Spider venom source protein and application thereof in cancer treatment

Abstract

The invention provides sequence identification, expression, anti-cancer activity and mechanism analysis of an anti-cancer large protein in spider venom, belongs to the technical field of biomedical proteins, and particularly relates to a method for extracting an anti-cancer component in the spider venom, performing RACE amplification through in-gel enzymolysis, transcriptome analysis and venom gland extraction to obtain a complete sequence of the anti-cancer component, and analyzing the anti-cancer activity and mechanism of the anti-cancer large protein in the spider venom. And carrying out protein prokaryotic expression by taking pCold II as a carrier, carrying out molecular sieve purification to obtain a high-purity sample, and carrying out anti-cancer functional research. The novel high-cytotoxic-activity protein is identified from spider venom, efficient expression of the novel high-cytotoxic-activity protein is achieved, operation is easy and convenient, reference is provided for identification and expression of natural large protein, and the protein has high cytotoxic activity, meanwhile has certain selectivity to cancer cells and normal cells, has the cancer drug research and development prospect and has good application prospects. Particularly, the polypeptide has potential application value in the aspect of research and development of cancer drugs, and is expected to provide new drug targets and treatment strategies for cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical proteins, and specifically relates to the identification and expression of a strong cytotoxic component in the spider Macrothele rabatii, and studies on its anti-cancer activity and mechanism. Background Art

[0002] Cancer poses a serious threat to global public health and is one of the leading causes of death. Current mainstream treatments include surgery, radiotherapy, targeted therapy, immunotherapy, and chemotherapy. Chemotherapy is widely used due to its low cost, broad spectrum, and compatibility with various treatment modalities. Currently, 80% of chemotherapeutic drugs are derived from natural products, which are crucial resources for the development of anticancer drugs. The venom of venomous animals such as spiders, snakes, scorpions, and centipedes is a unique natural product that contains a variety of anticancer active ingredients, many of which have anticancer activity. Spiders are diverse in species, and their venom is complex, containing a variety of proteins, peptides, enzymes, and other small molecules. The venom of different spider species varies significantly in composition and function, providing a rich resource for the screening of active anticancer compounds. However, research on the active anticancer compounds in spider venom is still in its early stages, and the anticancer potential of most spider toxins has not been fully explored. Furthermore, relatively few technologies exist for isolating and identifying active anticancer compounds from spider toxins and developing them into effective anticancer drugs. Therefore, the identification and expression of spider macroprotein is of great significance for its related research.

[0003] Our laboratory has long been dedicated to the study of spider toxins. Early studies have revealed that a certain protein in the venom of the spider Macrothele rabies has extremely strong anti-cancer toxicity, which mainly causes cell death through multi-modal mechanisms such as inducing apoptosis and ferroptosis. However, the identification and expression of large natural proteins has always been a relatively difficult research. Their complex spatial structure, harsh expression conditions, and the high incidence of degradation make it difficult for traditional technologies to achieve ideal results when processing large natural proteins. In response to the above difficulties, the present invention determined the full-length sequence of this highly cytotoxic protein through in-gel enzymatic hydrolysis, venom gland transcriptomics, and RACE amplification technology, and obtained a high-purity sample of the protein through prokaryotic expression, laying the foundation for the subsequent development of anti-cancer drugs. Summary of the Invention

[0004] The present invention aims to provide a technical route for identifying spider natural large proteins through in-gel enzymatic hydrolysis, venom gland transcriptomics and RACE amplification technology, and efficiently preparing spider venom proteins using the pCold II prokaryotic expression system, and to study its application in cancer treatment and anti-cancer mechanisms.

[0005] The above-mentioned invention object is achieved through the following technical solutions: (1) The effective components separated from the crude toxin are subjected to in-gel enzymatic hydrolysis in the electrophoresis gel. The detection values ​​of most peptide segments are concentrated in the range of -3-3 ppm, indicating that the data obtained by this in-gel enzymatic hydrolysis are relatively accurate and reliable.

[0006] (2) 96.17% of the base numbers in the transcriptome sequencing of the venom gland of the spider Macrothele raegeri met the Q30 standard, and the GC content in the raw data remained highly stable with an error rate of 0.02%. Transdecoder software was used to extract the protein coding region sequence, and then a database of venom proteins of the spider Macrothele raegeri was constructed. The in-gel enzymatic peptide data were searched and matched in the above database, and two fragments with high confidence were obtained, ILVSNHG and EVYLDG, whose corresponding amino acid sequences in the transcriptome were: ATCTTAGTTTCAAATCATGGT and GAAGTTTACTTAGACGGTGG.

[0007] (3) The venom gland mRNA of Macrothele rabat was extracted, and two high-confidence fragment sequences were used as specific primers for 3'RACE for PCR amplification and gel running. They were then imported into the transcriptome database for matching, and the base sequence of 3'RACE was obtained: AGTTTCAAATCATGGTGGAAGGCAACTAGATGACGTACCTGCCACTATTGATGCCCTTCAAGAAGTTGTAAATGCGGTCAAAGGCAGAAAGATTGAAGTGTATCTGGATGGAGGCGTACGAAATGGAACTGATGTTTTCAAAGCTTTAGCTATAGGAGCAAAGGCTGTTTTTGTTGGAAGACCAAACCTGTGGGGACTCTCTGCTGGAGGTGAAAAGGGATCACGAAGGGTTCTAGAATTACTGAGAACAGAATTTGACATGGCCCTAGGACTTGCAGGTCTCAACTCTGCGGCCGCTATCACACCAGACTATGTGGCTCGGAAATCATTTTATGAGGGACAGTGCCATTCATCGTTCCTT.

[0008]

[0009] In some embodiments, the spider toxin protein can directly act on cancer cells and kill specific cancer cells through its high cytotoxic activity and selectivity, thereby reducing the damage of cancer cells to the human body and achieving the purpose of cancer treatment.

[0010] In some embodiments, the spider toxin protein can be designed into drugs such as ADC. ADC (Antibody-Drug Conjugate) drugs are composed of three parts: a monoclonal antibody, a linker, and a cytotoxin. Antibodies achieve precise targeting by recognizing specific antigens on the surface of tumor cells (such as HER2, Trop-2, CD19, etc.); linkers (such as cleavable or non-cleavable types) are degraded in the acidic environment of lysosomes and release cytotoxins (such as microtubule inhibitors or DNA damaging agents) to directly kill tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : The cDNA sequence of the toxic protein SVHAO and its corresponding amino acid sequence.

[0012] Figure 2 :SDS-PAGE image of SVHAO protein after prokaryotic expression and purification.

[0013] Figure 3 :Toxic activity of SVHAO protein on different cell lines.

[0014] Figure 4 : The cytotoxic activity of SVHAO protein after treatment of cells with inhibitors of various death modes. DETAILED DESCRIPTION

[0015] The spider venom protein full-length cDNA sequence (1194 bp) disclosed in the present invention encodes 398 amino acids with a molecular weight of 43.37 kDa. After prokaryotic expression, it can be stored in a 500 mM sodium chloride solution at 4°C for a long time. Therefore, the present invention mainly includes the solution form. The obtained protein sequence is as follows Figure 1 The protein sample was obtained by prokaryotic expression and purified by molecular sieve separation and SDS-PAGE gel run to obtain the pure protein. Figure 2As shown. In this study, a variety of cell lines were selected to measure the IC50 values ​​of SVHAO, in order to further judge the strength of the cytotoxic activity of SVHAO, and also to detect its activity on normal cell lines, in order to analyze the selectivity of SVHAO. In this study, a total of 7 normal cell lines (BEAS-2B, HUVEC, HEK293, L02, AML12, CHO and L929) and 5 tumor cell lines (4T1, B16-F10, K562, SW620 and HepG2) were selected for CCK-8 cytotoxic activity detection. As shown Figure 3 As shown, CCK-8 cytotoxicity assays showed that the IC50 of SVHAO against cancer cell lines was 0.1331-2.8870 μg / mL (3.07-66.6 nM), and the IC50 against seven normal cell lines was 1.214-10.160 μg / mL (70.8-234 nM).

[0016] Programmed cell death is an orderly cell death process controlled by genes, which is crucial for maintaining the normal physiological functions and internal environment stability of the body. To further explore the way in which SVHAO induces cell death, we selected 12 inhibitors, including ferroptosis, apoptosis, pan-apoptosis, pyroptosis, autophagy and necrosis. Under the intervention of these inhibitors, the changes in the cytotoxic activity of SVHAO were measured to lay the foundation for determining its way of inducing cell death. Figure 4 As shown, we tested the toxicity of these inhibitors on K562 cells. According to the toxicity curve, we selected the inhibitor concentrations that interfered with the activity of SVHAO cells, including Lip-1 (Liproxstatin-1) 10 μM, DFO (Deferoxamine) 10 μM, Fer-1 (Ferrostatin-1) 10 μM, Z-DEVD-FMK 20 μM, VX-7655 μM, Z-VAD-FMK 20 μM, Nec-1 (Necrostatin-1) 50 Necrostatin-1, Wor (Wortmannin) 5 μM, LDC (LDC7559) 2.5 μM, M-β-C (Methyl-β-cyclodextrin) 10 μM, F-68 (Pluronic F-68) 50 μg / mL, and BFA (Brefeldin A) 100 nM. Figure 3As shown in the results, after treatment with 0.125 and 0.25 μg / mL of SVHAO, the viability of K562 cells was about 15%. The addition of VX-765, Wor, M-β-C, F-68 or BFA inhibitors had no obvious rescue effect on its cell viability. After treatment with 10 μM Lip-1, 10 μM DFO or 50 μM Nec-1, the viability of K562 cells was significantly restored. On the other hand, the toxic activity of 0.125 μg / mL of SVHAO was significantly inhibited after the addition of 10 μM Fer-1, 20 μM Z-DEVD-FMK, 20 μM Z-VAD-FMK or 2.5 μM LDC, while the toxicity of high concentration 0.25 μg / mL of SVHAO was not significantly affected. The above results indicate that SVHAO can induce ferroptosis, apoptosis, necroptosis and pyroptosis in cells and has a multi-mode anti-cancer mechanism.

[0017] Research Methods 1. In-gel enzymatic hydrolysis.

[0018] (1) Cut the SDS-PAGE stained band from the electrophoresis gel and rinse with deionized water. Repeat three times.

[0019] (2) Let the prepared decolorizing solution completely soak the electrophoresis gel to decolorize, and then wash it with clean water three times.

[0020] (3) Wash the electrophoresis gel with ammonium bicarbonate (25 mM) and different concentrations of acetonitrile until the gel dehydrates and turns white.

[0021] (4) Add DTT (10 mM) solution to the treated strips and incubate in a 56°C water bath for 30 min. (5) When the temperature drops to 25 °C, add an equal volume of AA (50 mM) solution and keep in the dark for 15 min.

[0022] (6) Wash the electrophoresis strips with ammonium bicarbonate solution (25 mM) and acetonitrile until they are dehydrated and turn white.

[0023] (7) Prepare a box of ice and perform the procedure on ice. Add trypsin (0.01 μg / mL) followed by a 50 mM NH4HCO3 solution containing 10% acetonitrile.

[0024] (8) Digest in a 37°C water bath for 16-18 h.

[0025] (9) After removing the sample, centrifuge at 10,000 g for 3 min. Transfer the supernatant and add the extract (2.5% TFA, 67% ACN) to the remaining gelatin. Incubate at 37 °C for 30 min and ultrasonicate. Centrifuge at 10,000 g for 1 min, mix with the supernatant, and dry by centrifugation for subsequent experiments.

[0026] 2. LC-MS / MS analysis.

[0027] The in-gel digested sample was fully dissolved in the Nano-LC mobile phase, and 2 μL of the sample was added to the pre-column for thorough washing and desalting. The sample was then separated on a C18 reversed-phase analytical column, with the concentration of mobile phase B gradually increased from 5% to 38%. Finally, mass spectrometry detection was performed.

[0028] 3. Venom gland transcriptome analysis.

[0029] DNA from the venom gland of the spider Macrothele rabat was digested with DNase and enriched using magnetic beads with oligo(dT) residues. Short mRNA fragments were then collected and used as templates for the synthesis of first- and second-strand cDNAs, which were then purified. These fragments were then end-repaired, A-tailed, and amplified by PCR. The quality of the cDNAs was assessed using an Agilent 2100 Bioanalyzer and sequenced using Illumina HiSeq X Ten and Illumina HiSeq™ 2500 systems, yielding paired-end data of 150 and 125 bp, respectively.

[0030] 4. 3'RACE and 5'RACE.

[0031] We collected Macrothele rae's spiders and, after feeding them for a week, extracted their venom using electrical stimulation. The venom glands were then removed and stored at -80°C. A high-confidence base sequence identified by in-gel digestion was used to identify the corresponding amino acid sequence in the transcriptome. This sequence was then used as a primer for a 3' RACE amplification experiment. Based on this result, 5' RACE amplification was performed and the amino acid sequence was mapped to the library to ensure sequence accuracy and authenticity. The specific experimental steps are as follows: (1) RNA was extracted using a Trizol extraction kit, 1.5% agarose, and 1× TAE electrophoresis buffer. The cells were observed and photographed under ultraviolet light.

[0032] Design primers based on existing high-confidence sequences: 3'RACE specific primers: RC1002-AF1 GAAGTTTACTTAGACGGTGG RC1002-AF2 AGTTTACTTAGACGGTGGGGT RC1002-BF1 ATCTTAGTTTCAAATCATGGT RC1002-BF2 AGTTTCAAATCATGGTGGAAG 5'RACE reverse transcription primer and specific primer: RC1002-RT3 TGTGAAGATTTGAACGGTGTCG RC1002-RT4 AGAACTATTCCGATGTAGGAACCTG RC1002-R4TTTTGTAAATTGCTCCATTTCTCAT RC1002-R5CATGTCTCCACCTGGTCCATAAT Designed according to AF2 RC1002-NRT1GAACACAGTCTTCGTTTATTCAAATC RC1002-NRT2 TAATTCTGGAACTCATTTCTTTTGG RC1002-NR1 GGCCATGTCAAATTCTGTTCTC RC1002-NR2 CTTTTCACCTCCAGCAGAGAGT Designed according to BF2 RC1002-NRT3ACAAAAACAGCCTTTGCTCCTAT RC1002-NRT4TACGCCTCCATCCAGATACACTT RC1002-NR5 GGGTAATTTGTATTTATTCCTCACG RC1002-NR6GGCTTTGTATCCGGCATTCTC First-strand cDNA synthesis: Mix 1 μL of reverse transcription primer (5'RACE reverse transcription primer, 3'adaptor Primer (10 μM) for 3'RACE), RNA, and RNase-free water, centrifuge, incubate in a 65°C water bath for 5 min, immediately place on ice for 2 min, add Reverse Transcriptase Mix (RNase H-), mix, and centrifuge. Incubate in a 50°C water bath for 30 min, heat in a metal bath at 85°C for 1 min, and store at -20°C until use.

[0033] 3'RACE: PCR electrophoresis and recovery using the 3'adaptor as the reverse primer and cDNA as the template.

[0034] 3'RACE PCR reaction system.

[0035]

[0036] 3'RACE PCR cycling conditions.

[0037]

[0038] 5'RACE: The cDNA obtained by reverse transcription using the A first-strand synthesis 5'RACE reverse transcription primer is processed: RNase H digestion, cDNA purification and recovery, TdT addition and C tailing.

[0039] 5'RACE PCR reaction system.

[0040]

[0041] 5'RACE PCR cycling conditions.

[0042]

[0043] Cloning and sequencing: Place 100 μL of competent cells on ice. After complete thawing, gently resuspend the cells evenly. Add 10 μL of ligation buffer and mix gently. Place on ice for 30 minutes, then heat shock in a 42°C water bath for 60 seconds. Place on ice for 10-15 minutes, add 400 μL of LB medium, and culture at 37°C with shaking at 200-250 rpm for 1 hour. Centrifuge at 4000 rpm for 5 minutes at room temperature. Aspirate 400 μL of supernatant with a pipette tip and resuspend the cells in the remaining medium. Plate the bacteria onto ampicillin plates pre-coated with 20 μL of 100 mM IPTG and 100 μL of 20 mg / mL X-gal. Incubate inverted overnight. PCR was performed using the universal primers M13+(-47): aggttttcccagtcacg and M13-(-48): gagcggataacaatttcacac carried on Pmd18-T.

[0044] Cloning and sequencing colony PCR reaction system.

[0045]

[0046] Cycling conditions for clone sequencing colonies.

[0047]

[0048] 5. Induced expression of protein.

[0049] (1) Based on the colonies on the plate, select the appropriate single clone and place it in 1 mL of LB liquid culture medium containing Amp+ resistance, and culture with shaking for about 6 hours.

[0050] (2) After adding an appropriate amount of Amp+, gradually expand the cultured single clone to 1 L of LB medium.

[0051] (3) Detect the OD600 value of the culture medium. When the OD600 value is 1.0, add IPTG to a final concentration of 0.5 mM for induction. Set the temperature to 18 °C and the rotation speed to 150 rpm and culture overnight.

[0052] (4) Collect the induced bacterial solution and centrifuge it at 8000 g for 10 min, then discard the supernatant.

[0053] (5) Use an appropriate amount of 1× Binding Buffer solution to fully resuspend the bacteria.

[0054] (6) Turn on the homogenizer and wait for it to be prepared. Then, break the bacteria 4-5 times at 30 kPa, collect the bacterial liquid, place it at 4°C, and centrifuge it at 12,000 g for 20 min.

[0055] (7) After equilibration of nickel beads with 1× Binding Buffer for 2-3 column volumes, the supernatant of the centrifuged bacterial solution was repeatedly passed through the column until the nickel beads turned completely gray.

[0056] (8) Wash the nickel beads with 1× Washing Buffer solution.

[0057] (9) Elute the protein with 1×Elusion Buffer. Add the eluted solution to a 40 kDa ultrafiltration tube and ultrafilter at 4500 g for 45 min. Then, add an appropriate amount of NaCl to a final concentration of 0.5 M for protein preservation. The protein concentration is then measured using Nanodrop.

[0058] 6. SDS-PAGE electrophoresis.

[0059] (1) Clean the glue holder, long glue board, short glue board and 10-hole comb with detergent, and then rinse them with deionized water three times.

[0060] (2) After the glass gel plate is completely dry, assemble the electrophoresis apparatus and prepare the gel. The lower separation gel needs to be pressed with isopropyl alcohol. After the lower separation gel solidifies, wash off the isopropyl alcohol with deionized water. Gently add the upper concentration gel and slowly insert the comb. Let it stand for about 30 minutes until the gel is fully solidified before use.

[0061] (3) Add 10 μL of 5×SDS Loading Buffer to 40 μL of the expressed protein sample, mix thoroughly, and then place in a 100°C metal bath and boil for 10 min to fully denature the protein. Place the treated sample in a 4°C centrifuge at maximum speed for 5 min and collect the supernatant.

[0062] (4) Remove the comb, add 5.5 μL of marker, and load the protein sample to ensure the same quality in each well.

[0063] (5) Turn on the electrophoresis apparatus, place the electrophoresis tank in ice, and power it on. Run at a constant voltage of 80 V for 30 minutes, then switch to 120 V to run the entire process.

[0064] (6) After the electrophoresis is completed, remove the gel and clean the electrophoresis solution on the gel. Then place it in Coomassie Brilliant Blue staining solution R250 for staining for 1-4 hours. Prepare a destaining solution to destain the stained gel. After the gel base color becomes transparent, scan the gel.

[0065] 7. Molecular sieve chromatography.

[0066] Prepare a 0.5 M NaCl solution and adjust its pH to 6.2-6.3. After preparation, filter the solution using a 0.22 μm filter membrane and then degas. After turning on the equipment, carefully check the system pipes and valves to ensure that there are no bubbles. Next, install the molecular sieve chromatography column. After installation, set the system alarm pressure to 0.25 MPa. Before performing sample analysis, the column needs to be equilibrated. First, use degassed deionized water to equilibrate the column at a flow rate of 1 mL / min, and set the equilibration time to 120 minutes. After completing the first equilibration step, use a 0.5 M NaCl solution at a flow rate of 1 mL / min to continue to maintain the column equilibrium state. The equilibration time for this step is also 120 minutes. Closely observe the changes in the baseline during this process. Set the detection wavelength to 280 / 215 nm and collect each peak.

[0067] 8. Cytotoxic activity detection.

[0068] This study used the Cell Counting Kit-8 (CCK-8) reagent to assess the cytotoxic activity of proteins against CHO, B16-F10, L02, K562, BEAS-2B, Hep-G2, HUVEC, MBA-MD231, and HEK293 cells. After drug administration, 10% CCK-8 was added to the cells, and the cells were incubated at 37°C for 1 hour. OD values ​​at 450 nm were measured using a microplate reader to determine cell viability.

[0069] 9. Effects of inhibitors on the cytotoxic activity of SVHAO.

[0070] K562 cells in good condition after resuscitation and passage 3 were collected by centrifugation at 1200 rpm at 25°C. The cells were thoroughly mixed with 1 mL of serum-free 1640 medium and seeded into a large dish. Once the cells were evenly distributed, they were seeded into 96-well plates, ensuring approximately 2000 cells per well for cell concentration adjustment. 1 mL of medium containing K562 cells was taken from the large dish and various inhibitors were added to achieve the maximum concentration that did not affect normal cell growth. 100 μL of the K562 cells per well of the 96-well plate was then seeded and incubated at 37°C in a 5% CO2 incubator for 30 min. 1 mL of serum-free 1640 medium was removed and the same concentrations of inhibitors as in step 2 were added, along with proteins at final concentrations of 0.125 μg / mL and 0.25 μg / mL. 100 μL of the medium was again added to the 96-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. Finally, cell viability was assessed.

Claims

1. A spider venom-derived anticancer protein, full-length cDNA sequence (1194 bp), containing 398 amino acids, with a molecular weight of 43.37 kDa, the sequence is: GNKICEAGCVCYEKEIRGCSPLDNKLTDMVSMQDFENSALESLDKNARDYYRSGANLEHTLRDNIEAFKRYKIRPRVLRDVSSRKLCVTVLGSKISFPVGIAPSAAQRMASDDGEIGTARAAGSAGTVMILSTLSSTRLEDVAKQYPNSPKWFQLYVFRDLTLTRKLVRRAENAGYKALVVTVDTPFFGRRIADV RNKYKLPSHLQFANFEQGDLDPGSLQSEGDSVLAAVSKLIDPSLTWSNIEWLKSISRLPIVVKGVMTGEDARIAADKGCGGILVSNHGGRQLDDVPATIDAL QEVVNAVKGRKIEVYLDGGVRNGTDVFKALAIGAKAVFVGRPNLWGLSAGGEKGSRRVLELLRTEFDMALGLAGLNSAAAITPDYVARKSFYEGQCHSSFL.

2. The anticancer protein according to claim 1, characterized in that The invention can be prepared into medicine for treating cancer, including breast cancer, melanoma, leukemia, colon cancer and liver cancer.

3. The anticancer protein according to claim 1, characterized in that It can be coupled with tumor-specific targeting compounds including but not limited to antibodies, peptides and liposomes to develop specific conjugated drugs.

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