Preparation method of human recombinant SPHK2 protein

Abstract

The invention provides a preparation method of a human recombinant SPHK2 protein, and belongs to the technical field of biology. The amino acid sequence of the human recombinant SPHK2 protein prepared by the invention is as shown in SEQ ID NO. 1. The human-derived recombinant SPHK2 protein prepared by the preparation method disclosed by the invention is good in expression quantity, and the obtained human-derived recombinant SPHK2 protein is soluble protein, relatively high in purity and good in stability. The human-derived recombinant SPHK2 protein prepared by the invention can be used for screening and evaluating inhibitors of the human-derived recombinant SPHK2 protein, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for preparing human recombinant SPHK2 protein. Background Art

[0002] Sphingosine-1-phosphate (S1P) is involved in regulating cardiovascular function, neuronal development, immune cell transport and other physiological processes, and is associated with various pathophysiological conditions, including cardiovascular disease, autoimmune / inflammatory diseases, neuropathy and cancer. Studies have shown that unlike sphingosine (Sph) and ceramide (Cer), which induce apoptosis, senescence and growth arrest, S1P can promote cell proliferation, migration and survival. Its disorder can promote the occurrence and development of tumors, stimulate angiogenesis and regulate vascular integrity. The accumulation of S1P is associated with the development of cancer and various other diseases, including asthma, inflammatory bowel disease, rheumatoid arthritis and diabetic nephropathy. Therefore, research on the S1P signaling pathway is an option for treating many diseases.

[0003] Inhibition of sphingosine kinases (SPHKs) is a key approach to targeting the S1P signaling pathway. SPHKs are key kinases in the S1P signaling pathway and can regulate the balance between Sph, Cer, and S1P. There are two isoforms of SPHKs, SPHK1 and SPHK2. These two isoforms are encoded by separate genes and exhibit different substrate preferences and subcellular localization. SPHK1 is mainly located in the cytoplasm and can be transferred to the plasma membrane, while SPHK2 is mainly located in the nucleus, endoplasmic reticulum, and mitochondria. SPHK1 is 270 amino acids shorter than SPHK2, and they share approximately 50% sequence identity. The overlapping regions of the two proteins are highly conserved, but their N-terminal and central regions are different.

[0004] At present, researchers have developed a variety of SPHK1 inhibitors, SPHK2 inhibitors and dual inhibitors for the treatment of various related diseases. However, the crystal structure of SPHK2 has not yet been solved, and most reported SPHK2 inhibitors have low potency and specificity. In addition, the number of SPHK2 inhibitors is small, and their clinical use is limited. Therefore, it is necessary to develop a low-cost method that can obtain stable SPHK2 protein to help obtain diverse qualitative and quantitative data for the study of SPHK2 drug targets, which is conducive to determining the crystal structure and mechanism of SPHK2, clarifying the interaction between ligands and binding sites, and providing data to drive chemical design, which provides practical guidance for the further design of new inhibitors. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing human recombinant SPHK2 protein.

[0006] The present invention provides a human recombinant SPHK2 protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a gene fragment for preparing the aforementioned human recombinant SPHK2 protein, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0008] The present invention also provides a plasmid for preparing the aforementioned human recombinant SPHK2 protein, wherein the plasmid is a recombinant plasmid containing a gene fragment having a nucleotide sequence as shown in SEQ ID NO.4.

[0009] The present invention also provides the use of the aforementioned gene fragment or the aforementioned plasmid in preparing the aforementioned human recombinant SPHK2 protein.

[0010] The present invention also provides a method for preparing the aforementioned human recombinant SPHK2 protein, which comprises the following steps:

[0011] (1) Construction of cell expression vector: The target gene fragment with the nucleotide sequence shown in SEQ ID NO.4 was connected to the pfastbac1 plasmid to obtain the recombinant plasmid pfastbac1-N-his-TEV-SPHK2;

[0012] (2) Expression of human recombinant SPHK2 protein: The Bac-to-Bac eukaryotic expression system was constructed using the recombinant plasmid pfastbac1-N-his-TEV-SPHK2 and insect cells to express human recombinant SPHK2 protein;

[0013] (3) Purification of human recombinant SPHK2 protein: Human recombinant SPHK2 protein was purified by Ni-NAT affinity chromatography and gel filtration chromatography.

[0014] Further,

[0015] In step (2), the expression of human recombinant SPHK2 protein is achieved by using recombinant bacilli to transfect insect cells and prepare virus bacilli, and then subculture to prepare P2 virus; P2 virus is harvested and P3 virus is prepared in the same way; and P3 virus is used to infect cells to express human recombinant SPHK2 protein.

[0016] Furthermore, the recombinant bacmid is constructed by transforming the recombinant plasmid pfastbac1-N-his-TEV-SPHK2 into DH10bac competent cells, and then extracting the recombinant bacmid from the DH10bac competent cells.

[0017] Furthermore, when the P3 virus infects cells, the virus-to-cell infection ratio is 1:100 v / v;

[0018] And / or, the P3 virus infects cells for 48-72 hours;

[0019] And / or, the cells infected by the P3 virus are SF9 cells or H5 cells.

[0020] Further,

[0021] In step (3), the purification of human recombinant SPHK2 protein is to resuspend the cells infected with P3 virus in step (2) with lysis buffer, lyse and break the cells, collect the supernatant and purify it with a nickel column; the sample after nickel column purification is enzymatically digested with TEV protease, the histidine tag on the protein is removed, and then a secondary nickel column purification is performed to remove foreign proteins that non-specifically bind to the nickel column; the protein sample after the secondary nickel column purification is purified with a molecular sieve column; and finally the human recombinant SPHK2 protein is obtained.

[0022] The present invention also provides use of the aforementioned human recombinant SPHK2 protein in screening and evaluating SPHK2 inhibitors.

[0023] The present invention provides a method for preparing a human recombinant SPHK2 protein, wherein the method has a good expression amount, and the obtained human recombinant SPHK2 protein is a soluble protein with high purity and good stability. The human recombinant SPHK2 protein prepared by the present invention can be used for screening and evaluation of its inhibitors, and has good application prospects.

[0024] The present invention utilizes the Bac-to-Bac eukaryotic expression system to obtain a recombinant baculovirus capable of expressing the SPHK2 protein, and then utilizes the presentation effect of the virus to infect insect cells again and express the SPHK2 protein. During the expression process of this system, the structure and characteristics of the SPHK2 protein can be post-transcriptionally modified, which is closer to the original function and is superior to the Escherichia coli expression system. SF9 insect cells are selected as expression cells, which have fast reproduction, short culture cycle and low culture conditions, thus providing support for scientific research or large-scale production.

[0025] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0026] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The expression condition verification results of human recombinant SPHK2 protein; in the figure, M: protein molecular weight standard; 1: supernatant of SF9 cells expressed for 48 hours; 2: supernatant of SF9 cells expressed for 72 hours; 3: supernatant of H5 cells expressed for 48 hours; 4: supernatant of H5 cells expressed for 72 hours; the virus infection ratios in the figures are all 1:100 (v / v).

[0028] Figure 2 The results of SDS-PAGE protein electrophoresis after the first nickel column purification of human recombinant SPHK2 protein; in the figure, M: protein molecular weight standard; 1: whole cell portion of protein; 2: protein supernatant portion; 3: protein supernatant flows through the nickel column; 4: binding solution passes through the nickel column; 5: cleaning solution passes through the nickel column (20mM imidazole); 6: cleaning solution passes through the nickel column (30mM imidazole); 7: eluent passes through the nickel column (250mM imidazole).

[0029] Figure 3 The results of SDS-PAGE detection of human recombinant SPHK2 protein after TEV digestion and secondary nickel column purification; in the figure, M: protein molecular weight standard; 1: protein after digestion; 2: protein sample after digestion flowing through the nickel column.

[0030] Figure 4 The test results of the final product of human recombinant SPHK2 protein: a. Human recombinant SPHK2 protein was tested by superdexS200 TM Increase 10 / 300GL column purification map; b is the SDS-PAGE protein electrophoresis result of the final product of human recombinant SPHK2 protein, M: protein molecular weight standard; l: sample.

[0031] Figure 5 The SDS-PAGE protein electrophoresis detection results after purification of the human recombinant SPHK2 protein prepared by a plasmid containing the nucleotide sequence shown in SEQ ID NO.5 and the SDS-PAGE protein electrophoresis detection results after freeze-thaw cycles: a is the SDS-PAGE protein electrophoresis detection result after purification, M: protein molecular weight standard, 1: sample without freeze-thaw cycles; b is the SDS-PAGE protein electrophoresis detection result after freeze-thaw cycles, M: protein molecular weight standard, 1: sample after freeze-thaw cycles.

[0032] Figure 6The results of SDS-PAGE protein electrophoresis after nickel column purification of the human recombinant SPHK2 protein expressed in SF9 cells and H5 cells with the amino acid sequence shown in SEQ ID NO.3: a is the detection result of SF9 cell expression, M: protein molecular weight standard, 1: protein whole cell fraction, 2: protein precipitate fraction, 3: protein supernatant passing through nickel column, 4: binding solution passing through nickel column, 5: cleaning solution passing through nickel column (20mM imidazole), 6: eluate passing through nickel column (250mM imidazole); b is the detection result of H5 cell expression, M: protein molecular weight standard, 1: protein whole cell fraction, 2: protein precipitate fraction, 3: protein supernatant passing through nickel column, 4: binding solution passing through nickel column, 5: cleaning solution passing through nickel column (20mM imidazole), 6: eluate passing through nickel column (250mM imidazole). DETAILED DESCRIPTION

[0033] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0034] Example 1. Preparation of human recombinant SPHK2 protein of the present invention

[0035] Step 1: Design of expression length of human recombinant SPHK2 protein

[0036] According to the sequence of human recombinant SPHK2 protein, some amino acids in the disordered region were deleted, and the amino acid sequences of human recombinant SPHK2 protein as shown in SEQ ID NO.1 (AA.48-399, 509-654), SEQ ID NO.2 (AA.72-654) and SEQ ID NO.3 (AA.72-399, 509-654) were designed. The sequence includes the TEV-His sequence at the N-terminus for protein purification. Through analysis, the obtained human recombinant SPHK2 protein can meet its biological function.

[0037] SEQ ID NO.1:

[0038]

[0039] SEQ ID NO.2:

[0040]

[0041]

[0042] SEQ ID NO.3:

[0043]

[0044] Description: Ala(A), Glu(E), Met(M), Tyr(Y), Arg(R), Gly(G), Phe(F), Val(V), Asn(N), His(H), Pro(P ), Pyl(O), Asp(D), Ile(I), Ser(S), Sec(U), Cys(C), Leu(L), Thr(T), Gln(Q), Lys(K), Trp(W).

[0045] Step 2: Construction of cell expression vector

[0046] The codons of the human recombinant SPHK2 gene were optimized to synthesize the plasmid pfastbac1-N-his-TEV-SPHK2 containing the nucleotide sequence of the human recombinant SPHK2. The nucleotide sequence of the target gene obtained after the codon optimization of the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.4, the nucleotide sequence of the target gene obtained after the codon optimization of the amino acid sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.5, and the nucleotide sequence of the target gene obtained after the codon optimization of the amino acid sequence shown in SEQ ID NO.3 is shown in SEQ ID NO.6. The obtained target gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd. to obtain the pfastbac1-N-his-TEV-SPHK2 plasmid.

[0047] SEQ ID NO.4:

[0048]

[0049]

[0050] SEQ ID NO.5:

[0051]

[0052]

[0053] SEQ ID NO.6:

[0054]

[0055]

[0056] Step 3: Expression of human recombinant SPHK2 protein

[0057] 3.1 Construction and identification of the recombinant baculovirus shuttle vector reBacmid-SPHK2

[0058] 3.1.1 Transformation: The pfastbac1 plasmid containing the SPHK2 gene (taking the plasmid constructed with the nucleotide sequence shown in SEQ ID NO.4 as an example) was transformed into DH10bac competent cells.

[0059] 1) Take 10 ng of pfastbac1 plasmid and add it to 20 μl of commercial DH10bac competent cells, incubate on ice for 30 min, heat shock at 42°C for 45 s, incubate on ice for 2 min, add 200 μl of LB medium, and culture at 37°C, 220 rpm with shaking for 4 h.

[0060] 2) Take 2 μl of the liquid obtained in step 1), mix it evenly with 100 μl LB medium, 100 μl X-gal solution, and 2 μl IPTG solution, take 50 μl of the mixed solution to spread on an agar plate (the agar plate contains 50 μg / mL kanamycin, 10 μg / mL tetracycline, and 7 μg / mL gentamicin), and culture it at 37°C in the dark for 72 h.

[0061] 3) After 72 hours, the blue-white spots become obvious.

[0062] 3.1.2 Extract reBacmid-SPHK2. Extract reBacmid-SPHK2 from competent cells.

[0063] 1) Pick a white spot and place it in 5 ml of LB medium containing antibiotics (kanamycin 50 μg / mL, tetracycline 10 μg / mL, gentamicin 7 μg / mL), and culture at 37°C and 220 rpm with shaking for about 16 hours.

[0064] 2) Centrifuge at 4000 rpm for 15 min, discard the supernatant, add 200 μl P1 to resuspend, and let stand for 5-10 min.

[0065] 3) Add 200 μl of P2, mix gently, and let stand for no more than 5 min to observe the solution to clarify.

[0066] 4) Add 200 μl P3, invert the centrifuge tube 6-8 times, and immediately place in a water bath for 5-10 minutes.

[0067] 5) Centrifuge the sample at 4°C, 14,000 g for 10 min; take another 550 μl of isopropanol and place it on ice.

[0068] 6) After centrifugation, take the supernatant and add it to pre-cooled isopropanol, place it in an ice bath for 10 minutes, and centrifuge it at 14000g for 10 minutes at room temperature.

[0069] 7) Discard the supernatant, add 500 μl of 70% ethanol, invert the centrifuge tube several times, wash the precipitate, and centrifuge at 14,000 g for 5 minutes at room temperature.

[0070] 8) Repeat step 7) once.

[0071] 9) Discard the supernatant and leave the centrifuge tube open for about 10 minutes to dry the ethanol. The precipitate will become transparent and the resulting precipitate is reBacmid-SPHK2.

[0072] 10) Add 60 μl TE to dissolve the precipitated reBacmid-SPHK2 for later use.

[0073] In the above steps, the buffer formulas and solvents are all pure water:

[0074] P1: 15mM Tris-HCl (pH 8.0), 10mM EDTA, 100μg / ml RNase.

[0075] P2: 0.2 M NaOH, 1% SDS.

[0076] P3: 3 M KAc, pH 5.5.

[0077] TE: 10mM Tris, pH 8.0, 10mM EDTA.

[0078] 3.1.3 Identification: The recombinant bacmid DNA was analyzed by PCR using universal primers for Bac to Bac baculovirus vectors. The PCR reaction system is shown in Table 1.

[0079] Table 1. PCR reaction system of recombinant bacmid DNA

[0080]

[0081] Carry out PCR reaction under the following conditions:

[0082] 98℃30s, 1 cycle; 98℃10s, 58℃5s, 72℃30s, 35 cycles in total; extension at 72℃ for 2min; finally storage at 16℃.

[0083] 3.2 Expression of human recombinant SPHK2 protein in insect cells

[0084] 3.2.1 Transfection: Steps for transfecting SF9 cells with reBacmid-SPHK2

[0085] To improve transfection efficiency, transfection should be performed promptly after reBacmid-SPHK2 is extracted.

[0086] 1) Preparation of transfection complexes

[0087] Solution A: 10 μg reBacmid-SPHK2 + SF9 culture medium = 100 μl;

[0088] Solution B: 8 μl transfection reagent (LipoInsect TM , C0551-1, purchased from Biotime Biotechnology Co., Ltd.) + 92 μl SF9 culture medium = 100 μl.

[0089] Mix solutions A and B to form a transfection complex and let it stand for 30 minutes.

[0090] 2) Plate 0.8*10^6 SF9 cells in each well of a six-well plate and wait for the cells to adhere to the bottom (about 15 minutes).

[0091] 3) After 30 minutes, the cells in the six-well plate have completed their attachment and the supernatant is aspirated. After adding 800 μl of SF9 culture medium, the transfection complex is gently added dropwise to the six-well plate and placed at 27° C. for 4 hours.

[0092] 4) After 4 hours, remove the supernatant and gently add 3 ml of fresh SF9 medium along the well wall and place at 27°C for 4 days.

[0093] 5) After 4 days, aspirate the supernatant, centrifuge at 800g for 3 minutes, collect the supernatant, and add 4% serum, which is the P1 virus.

[0094] 3.2.2 Amplification of P2 virus

[0095] 1) Take 1 ml of P1 virus and add it to 50 ml of SF9 cells (density is 2*10^6), and culture it at 27℃, 110rpm with shaking for 7 days. Store the remaining P1 at 4℃.

[0096] 2) After 7 days, centrifuge at 2000 rpm for 15 min, collect the supernatant, and add 4% serum, which is the P2 virus.

[0097] 3.2.3 Amplification of P3 virus

[0098] 1) Take 1 ml of P2 virus and add it to 50 ml of SF9 cells (density is 2*10^6), shake and culture at 27℃, 110rpm for 1 day. Store the remaining P2 at 4℃.

[0099] 2) After 7 days, centrifuge at 2000 rpm for 15 min, collect the supernatant, add 4% serum, and store at 4°C. This is the P3 virus.

[0100] 3.3 Optimization of cell infection conditions

[0101] The conditions for expressing human recombinant SPHK2 protein by infecting cells with P3 virus were optimized. The optimized conditions are shown in Table 2.

[0102] Table 2. Conditions for expressing human recombinant SPHK2 protein

[0103]

[0104] After expression under each condition, samples were taken for Western Blot detection to compare the protein expression under different conditions. The results of the expressed human recombinant SPHK2 protein are as follows Figure 1 shown. Figure 1 The results showed that when the virus infection ratio was 1:100 (v / v), human recombinant SPHK2 protein could be expressed, but when the virus infection ratio was 1:1000 (v / v), human recombinant SPHK2 protein could not be expressed. The optimal conditions were finally selected as follows: virus infection ratio 1:100 (v / v), infection time 48h, and expression cells SF9 cells. Under such conditions, human recombinant SPHK2 protein can be successfully expressed in a short time. Since SF9 cells are used for virus preparation, there is no need to replace cells and culture medium at this time, which is more convenient.

[0105] Step 4: Purification of human recombinant SPHK2 protein

[0106] 4.1 First step of nickel column purification of human recombinant SPHK2 protein

[0107] Lysis buffer: 250 mM NaCl, 20 mM Tris-HCl (pH 8.0), 10% glycerin, the solvent is pure water;

[0108] Binding buffer: 250 mM NaCl, 20 mM Tris-HCl (pH 8.0), 10% glycerin, 10 mM Mimidazole, the solvent is pure water;

[0109] Washing buffer: 250 mM NaCl, 20 mM Tris-HCl (pH 8.0), 10% glycerin, 20 / 30 mM Mimidazole, the solvent is pure water;

[0110] Elution buffer: 250 mM NaCl, 20 mM Tris-HCl (pH 8.0), 10% glycerin, 250 mM imidazole, the solvent is pure water;

[0111] 1) Add 1% (v / v) P3 virus to 1L SF9 cells (density 2*10^6), shake and culture at 27°C, 110 rpm for 2 days. Centrifuge at 3500 rpm, 15 min to collect the precipitate. Resuspend in lysis buffer.

[0112] 2) After the cells were disrupted by high-pressure disruptor, centrifugation was performed at 15,000 rpm and 4°C for 50 min to collect the supernatant.

[0113] 3) The supernatant and 3 ml of pre-equilibrated nickel filler were mixed and incubated at 4°C for 2 hours, followed by low-speed centrifugation (centrifugal speed of 2500 rpm / min, time of 3-5 min) to discard the supernatant and collect the filler. The nickel filler was balanced by washing the nickel filler with 30-60 mL of pure water and then adding 3-5 mL of binding buffer for balance.

[0114] 4) Use binding buffer to evenly disperse the filler, then centrifuge at low speed (centrifugal speed is 2500 rpm / min, time is 3-5 min), discard the supernatant and collect the filler, and repeat 5 times.

[0115] 5) Use 50 mL of washing buffer containing 20 mM imidazole to evenly disperse the filler, then centrifuge at low speed (centrifugal speed is 2500 rpm / min, time is 3-5 min), discard the supernatant and collect the filler, repeat 4 times, and transfer the filler to the purification column.

[0116] 6) Wash the filler with 50 mL of washing buffer containing 30 mM imidazole.

[0117] 7) Elute the human recombinant SPHK2 protein with an elution buffer.

[0118] 8) The results of the first nickel column purification of human recombinant SPHK2 protein by SDS-PAGE protein electrophoresis are as follows Figure 2 As shown, according to the electrophoresis results, the human recombinant SPHK2 protein was mainly eluted in 250 mM imidazole buffer.

[0119] 4.2 TEV protease cleavage of human recombinant SPHK2 protein

[0120] According to conventional methods in the art, human recombinant SPHK2 protein was digested with TEV protease to remove the histidine tag on the protein molecule (the digestion ratio was TEV: human recombinant SPHK2 protein = 1:100, mass ratio). 1 mM DTT was added to the lysate at 4°C for overnight digestion and dialysis.

[0121] 4.3 Secondary purification of human recombinant SPHK2 protein after TEV digestion using nickel column.

[0122] 1) Equilibrate a nickel column containing 1 ml of nickel packing with the lysate.

[0123] 2) Add the human recombinant SPHK2 protein solution after enzyme digestion and collect the flow-through.

[0124] 3) Wash the medium with a small amount of binding buffer.

[0125] 4) Wash the filler with a small amount of eluent.

[0126] like Figure 3 The flow-through was concentrated to less than 1 mL by centrifugation according to the SDS-PAGE electrophoresis results, and then further purified by molecular sieve using an S200 column. The flow-through contained human recombinant SPHK2 protein.

[0127] 4.4 Molecular sieve purification of human recombinant SPHK2 protein

[0128] Preparation buffer: 25mM HEPES, 300mM NaCl, pH7.5, 5% glycerol, 2mM DTT, 0.04% Triton-100, the solvent is pure water.

[0129] The concentrated human recombinant SPHK2 protein sample was washed with a molecular sieve column (superdex S200 TM Increase 10 / 300GL, 24mL, GE) for purification. During purification, the injection flow rate was 0.5mL / min and the collection volume was 0.5mL. The sample was passed through superdex S200 TM Increase 10 / 300GL column purification chart Figure 4 (a) as shown; the SDS-PAGE test results of the collected samples are as follows Figure 4 (b). Figure 4 (b) The samples in the collection tubes are combined to obtain the final product of human recombinant SPHK2 protein.

[0130] According to the method for preparing human recombinant SPHK2 protein using the plasmid constructed by the nucleotide sequence of SEQ ID NO.4, the plasmid constructed by the nucleotide sequence of SEQ ID NO.5 was used to prepare the human recombinant SPHK2 protein, which was denoted as SPHK2 (72-654) and whose amino acid sequence was shown in SEQ ID NO.2. The results of SDS-PAGE protein electrophoresis after purification of the obtained human recombinant SPHK2 protein were as follows: Figure 5 The prepared human recombinant SPHK2 protein was subjected to freeze-thaw cycles according to conventional methods in the art, and then subjected to SDS-PAGE protein electrophoresis detection. The results are shown in Figure 5 As shown in b. Figure 5 a and Figure 5 bIt can be seen that: Figure 5 b 1 sample ratio Figure 5 There is an extra band in sample 1 in a, which proves that the protein is degraded after freeze-thaw cycles, indicating that the protein expressed by the amino acid sequence such as SEQ ID NO.2 has a high purity after purification, but the protein is degraded after freeze-thaw cycles, and the protein stability is poor. The protein expressed by the SEQ ID NO.1 sequence is not only high in purity after purification, but also has good protein stability.

[0131] According to the method for preparing human recombinant SPHK2 protein using the plasmid constructed by the nucleotide sequence of SEQ ID NO.4, the plasmid constructed by the nucleotide sequence of SEQ ID NO.6 was used to prepare human recombinant SPHK2 protein, which was recorded as SPHK2 (72-399, 509-654), and its amino acid sequence was shown in SEQ ID NO.3. The results of SDS-PAGE protein electrophoresis after purification were as follows: Figure 6 As shown. Figure 6 It can be seen that when SEQ ID NO.3 was expressed in two cells, SF9 / H5, most of the target protein was precipitated, and the target band was observed to be thinner than the impurity protein, indicating that the expression level was low. This shows that SEQ ID NO.3 can express the target protein, but the expression level is low.

[0132] According to the results, the human recombinant SPHK2 proteins expressed by SEQ ID NO. 2 and SEQ ID NO. 3 have certain problems. Only the human recombinant SPHK2 protein expressed by SEQ ID NO. 1 has high expression level, good protein stability and excellent effect.

[0133] In summary, the present invention provides a method for preparing a human recombinant SPHK2 protein, which has a good expression amount, and the obtained human recombinant SPHK2 protein is a soluble protein with high purity and good stability. The human recombinant SPHK2 protein prepared by the present invention can be used for the screening and evaluation of its inhibitors, and has good application prospects.

Claims

1. A human recombinant SPHK2 protein, Features: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A gene fragment for preparing the human recombinant SPHK2 protein according to claim 1, Features: Its nucleotide sequence is shown in SEQ ID NO.

4.

3. A plasmid for preparing the human recombinant SPHK2 protein according to claim 1, Features: The plasmid is a recombinant plasmid containing a gene fragment having a nucleotide sequence as shown in SEQ ID NO.

4.

4. Use of the gene fragment according to claim 2 or the plasmid according to claim 3 in preparing the human recombinant SPHK2 protein according to claim 1.

5. A method for preparing the human recombinant SPHK2 protein according to claim 1, Features: It includes the following steps: (1) Construction of cell expression vector: The target gene fragment with the nucleotide sequence shown in SEQ ID NO.4 was connected to the pfastbac1 plasmid to obtain the recombinant plasmid pfastbac1-N-his-TEV-SPHK2; (2) Expression of human recombinant SPHK2 protein: The Bac-to-Bac eukaryotic expression system was constructed using the recombinant plasmid pfastbac1-N-his-TEV-SPHK2 and insect cells to express human recombinant SPHK2 protein; (3) Purification of human recombinant SPHK2 protein: Human recombinant SPHK2 protein was purified by Ni-NAT affinity chromatography and gel filtration chromatography.

6. The method according to claim 5, Features: In step (2), the expression of human recombinant SPHK2 protein is achieved by using recombinant bacilli to transfect insect cells and prepare virus bacilli, and then subculture to prepare P2 virus; P2 virus is harvested and P3 virus is prepared in the same way; and P3 virus is used to infect cells to express human recombinant SPHK2 protein.

7. The method according to claim 6, Features: The method for constructing the recombinant bacmid is as follows: transforming the recombinant plasmid pfastbac1-N-his-TEV-SPHK2 into DH10bac competent cells, and then extracting the recombinant bacmid from the DH10bac competent cells.

8. The method according to claim 6, Features: When the P3 virus infects cells, the virus-to-cell infection ratio is 1:100 v / v; And / or, the P3 virus infects cells for 48-72 hours; And / or, the cells infected by the P3 virus are SF9 cells or H5 cells.

9. The method according to claim 6, Features: In step (3), the purification of human recombinant SPHK2 protein is to resuspend the cells infected with P3 virus in step (2) with lysis buffer, lyse and break the cells, collect the supernatant and purify it with a nickel column; the sample after nickel column purification is enzymatically digested with TEV protease, the histidine tag on the protein is removed, and then a secondary nickel column purification is performed to remove foreign proteins that non-specifically bind to the nickel column; the protein sample after the secondary nickel column purification is purified with a molecular sieve column; and finally the human recombinant SPHK2 protein is obtained.

10. Use of the human recombinant SPHK2 protein according to claim 1 in screening and evaluating SPHK2 inhibitors.

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