCTAGCAAGGAATGCTTC。.

[0042] Amino acid sequence of recombinant sST2 protein mutant 1 SEQ ID NO.3: MGFWILAILTILMYSTAAKFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQLLKFLPAAVADSGIYTCIVRSPTFNRTGYAVVTIYKKQSDCNVPDYLMYSTVSGSEKNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAHKSFLVIDNVMTEDAGDYTCKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEVEIGKNANLTCSACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNPSKECF。

[0043] The nucleotide sequence encoding the recombinant sST2 protein mutant 1 is SEQ ID NO.4:ATGG GCTTTTGGATCCTGGCCATCCTGACAATCCTGATGTACAGCACCGCCGCCAAGTTCAGCAAGCAGTCTTGGGGCCTCGAGAACGAGGCCCTGATCGTCAGATGTCCCAGACAGGGCAAGCCCAGCTACACCGTGGATTGGTACTACAGCCAGACCAACAAGAGCATCCCCACACAAGAGCGGAACCGGGTGTTCGCTTCTGGCCAGCTGCTGAAGTTTCTGCCTGCCGCCGTTGCCGACAGCGGCATCTACACATGTATTGTGCGGAGCCCCACCTTCAACAGAACCGGCTACGCCGTCGTGACCATCTACAAGAAGCAGAGCGACTGCAACGTGCCCGACTACCTGATGTATTCCACCGTGTCCGGCAGCGAGAAGAACAGCAAGATCTACTGCCCCACCATCGACCTGTACAACTGGACAGCCCCTCTGGAATGGTTCAAGAACTGTCAGGCCCTCCAGGGCAGCAGATACAGAGCCCACAAGAGCTTTCTGGTCATCGACAACGTGATGACCGAGGACGCCGGCGACTACACATGCAAGTTCATCCACAACGAGAACGGCGCCAACTACAGCGTGACCGCCACCAGATCCTTTACCGTGAAGGACGAGCAGGGCTTCTCTCTGTTCCCTGTGATTGGAGCCCCTGCTCAGAACGAGATCAAAGAGGTGGAAATCGGCAAGAACGCCAATCTGACCTGCAGCGCCTGCTTCGGCAAGGGCACACAATTTCTGGCTGCCGTGCTGTGGCAGCTGAACGGCACCAAGATCACCGATTTCGGCGAGCCCCGGATCCAGCAAGAGGAAGGCCAGAATCAGAGCTTCAGCAACGGCCTGGCCTGCCTGGATATGGTGCTGAGAATCGCCGACGTGAAAGAAGAGGACCTGCTGCTGCAGTACGACTGTCTGGCCCTGAATCTGCACGGCCTGAGAAGGCACACAGTGCGGCTGAGCAGAAAGAACCCCAGCAAAGAGTGCTTC。.

[0044] Amino acid sequence of recombinant sST2 protein mutant 2, SEQ ID NO.5: MGFWILAILTILMYSTAAKFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQLLKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLMYSTVSGSEKNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAHKSFLVIDNVMTEDAGDYTCKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEVEIGKNANLTCSACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSYSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNPSKECF。

[0045] The nucleotide sequence encoding the recombinant sST2 protein mutant 2 is SEQ ID NO.6:ATGG GCTTTTGGATCCTGGCCATCCTGACAATCCTGATGTACAGCACCGCCGCCAAGTTCAGCAAGCAGTCTTGGGGCCTCGAGAACGAGGCCCTGATCGTCAGATGTCCCAGACAGGGCAAGCCCAGCTACACCGTGGATTGGTACTACAGCCAGACCAACAAGAGCATCCCCACACAAGAGCGGAACCGGGTGTTCGCTTCTGGCCAGCTGCTGAAGTTTCTGCCTGCCGCCGTTGCCGACAGCGGCATCTACACATGTATTGTGCGGAGCCCCACCTTCAACAGAACCGGCTACGCCAACGTGACCATCTACAAGAAGCAGAGCGACTGCAACGTGCCCGACTACCTGATGTATTCCACCGTGTCCGGCAGCGAGAAGAACAGCAAGATCTACTGCCCCACCATCGACCTGTACAACTGGACAGCCCCTCTGGAATGGTTCAAGAACTGTCAGGCCCTCCAGGGCAGCAGATACAGAGCCCACAAGAGCTTTCTGGTCATCGACAACGTGATGACCGAGGACGCCGGCGACTACACATGCAAGTTCATCCACAACGAGAACGGCGCCAACTACAGCGTGACCGCCACCAGATCCTTTACCGTGAAGGACGAGCAGGGCTTCTCTCTGTTCCCTGTGATTGGAGCCCCTGCTCAGAACGAGATCAAAGAGGTGGAAATCGGCAAGAACGCCAATCTGACCTGCAGCGCCTGCTTCGGCAAGGGCACACAATTTCTGGCTGCCGTGCTGTGGCAGCTGAACGGCACCAAGATCACCGATTTCGGCGAGCCCCGGATCCAGCAAGAGGAAGGCCAGAATCAGAGCTACAGCAACGGCCTGGCCTGCCTGGATATGGTGCTGAGAATCGCCGACGTGAAAGAAGAGGACCTGCTGCTGCAGTACGACTGTCTGGCCCTGAATCTGCACGGCCTGAGAAGGCACACAGTGCGGCTGAGCAGAAAGAACCCCAGCAAAGAGTGCTTC。.

[0046] The above three nucleotide sequences (SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6) were all synthesized by a biological company.

[0047] S2. Construction of recombinant plasmid

[0048] The three nucleotide sequences obtained in step S1 (SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6) were sequentially inserted into the vector pcDNA3.1 (Invitrogen) to form 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 plasmids and test reports were returned. The plasmid map is shown in Figure 2. Figure 1 As shown; wherein, the sST2 sequence can be the original sequence, mutant 1 and mutant 2 respectively.

[0049] S3. Extraction of recombinant plasmid

[0050] 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 Aibotec) to obtain single colonies. Single colonies were picked and cultured overnight in LB medium. The corresponding endotoxin-free pcDNA3.1-sST2-1, pcDNA3.1-sST2-2, and pcDNA3.1-sST2-3 recombinant plasmids were extracted using an endotoxin-free plasmid miniprep kit (Cat. No. DP118-02, Beijing Tiangen Biochemical). The specific steps are as follows:

[0051] (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 minutes, then heat them in a 42°C hot water bath for 90 seconds (strictly control the time), and treat them in an ice bath for 5 minutes; 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 minutes; centrifuge at 6000 rpm for 1 minute, 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, shake the plate gently to evenly spread the bacterial solution, pour out the glass beads, and place the plate upside down in a 37°C incubator for overnight culture.

[0052] (2) Endotoxin-free plasmid extraction: Single colonies were picked from the above plates and cultured overnight in 10 mL of LB medium containing ampicillin. The next morning, the culture was centrifuged at 12,000 rpm for 10 min. The recombinant plasmids were extracted according to the instructions of the endotoxin-free plasmid miniprep kit to obtain the corresponding endotoxin-free recombinant pcDNA3.1-sST2-1, pcDNA3.1-sST2-2, and pcDNA3.1-sST2-3 plasmids.

[0053] S3. Expression and purification of recombinant sST2 protein

[0054] 1. Transient Transfection

[0055] 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:

[0056] (1) Before transfection, subculture and expand HEK293 cells until the cell density reaches about 3-4×10 6 viable cells / mL, with a viability ≥ 95%.

[0057] (2) One day before transfection, the cells in step (1) were cultured at a rate of 1.5 to 1.8 × 10 6 The cells were seeded at a density of 100 cells / mL.

[0058] (3) On the day of transfection, the cell density was adjusted to 3.0×10 6 100 μg / mL, adjust the volume to 25.5 mL, and the cell viability for transfection should be ≥95%. Note: Discard the remaining cells and do not reuse high-density cells for routine subculture.

[0059] (4) Dilute 30 μg of plasmid DNA with 1.5 mL of OptiMEM medium and mix by rotating and / or inverting the tube.

[0060] (5) Dilute 75 μL of PEIMAX (1 mg / mL, pH 7.1) with 1.5 mL of OptiMEM medium and mix by rotating and / or inverting the tube.

[0061] (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.

[0062] (7) Slowly add the complex to the cell shake flask in step (3), and gently shake the shake flask during the addition process.

[0063] (8) Place the shake flask back into the 37°C incubator and incubate under the following conditions: 125 rpm, 8% CO2, 37°C, and 95% humidity.

[0064] (9) On the first day, 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.

[0065] (10) On day 3, during transient expression, the glucose concentration was maintained above 4 g / L, and cells were harvested when the cell viability was less than 70%.

[0066] 1.5 mL of feed was added for the first time 16-20 hours after transfection; cell viability was measured for 3 consecutive days, as shown in the following table:

[0067] Number of days 0d (the day of transfection) 1d 2d 3d Cell viability 99.24％ 98.44％ 90.44％ 68.91％

[0068] 2. Western Blot (WB) Detection

[0069] (1) Take 5 μL of the supernatant and perform SDS-PAGE electrophoresis. Use constant voltage mode: 80V for 5% stacking gel. When the marker begins to separate for about 25 minutes, adjust the voltage to 120V and terminate the electrophoresis when bromophenol blue reaches the bottom of the separating gel.

[0070] (2) Transfer assembly sequence: black side of transfer chuck (negative electrode) - sponge pad - 3 layers of filter paper - glue - membrane - 3 layers of filter paper - sponge pad - red side (positive electrode). Transfer time: 200 mA, 90-180 min.

[0071] (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;

[0072] (4) Flag-tag primary antibody incubation: After blocking, discard 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, discard the primary antibody solution; rinse the membrane 4 times with TBST, 5 minutes each time.

[0073] (5) Secondary antibody incubation: Before the primary antibody incubation is completed, dilute the enzyme-labeled secondary antibody corresponding to the primary antibody species at a dilution ratio of 1:5000 to the required amount 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 60-80 minutes. After the secondary antibody incubation is completed, discard the secondary antibody solution and rinse the membrane four times with TBST for 5 minutes each time.

[0074] (6) Exposure: Remove the membrane from the TBST solution with tweezers, drain thoroughly, and place it on a gel tray. Mix equal volumes of ECL Solution I and Solution II and apply evenly to the membrane, completely covering it. Allow the substrate to react with the membrane for approximately 30 seconds, then place the membrane on a chemiluminescence imaging system. Set the exposure time to 3 seconds, 10 seconds, 30 seconds, 60 seconds, and 120 seconds.

[0075] (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.

[0076] In this example, HEK293F cells were used as host cells, and the recombinant plasmids pcDNA3.1-sST2-1, pcDNA3.1-sST2-2, and pcDNA3.1-sST2-3 were transiently introduced into the host cells to obtain cells expressing the recombinant sST2 protein. This method has the advantages of a short transfection cycle and rapid production of the target protein, making it suitable for both exploratory research and large-scale high-throughput protein screening.

[0077] S4. Recombinant protein purification

[0078] (1) Cell lysis

[0079] Ultrasonic lysis: power 250 W, time 5 min, 3s / 3s (ultrasonication 3s, stop 3s), twice.

[0080] Lysis solution: 50mM Tris-HCl pH7.5, 300mM NaCl, 0.1*cOmplete TM Protease InhibitorCocktail, 0.1mM AEBSF, 1% TritonX-100.

[0081] After breaking the bacteria, centrifuge at 12000 rpm and 4°C for 15 min and collect the supernatant.

[0082] 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 poured into a new centrifuge tube after the second centrifugation.

[0083] (2) Affinity chromatography

[0084] Matrix: SIGMA Anti-DYKDDDDK Affinity Purification Gel.

[0085] Incubation: Place on a rotating incubator, 20 rpm, 4°C and incubate for 4 hours.

[0086] A. Column flow penetration:

[0087] After incubation, balance the tube and centrifuge at 4°C, 600 rpm for 10 minutes. Pour the supernatant into a new tube, which is the flow-through. Reserve approximately 5 mL of supernatant for suspending the matrix. Transfer the matrix to the purification column tube and collect the flow-through (FT) after the flow-through is complete.

[0088] B. Cleaning and elution

[0089] a. Add 10 mL of Washing Buffer to the purification column to wash away any contaminants in the matrix. Allow gravity flow and collect the flow-through in a sterile 5 mL EP tube. Keep the tube on ice to keep it cold. After flow-through, detect the column using a G250 microscope (place 100 μL of G250 in a 96-well plate and add 10 μL of the eluate). If the G250 turns blue, continue washing the column by adding 5 mL of Washing Buffer until the G250 turns blue, completing the Washing Buffer pre-elution.

[0090] b. Add 1 mL of Elution Buffer to wash the column and remove matrix-bound proteins. Allow gravity flow and collect the effluent in a 1.5 mL endotoxin-free EP tube. Place the tube on ice to keep it cold. After the flow-through, detect the column using a G250 (place 100 μL of G250 in a 96-well plate and add 10 μL of the dripping eluent). If the G250 turns blue, continue elution until the G250 no longer turns blue, indicating the end of the Elution Buffer elution.

[0091] c. Prepare samples of the collected flow-through and eluate, usually using 6× loading buffer, i.e., 20 μL protein + 5 μL 6× loading buffer, for SDS PAGE electrophoresis.

[0092] The buffer used in the affinity chromatography process is as follows:

[0093] Balance buffer: 50mM Tris-HCl, 300mM NaCl, pH7.5.

[0094] Washing buffer: 50mM Tris-HCl, 300mM NaCl, pH7.5.

[0095] Elution Buffer: 20 mM HEPES pH 8.0, 300 mM NaCl, 0.1 mg / mL synthetic peptide DYKDDDDK, 0.1*cOmplete TM Protease Inhibitor Cocktail, 0.1mM AEBSF.

[0096] 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.

[0097] (3) Ion exchange chromatography (Q column)

[0098] A. Desalination:

[0099] Take a suitable dialysis bag, rinse it clean, and add the protein collected in the previous step to the dialysis bag. Place the dialysis bag in the prepared desalting buffer (20mM HEPES, 50mM NaCl, pH 8.0). Generally, 50mL of sample is dialyzed using 2L of buffer at 4°C for 2h.

[0100] B. Equilibrate the medium and incubate:

[0101] Equilibrate the matrix with Binding Buffer (usually 6-10 column volumes). Mix the equilibrated matrix and desalted protein solution in a centrifuge tube and incubate the tube in a rotating incubator at 20 rpm and 4°C for 40 minutes.

[0102] C. Add 10 to 15 column volumes of Binding Buffer to the purification column to wash away weakly bound contaminants on the matrix. Control the flow rate to 2 mL / min. After the flow is complete, use G250 detection (take 100 μL of G250 in a 96-well plate and add 10 μL of the elution solution that is dripping). If the G250 turns blue, continue elution until the G250 does not turn blue. Collect the effluent. The collection tube needs to be placed on ice to keep it cold. Add Elution Buffer A to wash the column. Control the flow rate to 0.6 mL / min. Collect the effluent with an endotoxin-free tube. Collect each column volume of effluent in an EP tube (the collection tube should be placed on an ice box to keep it cold). After the flow is complete, use G250 detection (take 100 μL of G250 in a 96-well plate and add 10 μL of the elution solution that is dripping). If the G250 turns blue, continue elution until the G250 does not turn blue.

[0103] Add elution buffer B to wash the column, control the flow rate to 0.6 mL / min, collect the effluent with an endotoxin-free tube, and 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). After the flow is completed, use G250 to detect (take 100 μL G250 in a 96-well plate and add 10 μL of the dripping eluate). If the G250 turns blue, continue elution until the G250 does not turn blue.

[0104] Add Elution Buffer C to wash the column, control the flow rate to 0.6 mL / min, collect the effluent with an endotoxin-free tube, and 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). After the flow is completed, use G250 to detect (take 100 μL G250 in a 96-well plate and add 10 μL of the dripping eluate). If the G250 turns blue, continue elution until the G250 does not turn blue.

[0105] The buffer used for ion exchange is as follows:

[0106] Balance buffer: 20mM HEPES, 50mM NaCl, pH8.0.

[0107] Binding Buffer: 20mM HEPES, 50mM NaCl, pH8.0.

[0108] Elution Buffer A: 20 mM HEPES, 150 mM NaCl, 10% glycerol, pH 8.0.

[0109] Elution Buffer B: 20 mM HEPES, 300 mM NaCl, 10% glycerol, pH 8.0.

[0110] Elution Buffer C: 20 mM HEPES, 500 mM NaCl, 10% glycerol, pH 8.0.

[0111] The eluted samples were collected and prepared, generally using 6× loading buffer, i.e., 20 μL protein + 5 μL 6× loading buffer, for SDS PAGE electrophoresis.

[0112] 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.

[0113] (4) Dialysis fluid exchange and concentration

[0114] 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, pH 7.5, 1mM TCEP). Generally, 10mL of sample is dialyzed into 1L of buffer at 4℃ for 2h. Change the buffer once and dialyze again for 2h.

[0115] B. After changing the medium, use a 30kDa ultrafiltration tube to concentrate the protein to 4 mg / mL. Centrifuge at 4°C, 3000 rpm, 15 min / time. Repeat the centrifugation until the concentration reaches 4 mg / mL.

[0116] C. After concentration, aliquot into 100 μL / tube and store at -80℃.

[0117] After the above steps, the original sST2 protein, the recombinant sST2 protein mutant 1, and the recombinant sST2 protein mutant 2 are prepared.

[0118] Furthermore, the following experiments were conducted to understand the performance of the triple protein prepared above.

[0119] 1. Purity Verification

[0120] The specific purity verification method is as follows:

[0121] 1.1 Gel preparation

[0122] 1.1.1 Preparation of separation gel

[0123] 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.

[0124] 1.1.2 Preparation of stacking gel

[0125] 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.

[0126] 1.2 Preparation of test solution

[0127] Prepare the test solution by mixing the test solution with the reduced test buffer in a 3:1 volume ratio. Heat in a water bath or a 100°C block heater for 5 minutes and cool to room temperature. Repeat the same procedure for the standard solution.

[0128] 1.3 Electrophoresis

[0129] 1.3.1 Sample loading: After the stacking gel solution is polymerized, carefully pull out the sample comb and fill the electrophoresis tank with electrode buffer. When checking for purity and impurities, add no less than 10 μg of the test solution and standard solution to the sample well.

[0130] 1.3.2 Electrophoresis

[0131] 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.

[0132] 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.

[0133] 1.4 Fixation and staining

[0134] Coomassie Brilliant Blue staining method: Remove the electrophoresis gel slice and place it in the fixative for 60 minutes. Remove the film and place it in excess Coomassie Brilliant Blue staining solution for 1-2 hours. Discard the staining solution and place it in excess destaining solution. Change the destaining solution several times as needed. Destain until the gel background is transparent and then store in the preservation solution.

[0135] 1.5 Result determination

[0136] After the gel color development process was completed, it was photographed or scanned. The analysis results showed that the molecular weight was 45-50 KDa, and the protein purity of the recombinant proteins obtained from the three recombinant plasmids was >95%.

[0137] 2. Concentration Verification

[0138] The protein concentration of sST2 recombinant protein was determined using the Coomassie Brilliant Blue method before lyophilization. This method is based on the fact that Coomassie Brilliant Blue G250 binds to basic amino acids (arginine) and aromatic amino acids in protein molecules in an acidic environment to form a blue complex. The color intensity is proportional to the protein concentration within a certain range. A protein control solution was used as a standard curve, and the protein content in the test sample was determined by colorimetry. The protein concentrations were as follows: 3.7 mg / mL for the original sequence recombinant protein, 3.9 mg / mL for the mutant 1 recombinant protein, and 4.2 mg / mL for the mutant 2 recombinant protein.

[0139] 3. Activity Verification

[0140] The binding capacity of immobilized recombinant human IL-33 at 10 μg / mL was measured by functional ELISA with recombinant human sST2 protein (diluted 1000-fold) as follows:

[0141] 3.1 Coating: Add the antigen (here is IL-33) solution to the ELISA plate wells, add 100 μL per well, coat the antigen on the ELISA plate well wall, and incubate at 37°C for 1-2 hours or 4°C overnight.

[0142] 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 wash three times. Finally, gently absorb the residual liquid in the wells of the ELISA plate with a paper towel.

[0143] 3.3 Blocking: Add 200 μL of blocking solution to each well and incubate at 37°C for 30 minutes to block the remaining binding sites in the ELISA plate wells.

[0144] 3.4 Washing: Wash again 3 times using the same method as above.

[0145] 3.5 Sample addition: Add 100 μL of standard or sample to be tested (here, diluted sST2 recombinant protein) to each well and incubate at 37°C for 1 hour.

[0146] 3.6 Washing: After the last wash, spin dry and gently absorb the remaining liquid in the ELISA plate wells with a paper towel.

[0147] 3.7 Add enzyme-labeled antibody: Add 1:5000 diluted HRP-labeled sST2 antibody, add 100 μl to each well, and incubate at 37°C for 30 minutes;

[0148] 3.8 Washing: After washing, spin dry and gently absorb the residual liquid in the wells of the ELISA plate with a paper towel.

[0149] 3.9 Color development: Add colorants A and B. First add 50 μL of colorant A to each well, then add 50 μL of colorant B. Mix gently and incubate at 37°C for 10-15 minutes.

[0150] 3.10 Stop: Add 50 μL of stop solution to each well and mix gently.

[0151] 3.11 Determination: Measure the optical density of each well at a wavelength of 450 nm using a microplate reader and record the results.

[0152] The result of multiplying the dilution factor by 3.12 shows that the protein concentrations are 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.

[0153] 4. Stability and uniformity verification

[0154] Evaluate the storage time of quality control products prepared from recombinant proteins at -20°C and 37°C, as well as the storage time of the reconstituted protein solution at -20°C under sterile conditions; evaluate the homogeneity of quality control products within and between tubes.

[0155] 4.1 Stability of quality control products

[0156] Quality control samples were prepared using the three aforementioned sST2 recombinant proteins. After preparation, the company's selected measurement procedure was used: a commercially available soluble growth stimulatory gene 2 protein assay kit was used to assign values. The assigned values were then tested using the soluble growth stimulatory gene 2 protein (sST2) assay kit (fluorescence immunochromatography). Each concentration was tested three times, and the results were averaged. The tested quality control samples were aliquoted and stored at 37°C and -20°C, respectively. The same batch of soluble growth stimulatory gene 2 protein (sST2) assay kit (fluorescence immunochromatography) was used to test the quality control samples at 37°C for 3 days, 6 days, 9 days, and 12 days (see Table 1) and at -20°C for 1 month, 3 months, 6 months, and 12 months (see Table 2). Each concentration was tested three times, and the average was calculated. The test results were compared with the 0-day results at room temperature during preparation, and the deviation was calculated. A deviation within ±10% indicated that the quality control sample was stable. Specific results are shown in Table 1.

[0157] Table 1 Stability data analysis of sST2 quality control product at 37℃

[0158]

[0159]

[0160] Table 2 Stability data analysis of sST2 quality control product at -20℃

[0161]

[0162]

[0163] The results in Tables 1 and 2 show that the quality control prepared with recombinant protein ① is stable for 3 days at 37°C and 3 months at -20°C; the quality control prepared with recombinant protein ② is stable for 6 days at 37°C and 6 months at -20°C; and the quality control prepared with recombinant protein ③ is stable for 9 days at 37°C and 12 months at -20°C. The stability of the mutated recombinant protein at both 37°C and -20°C is significantly higher than that of the original protein.

[0164] 4.2 Uniformity of quality control products

[0165] For each quality control product, 5 tubes were selected and the test was repeated 5 times for each tube. The intra-tube CV and inter-tube CV were calculated respectively. The coefficient of variation (CV) should not be greater than ±10%.

[0166] 4.2.1. In-tube uniformity

[0167] 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%.

[0168] Table 3 sST2 quality control product homogeneity data analysis table

[0169]

[0170]

[0171] 4.2.2 Uniformity between tubes

[0172] 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%.

[0173] Table 4 sST2 quality control uniformity data analysis table

[0174] sample ① Mean of the results CV ②Result mean CV ③Result mean CV Quality control product 1 5.0 ng / mL 6.1％ 8.1 ng / mL 5.4％ 5.8 ng / mL 5.8％ Quality control product 2 99.6 ng / mL 5.9％ 154.4 ng / mL 4.9％ 159.0 ng / mL 4.8％ Quality control product 3 1002.5 ng / mL 5.6％ 807.7 ng / mL 5.8％ 940.0 ng / mL 5.8％

[0175] 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.

[0176] 5. Interoperability testing

[0177] Samples and quality control products close to the upper limit of the linear range of the soluble growth stimulating expression gene 2 protein (sST2) detection kit (fluorescence immunochromatography) were selected, and low-value samples close to the lower limit of the linear range were used for gradient dilution. The samples and quality control products obtained by gradient dilution were tested with the soluble growth stimulating 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 (results see Figure 4 ), indicating that the recombinant protein has good intercommunication with the natural protein.

[0178] In summary, the recombinant sST2 protein provided by the present invention has a high titer and is close to the natural antigen. It can be used as a raw material for diagnostic reagents (quality control products and preparation of quality control products) or to screen 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. Therefore, sST2 is regarded as a decoy receptor. On the contrary, sST2 acts as a decoy receptor, antagonizing the cardioprotective effect of 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. It can be used to study drug targets, efficacy, toxicity, etc., and to evaluate the correlation of sST2 recombinant protein concentration with cardiac damage, prognosis, cardiac function and inflammatory cell infiltration.

[0179] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0180] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection 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. An expression vector containing a nucleotide encoding the recombinant sST2 protein according to claim 1.

3. A host cell containing the expression vector according to claim 2.

4. 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, transform the vector into competent cells, and extract the endotoxin-free plasmid after culture; S2. The endotoxin-free plasmid is transfected into host cells, and after culture, the cells are subjected to cell lysis, affinity chromatography, washing and elution, ion exchange chromatography, dialysis and concentration to obtain the recombinant sST2 protein; 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.

5. The method according to claim 4, characterized in that In step S1, the vector is pcDNA3.1 and the competent cells are DH5α.

6. The method according to claim 4, characterized in that The matrix in the affinity chromatography is SIGMA Anti-DYKDDDDK affinity purification gel.

7. The method according to claim 4, 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.

8. The method according to claim 4, characterized in that The buffer used in the dialysis process was as follows: 20 mM Tris–HCl, 200 mM NaCl, 50% Glycerol, pH 7.5, 1 mM TCEP.

9. Use of the recombinant sST2 protein according to claim 1 in the preparation of a kit for detecting IL-33.

Images