CN109134611B - Polypeptide for inhibiting EGF (epidermal growth factor receptor) promoting tumor cell proliferation by specifically binding EGFR (epidermal growth factor receptor) - Google Patents

Abstract

The invention relates to a polypeptide for inhibiting EGF (epidermal growth factor receptor) from promoting tumor cell proliferation by competitively binding EGFR (epidermal growth factor receptor), belonging to the technical field of biological medicines; the invention takes the ectodomain EGFR-ECD of the epidermal growth factor receptor as a target molecule, and takes EGF as an effective component to specifically elute phage combined with the target molecule by panning of phage display technology, so as to obtain a bioactive polypeptide TUZG 14; the invention proves that the bioactive polypeptide TUZG14 obviously inhibits the proliferation process of stomach cancer cells caused by EGF; the polypeptide sequence of the invention is shorter, the synthesis and the realization of large-scale production are easy, the cancer promotion effect of EGF can be inhibited, the polypeptide has the potential of being subsequently developed into an anti-cancer medicament, and the polypeptide has important application value in the research and development aspects of the anti-cancer medicament.

Description

Polypeptide for inhibiting EGF (epidermal growth factor receptor) promoting tumor cell proliferation by specifically binding EGFR (epidermal growth factor receptor)

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a polypeptide which specifically binds to EGFR and inhibits EGF from promoting tumor cell proliferation.

Background

The epidermal Growth Factor receptor EGFR (epidermal Growth Factor receptor) belongs to

The epidermal growth factor family (i.e., the ErbB family). EGFR is a glycoprotein receptor on the surface of cell membranes, consists of 1186 amino acid residues, has a molecular weight of 170KD, and has tyrosine kinase activity. EGFR can be structurally divided into three parts: extracellular region: 621 amino acid residues, a region binding to its ligand; ② a transmembrane region: 23 amino acid residues form an alpha helical structure to fix the receptor on a cell membrane; ③ intracellular region: 542 amino acid residues, which contain the typical ATP binding site and tyrosine kinase phosphorylation site, are responsible for the activation of downstream pathways. In the ligand-free state, the EGFR exists on the surface of a cell membrane in a monomer form, when an extracellular region of the EGFR is combined with a ligand, the EGFR forms a homo-or heterodimer, a tyrosine kinase phosphorylation site of the EGFR is activated and is subjected to autophosphorylation, and a phosphorylated tyrosine anchoring site can activate downstream related proteins, provide division signals for cells and stimulate the growth and proliferation of the cells.

Epidermal Growth Factor (EGF) is one of the main members of the epidermal growth factor superfamily, is a single-chain low-molecular-weight polypeptide, contains 53 amino acid residues in the chain, has 3 pairs of disulfide bonds, has the molecular weight of 6045Da and the isoelectric point PI4.6, is stable to heat, and is one of the most stable proteins known at present. EGF is a multi-effect cytokine, and can promote cell proliferation and stimulate cell differentiation, and the EGF has the main functions of regulating differentiation and proliferation of epidermal cells, epithelial tissues and interstitium. EGF acts to stimulate cell growth by binding to the specific receptor EGFR on its cell membrane. Under normal physiological conditions, binding of EGF to EGFR is necessary to regulate proliferation, growth and differentiation of normal cells, but if EGF and EGFR-mediated signaling pathways are abnormal, the growth, division and differentiation of cells are changed from a controlled state to a uncontrolled state, resulting in tumor occurrence. Research shows that EGFR is highly expressed in many cancers, such as non-small cell lung cancer, breast cancer, gastric cancer, bladder cancer, colorectal cancer and the like, so that EGFR is an important target in tumor treatment.

A large number of researches show that EGFR is related to the differentiation degree of gastric cancer tissues, and the higher expression rate of EGFR indicates the higher malignancy degree of tumors. The gastric cancer is slow in progress but continues to develop, most of gastric cancer patients have a large amount of metastatic spread at the initial diagnosis, which is also a main reason for poor prognosis of the gastric cancer, and researches show that EGF can promote epithelial-mesenchymal transformation of gastric cancer cells and is beneficial to tumor cell infiltration and metastasis. The expression of EGF and EGFR is closely related to the differentiation degree, infiltration and transfer of gastric cancer, and is an ideal target for treating gastric cancer.

Drugs targeting the EGFR family include two broad classes: antibody drugs and small molecule tyrosine kinase inhibitors. Currently, EGFR antibody drugs on the market in China include cetuximab, panitumumab, nimotuzumab and the like, and although the drugs have obvious curative effects and fewer side effects, the drugs are very expensive, and patients cannot bear high cost for a long time, so that the application of the drugs is limited. The action mechanism of the small molecule inhibitor is different from that of the monoclonal antibody, the small molecule inhibitor is mainly used for blocking the interaction of the small molecule inhibitor and ATP by competitively binding with the phosphorylation site of EGFR intracellular tyrosine kinase and then inhibiting the tyrosine phosphorylation of EGFR and a series of downstream signal transduction, so that the small molecule inhibitor is inferior to the monoclonal antibody in specificity, and the side effects of diarrhea, rash, nausea and the like can occur in the using process of a patient.

In recent years, polypeptide drugs have become a hot spot in drug development. The polypeptide drug has small molecular weight, is easy to pass through human body barriers, has the quality control level close to that of the traditional micromolecule chemical drug, has the activity close to that of antibody drugs, combines the advantages of the traditional micromolecule chemical drug and protein drugs, has less waste discharged in the production process, belongs to the green pharmacy category, and has wide development prospect. The phage display technology fuses the gene of the exogenous polypeptide with the coat protein gene of the phage, so that random polypeptide is expressed on the surface of the phage to form a peptide library, and in the panning process, certain phage capable of being specifically combined with target molecules are retained, thereby obtaining the polypeptide sequence on the surface of the phage through sequencing. The phage display technology has strong purposiveness, can realize high-flux panning, is easy to produce polypeptide and low in cost, is widely applied to multiple fields of biological medicine research, and particularly has wide application prospects in the aspects of artificial antibody and vaccine development, polypeptide medicine development and the like.

Disclosure of Invention

The invention aims to provide a novel EGFR (epidermal growth factor receptor) targeted polypeptide, and particularly provides a polypeptide which is obtained by screening through a phage display technology and takes EGFR as a target spot and has anti-tumor activity.

The invention provides a polypeptide for inhibiting EGF (epidermal growth factor) to promote tumor cell proliferation, wherein the polypeptide is TUZG14, the amino acid sequence of the polypeptide is shown as SEQ.ID.NO.1, and the specific sequence information is as follows: lys Val Trp Val Val Pro Asn are provided.

Wherein the polypeptide inhibits EGF proliferation of tumor-promoting cells by specifically binding to EGFR; preferably, the tumor cell is a gastric cancer cell; preferably, the gastric cancer cell is an AGS cell.

The invention also provides application of the polypeptide for inhibiting EGF from promoting tumor cell proliferation in preparing a tumor-targeted drug.

Among them, preferably, the tumor is gastric cancer.

The present invention also provides a targeted drug for treating or preventing tumor, the drug comprising the polypeptide of claim 1; preferably, the tumor is gastric cancer.

The invention firstly introduces the gene of a target EGFR extracellular domain (EGFR-ECD) into a prokaryotic expression vector pET-30a, so that the recombinant protein EGFR-ECD with a label on the vector is expressed in a large quantity, and the high-purity target protein EGFR-ECD is obtained by purifying from an inclusion body.

And coating and fixing the EGFR-ECD, enabling the phage to be combined with the EGFR, competitively eluting the phage specifically combined with the target molecule by using a ligand EGF of the EGFR, and enriching to obtain the phage with high affinity through 3 rounds of panning. Amplifying the selected monoclonal phage, extracting DNA, sequencing the amplified phage and synthesizing the polypeptide TUZG14 with high frequency of occurrence.

The result of the verification of the in vitro activity of the polypeptide by the MTT method shows that the polypeptide TUZG14 has the inhibiting effect on EGF promoting the proliferation of gastric cancer cell AGS, and the amino acid sequence is as follows: lys Val Trp Val Val Pro Asn are provided.

The invention has the beneficial effects that:

the invention relates to a polypeptide TUZG14 which is screened by taking EGFR as a target and can specifically bind to EGFR and inhibit EGF from promoting tumor cell proliferation. In the invention, the gastric cancer cells are taken as a model, and the polypeptide TUZG14 is verified to have the activity of remarkably inhibiting EGF from promoting the growth of tumor cells, so that the polypeptide TUZG14 has clinical value of being an anti-tumor medicament. The polypeptide of the invention has short sequence, small molecular weight, easy realization of large-scale production, low production cost and wide market prospect.

Drawings

FIG. 1 is an amino acid sequence of a prokaryotic expression protein EGFR-ECD; wherein the shadow part is (His)₆The underlined part of the tag is the amino acid sequence of the target protein EGFR-ECD.

FIG. 2 shows the results of gel electrophoresis of the constructed protein expression vector pET-30 a/EGFR-ECD; wherein Panel A is a 1% agarose gel validation of PCR amplification products of EGFR-ECD; FIG. B shows the double restriction enzyme digestion verification of the recombinant plasmid pET-30a/EGFR-ECD, in which M is a DNA marker and 1 is Bam H of empty pET-30a vector

、Eco R

The double digestion result of (1) is that 2 is Bam H of EGFR-ECD

、Eco R

The double enzyme digestion result of (3) is the PCR amplification product of EGFR-ECD.

FIG. 3 shows the results of the expression and purification verification of the recombinant protein EGFR-ECD in BL21(DE 3); wherein, the graph A is a bacterial liquid which is not centrifuged after the ultrasonication, wherein M is a protein marker, 1 is a transformant for transforming an empty vector pET-30a, and 2 is a transformant for transforming a recombinant vector pET-30 a/EGFR-ECD; panel B is the supernatant after ultrasonication and centrifugation, where M is a protein marker, 1 is a transformant transforming an empty vector pET-30a, and 2 is a transformant transforming a recombinant vector pET-30 a/EGFR-ECD; and the figure C is a protein dialysis renaturation result, wherein 1 is a bacterial liquid which is not centrifuged after a transformant for transforming the pET-30a/EGFR-ECD recombinant vector is subjected to ultrasonic disruption, and 2 is a protein sample band after precipitation after centrifugation and denaturation and dialysis renaturation.

FIG. 4 shows the results of screening EGF-sensitive gastric cancer cell lines; wherein Panel A is the effect of EGF on BGC-803 cell survival; b is the effect of EGF on MGC-803 cell survival; c is the effect of EGF on AGS cell survival.

FIG. 5 shows the result of verifying the activity of the polypeptide TUZG 14; EGF concentration was 0.033 μ g/mL (5 nM), TUZG14 concentrations were 5nM, 15nM, 50nM, 150nM, pure Control without any substance (Control), cell viability of EGF and groups with EGF and varying amounts of TUZG 14; in the figure, P < 0.05;. and P < 0.01.

Detailed Description

In order to make the technical scheme of the invention better understood, the invention is further described below with reference to the drawings and examples, and reagents, materials and the like used in the invention are all generally commercially available without specific description.

Example 1: construction of pET-30a/EGFR-ECD protein expression vector

The sequence information of EGFR is obtained from Genebank, and the accession number is CCDS5514.1 and is 3363bp in total. The EGFR Extracellular Domain (Extracellar Domain) is a segment (aa 25-645) of 1863bp, a DNA sequence encoding an EGFR-ECD fragment is extracted from the whole sequence of the EGFR, and PCR primers are designed according to the sequence shown in SEQ ID No. 2:

forward primer (seq. id No. 3):

5’-GCTGATATCGGATCCATGCTGGAGGAAAAGAAAGT-3’

and a reverse primer (seq. id No. 4):

5’-ACGGAGCTCGAATTCTCAGGACGGGATCTT AGG-3', the underlined parts are the BamHI and Eco RI sites at both ends, respectively. After the PCR product of EGFR-ECD and the vector pET-30a (Novagen, # 69909-3) were digested with Bam HI and Eco RI at 37 ℃ for 1 h, they were ligated with T4DNA ligase at 16 ℃ for 12 h. And transforming the ligation product into DH5 alpha competent cells (all-type gold, CD 201), when a single colony grows on a kanamycin (50 mu g/mL) resistant LB plate, selecting the single colony, amplifying bacteria, extracting plasmids for enzyme digestion verification, sending the recombinant plasmids to Huada gene sequencing, and verifying the sequencing result that the plasmids with accurate ligation transformation are pET-30 a/EGFR-ECD.

FIG. 2 shows the results of gel electrophoresis of the constructed protein expression vector pET-30 a/EGFR-ECD; wherein, the graph A is the result of verifying the PCR amplification product of the EGFR-ECD by 1 percent agarose gel electrophoresis; FIG. B shows the results of double digestion verification of the recombinant plasmid pET-30 a/EGFR-ECD; wherein M is a DNA marker, 1 is a double enzyme cutting result of Bam HI and Eco RI of an empty pET-30a vector, 2 is a double enzyme cutting result of Bam HI and Eco RI of EGFR-ECD, and 3 is a PCR amplification product of EGFR-ECD. The experimental result shows that the EGFR-ECD is successfully amplified by PCR; and the recombinant plasmid pET-30a/EGFR-ECD is successfully constructed.

Example 2: induced expression and renaturation purification of EGFR-ECD

Inducible expression of EGFR-ECD: the successfully constructed recombinant plasmid pET-30a/EGFR-ECD is transformed into a host bacterium BL21(DE3) (full formula)Gold), recombinants were screened using kanamycin-resistant plates, and single colonies were picked and cultured in LB liquid medium containing kanamycin for 10 h. Inoculating the culture into LB liquid medium at a volume ratio of 1:100, and culturing at 37 deg.C with vigorous shaking to OD₆₀₀= 0.5-0.6, add 0.1M IPTG to final concentration 0.5 mM, induce 4 h at 25 ℃.

And (3) verifying the expression of the target protein: the induced bacterial liquid is centrifuged for 10 min at 5000 rpm, the supernatant is removed, the bacteria are resuspended in a lysis solution (50 mM Tris-HCl pH7.5, 500 mM NaCl, 10% glycerol, 1% TritonX-100, 1 mM protease inhibitor PMSF, 1 mg/mL lysozyme) according to the ratio of the volume of the bacterial liquid to the volume of the lysis solution = 20:1, and the bacterial liquid is placed on ice for ultrasonic treatment, ultrasonic treatment is carried out at 200W power for 3 s, and the interval is 5 s until the bacterial liquid is clarified. And centrifuging the ultrasonically crushed bacterial liquid at 15000 Xg for 30 min at 4 ℃ to obtain supernatant and precipitate. And (3) performing polyacrylamide gel electrophoresis on the bacteria liquid which is not centrifuged after the ultrasonication and the supernatant liquid which is centrifuged, and dyeing the bacteria liquid with Coomassie brilliant blue.

FIG. 3 shows the results of the expression and purification verification of recombinant protein EGFR-ECD in host bacteria BL21(DE 3); wherein, the picture A is the bacterial liquid which is not centrifuged after the ultrasonic disruption, wherein, M is a protein marker, 1 is a transformant for transforming empty pET-30a vector, and 2 is a recombinant vector for transforming pET-30 a/EGFR-ECD; panel B is the supernatant after ultrasonication and centrifugation, where M is a protein marker, 1 is a transformant for transforming an empty vector pET-30a, and 2 is a transformant for transforming a recombinant vector pET-30 a/EGFR-ECD; and C is a protein dialysis renaturation result, wherein 1 is a bacterial liquid which is not centrifuged after the transformant of the pET-30a/EGFR-ECD recombinant vector is subjected to ultrasonic disruption, and 2 is a protein sample band obtained after the precipitate of the transformant of the pET-30a/EGFR-ECD recombinant vector is centrifuged is subjected to denaturation and dialysis renaturation.

The experimental results show that in FIG. 2A, compared with the transformant transformed with the empty pET-30a vector, a large amount of EGFR-ECD appears in the bacterial lysate of the transformant transformed with the pET-30a/EGFR-ECD recombinant vector, which indicates that the EGFR-ECD is successfully expressed in BL21(DE 3); while FIG. 2B shows no EGFR-ECD band in the supernatant after centrifugation, demonstrating that EGFR-ECD is expressed as insoluble inclusion bodies in BL21(DE 3).

Therefore, in the invention, the soluble protein is washed away by using Buffer I, the fat-soluble protein is washed away by using Buffer T, the inclusion body is dissolved in the high-concentration urea Buffer solution to be denatured, and then dialysis is performed step by step to reduce the urea concentration and ensure the protein renaturation, thereby obtaining the target protein through purification.

Denaturation, renaturation and purification of the protein of interest EGFR-ECD: the centrifuged supernatant was discarded, and the pellet was resuspended by ultrasonic wave using buffer I (50 mM Tris-HCl pH8.0, 50 mM NaCl, 5% glycerol, 2M urea) and buffer T (50 mM Tris-HCl pH8.0, 50 mM NaCl, 5% glycerol, 1% TritonX-100) in the same volume as the lysate, and then centrifuged at 15000 Xg for 30 min at 4 ℃ after each resuspension, and the supernatant was discarded. Then passes through buffer

Ultrasonic resuspension (50 mM Tris-HCl pH8.0, 150 mM NaCl, 8M urea), standing overnight at 4 ℃, 15000 Xg, centrifuging for 30 min at 4 ℃, and discarding the precipitate to obtain the denatured protein EGFR-ECD. The supernatant fluid is buffer

The protein solution was diluted at a ratio (volume ratio) of 1:5, placed in a dialysis bag, and dialyzed sequentially with 6M, 4M, 2M, 1M, 0.5M, and 0M urea buffer to renature the protein. After dialysis, the mixture was centrifuged at 15000 Xg at 4 ℃ for 30 min to remove the precipitate. And (3) putting the supernatant into a 15 mL 30 KDa ultrafiltration tube, centrifuging at 5000 Xg and 4 ℃, and concentrating to about 1 mL to obtain the purified target protein EGFR-ECD.

Example 3: bioactive peptides specifically binding to EGFR by phage display panning

Target molecule immobilization: 100 muL of a target molecule EGFR-ECD protein solution (0.1M TBS pH7.4) with a concentration of 100 mug/mL was added to a 96-well plate, placed on a shaker for gentle shaking, and incubated overnight at 4 ℃ in a humidified container. The target molecule solution was removed and treated with TBST (50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.1% [ v/v ]]Tween-20) was washed 6 times. Finally, a blocking solution (0.1M NaHCO) is used₃ pH 8.6，5 mg/mL BSA，0.02% NaN₃) And sealing for 1 h.

Phage random peptide libraryBinding to a target molecule: the blocking solution was removed and treated with TBST (containing 0.1% [ v/v ]]Tween-20) was washed 10 times. Phage library (NEB (Beijing) Ltd., Ph.D. -7 phage display peptide library kit, cat # E8100S) or amplified phage by TBST (containing 0.1% [ v/v ]]Tween-20) the titer of the phage was 10⁹~10¹¹Adding the diluted phage to a six-hole plate to combine with the target molecule, and incubating at room temperature for 10-60 min.

Washing away unbound phage: TBST (0.1% in the first round followed by 0.5% [ v/v ] Tween-20) was washed 10 times, and the residual solution was removed by beating vigorously on sterilized filter paper after each washing.

Elution of specifically bound phage: 100 μ L of EGF (Biotechnology (Shanghai) GmbH, C610033) (100 μ g/mL) solution was added to a six-well plate (Corning Corp.) and gently shaken at room temperature for 10-60 min. Collecting the eluent to obtain the phage which is competitively eluted and specifically binds to the target molecule.

Phage amplification: the eluted phage was added to 20 mL of the ER2738 host strain (NEB (Beijing) Co., Ltd.) in the pre-log stage and shaken vigorously at 37 ℃ for 4.5 hours. The culture was transferred to a centrifuge tube and centrifuged at 12000 Xg for 10 min at 4 ℃ and the supernatant was transferred to another centrifuge tube and centrifuged repeatedly. The upper 80% of the supernatant was transferred to a fresh centrifuge tube and 1/6 volumes of PEG/NaCl (20% [ w/v ] PEG-8000, 2.5M NaCl) were added and precipitated overnight at 4 ℃. Centrifuging again, resuspending in 1 mL TBS, precipitating with PEG/NaCl for 60 min, centrifuging at 12000 Xg for 10 min, dissolving the precipitate in 200 μ L TBS at 14000 rpm, centrifuging for 1 min, and transferring the supernatant to another microcentrifuge tube to obtain the amplified eluate. And taking out 1 mu L for titer determination, and performing the next round of elutriation or preservation.

Extraction of monoclonal phage information: after 4 rounds of panning, the phage eluted in the last round were plated on LB/IPTG/Xgal plates after infecting the host bacterium ER2378 with the phage. After 13 h, the phage blue spots grew, and the blue spots were picked and subjected to monoclonal phage amplification. After 4 h, the culture is centrifuged at 14000 rpm for 30 s, the supernatant is taken, the process is repeated for 2 times, and finally 80% of the supernatant is taken as the amplified monoclonal phage. Designing a PCR primer forward primer (SEQ. ID. NO. 5) by taking the monoclonal phage as a template:

5’-TTATTCGCAATTCCTTTAG-3’

and a reverse primer (seq. id No. 6): 5'-CCCTCATAGTTAGCGTAACG-3'

Amplifying the sequence, sequencing the amplified product (Huada gene) to obtain a dominant polypeptide sequence, which is named as TUZG14, and then sending the polypeptide sequence to a company (Nanjing Kingsler Biotech Co., Ltd.) for synthesis. The sequence of the polypeptide TUZG14 is shown in SEQ ID No. 1: tung 14 (seq. id No. 1): lys Val Trp Val Val Pro Asn are provided.

Example 4: screening of EGF-sensitive cell lines

The invention selects a plurality of EGFR high-expression gastric cancer cell lines as experimental objects, namely AGS, BGC-803 and MGC-803 (American type culture Collection). The above 3 kinds of cells were cultured in RMPI-1640 medium containing 10% fetal bovine serum and penicillin-streptomycin, and the growth state of the cells was observed every day at regular intervals and passaged in time.

Plate preparation: from CO₂The cultured cells were removed from the incubator, the original medium was discarded, 1 mL of PBS was added for washing, and the supernatant was aspirated with a Pasteur tube. 0.5 mL of trypsin was added for digestion, transferred to a microcentrifuge tube, centrifuged at 200 Xg for 5 min, and the supernatant was aspirated by a Pasteur tube. Sucking 1 mL of RMPI-1640 culture medium containing 10% fetal calf serum and penicillin-streptomycin, blowing and mixing uniformly, and taking 10 mL of cell suspension by using a pipette gun to count on a cell counting plate. 4000 cells are planted in each hole, 6 groups of holes are formed in total, 5 holes are formed in each group, the required cell suspension is taken out and diluted to the corresponding volume, and the cell suspension is blown, beaten, mixed evenly and added into a 96-hole plate. Put in CO₂And (5) culturing the mixture in a constant-temperature incubator overnight for 12-16 hours until the mixture adheres to the wall.

Adding medicine: different concentrations of EGF were added. Dissolve 100 mg EGF in 1 mL ddH₂And O, preparing a 100 mug/mL stock solution. 100 μ g/mL EGF was diluted with culture medium (1% fetal bovine serum and penicillin-streptomycin) to the following 6 concentrations by gradient dilution: 0 [ mu ] g/mL (control), 0.01 [ mu ] g/mL, 0.033 [ mu ] g/mL, 0.1 [ mu ] g/mL, 0.33 [ mu ] g/mL, 1 [ mu ] g & gt & lt/mu ] g & gtmL, added to the corresponding wells, 5 replicates per concentration. Adding CO after the addition of the chemicals is finished₂The culture was carried out in a constant temperature incubator (37 ℃) for 48 hours.

MTT colorimetric method: taking out the cells after 48h of drug treatment, adding 10 mu L MTT (5 mg/mL) into each well, and adding CO₂Reacting in a constant temperature incubator (37 ℃) for 1-1.5 h, absorbing the culture medium after the cells are stained into blue-purple, adding 0.1 mL of DMSO, and slightly shaking to dissolve crystals. And (3) measuring the absorption value of each hole at the wavelength of 550 nm by using a microplate reader, recording the result, and calculating the cell survival rate: cell viability = (experimental OD value/control OD value) × 100%.

FIG. 4 shows the results of screening EGF-sensitive gastric cancer cell lines AGS, BGC-803 and MGC-803; wherein Panel A is the effect of EGF on BGC-803 cell survival; b is the effect of EGF on MGC-803 cell survival; c is the effect of EGF on AGS cell survival. EGF set the following concentrations: the method comprises the steps of 0 mug/mL, 0.01 mug/mL, 0.033 mug/mL, 0.1 mug/mL, 0.33 mug/mL and 1 mug/mL, and the influence of EGF on the survival rate of three cell lines is inspected after 48 hours. As can be seen from fig. 4A, B, EGF had no significant effect on the growth of BGC-803 cells and MGC-803 cells, and as can be seen from fig. 4C, EGF had a significant effect of promoting the proliferation of AGS cells.

Example 5: activity verification of polypeptides

Plate preparation: from CO₂The incubator removes the cultured AGS cells, discards the original medium, adds 1 mL PBS for washing, and aspirates the supernatant with a pasteur tube. 0.5 mL of trypsin was added for digestion, transferred to a microcentrifuge tube, centrifuged at 200 Xg for 5 min, and the supernatant was aspirated by a Pasteur tube. Sucking 1 mL of RMPI-1640 culture medium containing 10% fetal calf serum and penicillin-streptomycin, blowing, mixing uniformly, and taking 10 mu L of cell suspension by using a pipette gun to count on a cell counting plate. According to 4000 cells planted in each hole, 6 groups of holes are totally arranged, each group is provided with 5 multiple holes, the required cell suspension is taken out to be diluted to the corresponding volume, and the cell suspension is evenly blown and planted in a 96-well plate. Put in CO₂And (5) culturing the mixture in a constant-temperature incubator overnight for 12-16 hours until the mixture adheres to the wall.

Adding medicine: EGF was added with varying concentrations of the polypeptide. EGF solution at a concentration of 0.033 μ g/mL (5 nM) was prepared in a medium containing 1% fetal bovine serum, andthey were divided into 5 groups, to which different amounts of polypeptide solutions were added to prepare the following concentrations: 0nM, 5nM, 15nM, 50nM, 150 nM. The cells were treated in 6 groups, pure Control (Control) without any substance, EGF +5nM TUZG14, EGF +15nM TUZG14, EGF +50nM TUZG14, EGF +150nM TUZG14, respectively. Finally adding CO₂The culture was carried out in a constant temperature incubator (37 ℃) for 48 hours.

MTT colorimetric method: taking out the cells after 48h of drug treatment, adding 10 mu L MTT (5 mg/mL) into each well, and adding CO₂Reacting in a constant temperature incubator (37 ℃) for 1-1.5 h, absorbing the culture medium after the cells are stained into blue-purple, adding 0.1 mL of DMSO, and slightly shaking to dissolve crystals. And (3) measuring the absorption value of each hole at the wavelength of 550 nm by using a microplate reader, recording the result, and calculating the cell survival rate: cell viability = (experimental OD value/control OD value) × 100%.

FIG. 5 shows the result of verifying the activity of the polypeptide TUZG 20; cell viability was determined for the Control group without any substance (Control), the EGF group and the groups with EGF and various amounts of TUZG14 after 48h of culture by adding TUZG14 at a ratio of 1:1 (5 nM), 1:3 (15 nM), 1:10 (50 nM) and 1:30 (150 nM) at an effect concentration of EGF of 0.033 μ g/mL (5 nM), respectively. P < 0.01;. P < 0.05. As can be seen in fig. 5, the cell growth of the control group was slower compared to the EGF group, which indicates that EGF promoted the growth of AGS tumor cells, and after the addition of TUZG14, the growth of AGS cells gradually decreased with the increase of the concentration of TUZG14, and the growth of AGS tumor cells accelerated at the concentrations of 50nM and 150nM by the TUZG14 was significantly inhibited, indicating that the TUZG14 significantly inhibited the tumor cell proliferation promoting effect of EGF.

Sequence listing

<110> university of Jiangsu

<120> polypeptide inhibiting EGF tumor cell proliferation by binding specifically to EGFR

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Lys Val Trp Val Val Pro Asn

1 5

<210> 2

<211> 672

<212> PRT

<213> human (Homo sapiens)

<400> 2

Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser

1 5 10 15

Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp

20 25 30

Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile

35 40 45

Gly Ser Met Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn Lys

50 55 60

Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg

65 70 75 80

Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr

85 90 95

Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val

100 105 110

Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu

115 120 125

Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr

130 135 140

Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu Lys

145 150 155 160

Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val Arg

165 170 175

Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp Arg

180 185 190

Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe Gln

195 200 205

Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn Gly

210 215 220

Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile

225 230 235 240

Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser

245 250 255

Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu

260 265 270

Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys

275 280 285

Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln Met

290 295 300

Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Lys

305 310 315 320

Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg

325 330 335

Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg Lys

340 345 350

Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly

355 360 365

Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys

370 375 380

His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro

385 390 395 400

Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro

405 410 415

Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu

420 425 430

Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu

435 440 445

Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser

450 455 460

Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu

465 470 475 480

Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu

485 490 495

Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly

500 505 510

Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala

515 520 525

Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly

530 535 540

Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg

545 550 555 560

Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe

565 570 575

Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln

580 585 590

Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln

595 600 605

Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala

610 615 620

Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala

625 630 635 640

Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr

645 650 655

Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser

660 665 670

<210> 3

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gctgatatcg gatccatgct ggaggaaaag aaagt 35

<210> 4

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

acggagctcg aattctcagg acgggatctt agg 33

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ttattcgcaa ttcctttag 19

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccctcatagt tagcgtaacg 20

Claims (4)

1. The polypeptide for inhibiting EGF (epidermal growth factor) to promote gastric cancer AGS (epithelial cell growth factor) cell proliferation is characterized in that the amino acid sequence of the polypeptide is as follows: lys Val Trp Val Val Pro Asn are provided.

2. The polypeptide of claim 1, wherein said polypeptide inhibits EGF proliferation of gastric AGS cells by binding specifically to EGFR.

3. The use of the polypeptide of claim 1 in the preparation of a targeted medicament for the treatment of gastric cancer.

4. A targeted drug for treating or preventing gastric cancer, which comprises the polypeptide of claim 1.

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