A polypeptide targeting claudin 6 protein and preparation method and application thereof

Abstract

The application discloses a polypeptide targeting CLDN6 protein and a preparation method and application thereof, and belongs to the technical field of medicinal chemistry.The polypeptide targeting CLDN6 protein has an amino acid sequence structure general formula of X1IX2PYX3; wherein X1-X3 represents a sequence with a length of 1-7 amino acid residues, and the amino acid is any one of 21 natural amino acids and non-natural amino acids; the polypeptide can specifically target CLDN6 positive cells, has high selectivity, can be used as a delivery carrier of a drug, or is coupled with a known component to form a drug combination, so that a better treatment effect is obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a polypeptide targeting CLDN6 protein and a preparation method and application thereof. BACKGROUND

[0002] Claudin-6 (CLDN6) is a member of the claudin family of membrane proteins, which consists of 27 different proteins characterized by four transmembrane domains and two extracellular loops. Various claudin (CLDN) family members are located at tight junctions between epithelial cells, which are believed to play a key role in barrier function. Recently, CLDN6 has also been associated with some key intracellular signal transduction pathways, including the YAP1-snail1 axis, the ASK1-p38 / JNK MAPK secretory signal transduction pathway, and CLDN6 signaling has been shown to activate ERa transcriptional activity in a ligand-independent manner, thereby promoting tumor progression in endometrial cancer. CLDN6 is also highly expressed in undifferentiated mouse and human stem cells and is believed to contribute to the tumorigenic potential of human pluripotent stem cell-containing cultures. Although CLDN6 is widely expressed in cells and tissues during early stages of embryonic and fetal development, in adults, CLDN6 expression is mainly limited to malignant tissues, which is associated with the occurrence, progression and metastasis of certain cancers. It has been reported that it is abnormally expressed in ovarian cancer, gastric cancer, lung cancer, endometrial cancer and cervical cancer.

[0003] In addition, in patients with ovarian cancer and endometrial cancer, high levels of CLDN6 expression in malignant tissues are independent prognostic markers for poor progression-free survival and overall survival. Compound data showing higher expression in adult cancers than in normal tissues make CLDN6 an attractive target for developing polypeptide-based therapies. SUMMARY

[0004] In view of the deficiencies in the prior art, the purpose of the present application is to provide a polypeptide targeting CLDN6 protein and a preparation method and application thereof, which solves the problems in the prior art.

[0005] The purpose of the present application can be achieved by the following technical solutions:

[0006] A polypeptide targeting CLDN6 protein, the amino acid sequence structure general formula of which is: X1IX2PYX3; wherein X1-X3 represents a sequence of 1-7 amino acid residues in length, and the amino acids therein are any one of 21 natural amino acids and unnatural amino acids.

[0007] Further, the amino acid sequences of the polypeptide are shown in SEQ. ID NO 1-SEQ. ID NO 10, respectively.

[0008] Use of the polypeptide targeting CLDN6 protein in the preparation of a medicament or a drug carrier for treating a tumor with high expression of CLDN6 protein.

[0009] Further, the tumor with high expression of CLDN6 protein includes an ovarian tumor, an endometrial tumor, and a prostate tumor.

[0010] A medicament for treating a tumor with high expression of CLDN6 protein, comprising the polypeptide targeting CLDN6 protein.

[0011] A drug carrier for treating a tumor with high expression of CLDN6 protein, comprising the polypeptide targeting CLDN6 protein.

[0012] Further, the drug carrier is a polypeptide hydrogel prepared by coupling the polypeptide with any amino acid with electric charge or different hydrophilicity and hydrophobicity.

[0013] An immunoconjugate, comprising the polypeptide.

[0014] Further, the immunoconjugate further comprises a chemotherapeutic agent, a radioactive atom, a cell growth inhibitor, a cytotoxic agent, an immune checkpoint inhibitor, an antibody, or an antibody fragment.

[0015] A method for preparing the polypeptide targeting CLDN6 protein, comprising the following steps:

[0016] S1, weigh Fmoc-Rink amide-MBHA Resin, and after swelling in DCM, filter to remove DCM, then swell in NMP for 30 minutes, and finally wash with NMP, DCM, and NMP, respectively;

[0017] S2, place the swelled resin into a reactor, and add a piperidine / NMP solution containing HOBT to react, after the reaction, filter the solution, and wash with NMP;

[0018] S3, dissolve Fmoc-Arg(Pbf)-OH, HBTU, HOBT, and DIPEA in NMP, and then add the solution to the resin after removal of piperidine to react, after the reaction, filter the reaction solution, and wash with DCM and NMP;

[0019] S4, qualitatively detect the coupling efficiency of the resin by the ninhydrin method or the bromophenol blue method, and if the color reaction is negative, proceed to the next coupling cycle;

[0020] S5, according to the sequence of the polypeptide, repeat S2 and S3 to sequentially connect the corresponding amino acids, to obtain a resin with the polypeptide sequence connected thereto;

[0021] S6, add a cleavage agent to cleave the resin, to obtain the polypeptide.

[0022] The beneficial effects of the present application are as follows:

[0023] 1、The polypeptide of the present application can specifically target CLDN6 positive cells with high selectivity, and the polypeptide of the present application can be prepared by chemical synthesis method, has high purity, small molecular weight, strong specificity, no immunogenicity, and is safe and reliable.

[0024] 2、The polypeptide of the present application can be used as a drug delivery carrier, or coupled with known components to form a drug combination, so as to obtain better therapeutic effect. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below, and obviously, other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0026] Figure 1 is the mass spectrum of the amino acid sequence prepared in Example 1 in the present application;

[0027] Figure 2 is the mass spectrum of the amino acid sequence prepared in Example 2 in the present application;

[0028] Figure 3 is the mass spectrum of the amino acid sequence prepared in Example 3 in the present application;

[0029] Figure 4 is the mass spectrum of the amino acid sequence prepared in Example 4 in the present application;

[0030] Figure 5 is the mass spectrum of the amino acid sequence prepared in Example 5 in the present application;

[0031] Figure 6 is the mass spectrum of the amino acid sequence prepared in Example 6 in the present application;

[0032] Figure 7 is the mass spectrum of the amino acid sequence prepared in Example 7 in the present application;

[0033] Figure 8 is the mass spectrum of the amino acid sequence prepared in Example 8 in the present application;

[0034] Figure 9 is the mass spectrum of the amino acid sequence prepared in Example 9 in the present application;

[0035] Figure 10 is the mass spectrum of the amino acid sequence prepared in Example 10 in the present application;

[0036] Figure 11is the result chart of CCK-8 determination of the amino acid sequence prepared in the examples 1-10 of the present application.

[0037] Figure 12 is the immunofluorescence staining chart of internalization after the amino acid sequence prepared in the examples 1-10 of the present application is connected with FITC fluorescent group and then incubated with cells. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0039] A polypeptide targeting CLDN6 protein, the amino acid sequence structure general formula of which is X1IX2PYX3; wherein X1-X3 represents a sequence of 1-7 amino acid residues in length, and the amino acid therein is any one of 21 natural amino acids and unnatural amino acids.

[0040] The amino acid sequences of the polypeptides are respectively shown in SEQ.ID NO1-SEQ.ID NO10; specifically, IGRIWPYFGG, IGKIWPYYGG, IGRIKPYRGG, IGRILPYYGG, RIWPYR, GRIWPYFG, IGRIRPYRGG, KIWPYY, GLINPYNG, and GRIWPYRG.

[0041] The synthesis process of the above polypeptides (SEQ.ID NO1-SEQ.ID NO10) will be introduced below through examples 1-10;

[0042] Example 1

[0043] Solid-phase synthesis of IGRIWPYFGG (SEQ.ID NO.1);

[0044] (1) Resin swelling

[0045] Fmoc-Rink amide-MBHA Resin 50 mg (substitution amount 0.4 mmol / g) was weighed, swelled in 7 mL of dichloromethane (DCM) for 30 min, filtered to remove DCM, then swelled in 10 mL of N-methyl pyrrolidone (NMP) for 30 min, and finally washed with NMP and DCM 7 mL respectively.

[0046] (2) Removal of Fmoc protecting group

[0047] The swelled resin was put into a reactor, 7 mL of 25% piperidine / NMP (V / V) solution containing 0.1 M 1-hydroxybenzotriazole (HOBT) was added, and the reaction was carried out for 25 min; after the reaction was completed, the solution was filtered off, and the resin was washed with NMP until clean, to obtain the resin from which the Fmoc protecting group of the initial connection was removed.

[0048] (3) Synthesis of Fmoc-Gly-Rink amide-MBHA Resin

[0049] Fmoc-Gly-OH (0.04 mmol), O-benzotriazolyl-tetramethyluronium hexafluorophosphate (HBTU) (0.04 mmol), HOBT (0.04 mmol) and N,N-diisopropyl ethylamine (DIPEA) (0.08 mmol) were dissolved in 10 mL of NMP, and the solution was added to the resin obtained in step (2), and the reaction was carried out for 45 min; after the reaction was completed, the reaction solution was filtered off, and the resin was washed with 7 mL of DCM and NMP each for 3 times.

[0050] (4) Detection of coupling efficiency

[0051] The coupling efficiency of the resin was qualitatively detected by the ninhydrin method or the bromophenol blue method, and if the color reaction was negative, the next coupling cycle could be entered.

[0052] Ninhydrin method: a small amount of resin particles was washed with ethanol, and was put into a transparent vial, 5% ninhydrin ethanol, 80% phenol ethanol solution, 2 drops of KCN pyridine solution (2 mL of 0.001 M KCN diluted in 98 mL of pyridine) were added, and heating was carried out at 100°C for 5 min; if the resin showed blue color, it was positive.

[0053] Bromophenol blue method: a small amount of resin particles was washed with dimethylformamide, and was put into a transparent vial, 3 drops of 1% bromophenol blue dimethylformamide solution were added, and shaking was carried out at room temperature for 3 min; if the resin showed blue color, it was positive.

[0054] (5) Extension of the peptide chain

[0055] According to the sequence of the polypeptide, the above-mentioned deprotection and coupling steps were repeated to sequentially connect the corresponding amino acids, to obtain the resin connected with the IGRIWPYFGG (SEQ. ID NO. 1) polypeptide sequence.

[0056] (6) Cleavage of the polypeptide on the resin

[0057] The resin with the polypeptide sequence of IGRIWPYFGG (SEQ. ID NO. 1) obtained above was put into a reaction bottle, 10 mL of cleavage reagent Reagent K (TFA / benzyl methyl sulfide / water / phenol / EDT, 82.5:5:5:5:2.5, V / V) was added, and the mixture was shaken at 0°C for 30 min, and then reacted at room temperature for 3 h. After the reaction, the mixture was filtered, washed with a small amount of TFA and DCM for three times, and the filtrates were combined. The filtrates were added into a large amount of ice ethyl ether to precipitate a white flocculent precipitate, and the crude product of the target polypeptide was obtained by freezing and centrifugation.

[0058] The crude product of the polypeptide was dissolved in 2 mL of water, and directly subjected to preparative liquid chromatography purification. The chromatography conditions were as follows: C18 reverse phase column (320 mm x 28 mm, 5 μm); mobile phase A: 0.1% TFA / water (V / V), mobile phase B: 0.1% TFA / acetonitrile (V / V); mobile phase gradient: mobile phase B 20%-80% for 20 min; flow rate: 7 mL / min; detection wavelength: 214 nm; and the collected solution was lyophilized to obtain 25 mg of the pure product. The theoretical relative molecular mass was 1165.366. ESI-MS m / z: found [M+H]+=1166.

[0059] The mass spectrum of IGRIWPYFGG (SEQ. ID NO. 1) is shown in Figure 1 Figure 1 The (a) and (b) in the figure are the HPLC and mass spectrum of the peptide segment (SEQ. ID NO. 1), respectively. It can be seen from the figure that the purity of the polypeptide was 96.45%, and the molecular weight was the molecular weight of the target polypeptide.

[0060] Example 2

[0061] Solid phase synthesis of IGKIWPYYGG (SEQ. ID NO. 2)

[0062] The difference between this example and the synthesis step of Example 1 is only that the sequence of the synthesized polypeptide is different.

[0063] The solid phase synthesis of IGKIWPYYGG (SEQ. ID NO. 2) has a theoretical relative molecular mass of 1153.356. ESI-MS m / z: found [M+H]+=1154.

[0064] The mass spectrum of IGKIWPYYGG (SEQ. ID NO. 2) is shown in Figure 2 Figure 2 The (a) and (b) in the figure are the HPLC and mass spectrum of the peptide segment (SEQ. ID NO. 2), respectively. It can be seen from the figure that the purity of the polypeptide was 96.43%, and the molecular weight was the molecular weight of the target polypeptide.

[0065] Example 3​​

[0066] Solid phase synthesis of IGRIKPYRGG (SEQ. ID NO. 3)

[0067] The difference between this example and the synthesis step of Example 1 is that the sequence of the synthesized polypeptide is different.

[0068] The solid phase synthesis of IGRIKPYRGG (SEQ. ID NO. 3) has a theoretical relative molecular mass of 1116.336. ESI-MS m / z: found [M+H]+= 1117.

[0069] The mass spectrum of IGRIKPYRGG (SEQ. ID NO. 3) is shown in Figure 3 , Figure 3 (a) and (b) in the figure are the HPLC and mass spectrum of the peptide segment (SEQ. ID NO. 3), respectively. It can be seen from the figure that the purity of the polypeptide is 96.57%, and the molecular weight is the molecular weight of the target polypeptide.

[0070] Example 4

[0071] Solid phase synthesis of IGRILPYYGG (SEQ. ID NO. 4)

[0072] The difference between this example and the synthesis step of Example 1 is that the sequence of the synthesized polypeptide is different.

[0073] The solid phase synthesis of IGRILPYYGG (SEQ. ID NO. 4) has a theoretical relative molecular mass of 1108.306. ESI-MS m / z: found [M+H]+= 1109.

[0074] The mass spectrum of IGRILPYYGG (SEQ. ID NO. 4) is shown in Figure 4 , Figure 4 (a) and (b) in the figure are the HPLC and mass spectrum of the peptide segment (SEQ. ID NO. 4), respectively. It can be seen from the figure that the purity of the polypeptide is 98.21%, and the molecular weight is the molecular weight of the target polypeptide.

[0075] Example 5

[0076] Solid phase synthesis of RIWPYR (SEQ. ID NO. 5)

[0077] The difference between this example and the synthesis step of Example 1 is that the sequence of the synthesized polypeptide is different.

[0078] The solid phase synthesis of RIWPYR (SEQ. ID NO. 5) has a theoretical relative molecular mass of 890.05. ESI-MS m / z: found [M+H]+= 891.

[0079] The mass spectrum of RIWPYR (SEQ.ID NO.5) is as follows: Figure 5 As shown, Figure 5 (a) and (b) in the figure are the HPLC and mass spectra of the peptide (SEQ.ID NO.5), respectively. As can be seen from the figure, the purity of the peptide is 97.37%, and the molecular weight is the molecular weight of the target peptide.

[0080] Example 6

[0081] Solid-phase synthesis of GRIWPYFG (SEQ.ID NO.6)

[0082] The difference between this embodiment and Example 1 lies in the synthesis steps: the sequence of the synthesized polypeptide is different.

[0083] The theoretical relative molecular mass of GRIWPYFG (SEQ.ID NO.6) obtained through solid-phase synthesis is 995.148. ESI-MS m / z: found [M+H]+ = 996.

[0084] The mass spectrum of GRIWPYFG (SEQ.ID NO.6) is as follows: Figure 6 As shown, Figure 6 (a) and (b) in the figure are the HPLC and mass spectra of the peptide (SEQ.ID NO.6), respectively. It can be seen from the figure that the purity of the peptide is 96.01%, and the molecular weight is the molecular weight of the target peptide.

[0085] Example 7

[0086] Solid-phase synthesis of IGRIRPYRGG (SEQ.ID NO.7)

[0087] The difference between this embodiment and Example 1 lies in the synthesis steps: the sequence of the synthesized polypeptide is different.

[0088] The theoretical relative molecular mass of IGRIRPYRGG (SEQ.ID NO.7) for solid-phase synthesis is 1144.346. ESI-MS m / z:found [M+H]+=1145.

[0089] The mass spectrum of IGRIRPYRGG (SEQ.ID NO.7) is as follows: Figure 7 As shown, Figure 7 (a) and (b) in the figure are the HPLC and mass spectra of the peptide (SEQ.ID NO.7), respectively. It can be seen from the figure that the purity of the peptide is 96.01%, and the molecular weight is the molecular weight of the target peptide.

[0090] Example 8

[0091] Solid-phase synthesis of KIWPYY (SEQ.ID NO.8)

[0092] The difference between this embodiment and the synthesis step of Example 1 is that the sequence of the synthesized polypeptide is different.

[0093] The solid-phase synthesis of KIWPYY (SEQ. ID NO. 8) has a theoretical relative molecular mass of 869.03. ESI-MS m / z: found [M+H]+= 870.

[0094] The mass spectrum of KIWPYY (SEQ. ID NO. 8) is shown in Figure 8 , Figure 8 (a) and (b) in the figure are the HPLC and mass spectrum of the peptide segment (SEQ. ID NO. 8), respectively. It can be seen from the figure that the purity of the polypeptide is 96.49%, and the molecular weight is the molecular weight of the target polypeptide.

[0095] Example 9

[0096] Solid-phase synthesis of GLINPYNG (SEQ. ID NO. 5)

[0097] The difference between this embodiment and the synthesis step of Example 1 is that the sequence of the synthesized polypeptide is different.

[0098] The solid-phase synthesis of GLINPYNG (SEQ. ID NO. 9) has a theoretical relative molecular mass of 846.938. ESI-MS m / z: found [M+H]+= 848.

[0099] The mass spectrum of GLINPYNG (SEQ. ID NO. 9) is shown in Figure 9 , Figure 9 (a) and (b) in the figure are the HPLC and mass spectrum of the peptide segment (SEQ. ID NO. 9), respectively. It can be seen from the figure that the purity of the polypeptide is 97.34%, and the molecular weight is the molecular weight of the target polypeptide.

[0100] Example 10

[0101] Solid-phase synthesis of GRIWPYRG (SEQ. ID NO. 10)

[0102] The difference between this embodiment and the synthesis step of Example 1 is that the sequence of the synthesized polypeptide is different.

[0103] The solid-phase synthesis of GRIWPYRG (SEQ. ID NO. 10) has a theoretical relative molecular mass of 1004.158. ESI-MS m / z: found [M+H]+= 1005.

[0104] The mass spectrum of GRIWPYRG (SEQ. ID NO. 10) is shown in Figure 10 , Figure 10The HPLC and mass spectrum of (a) and (b) in Figure 2 are peptide segments (SEQ. ID NO. 10), respectively. It can be seen from the figure that the purity of the polypeptide is 97.09%, and the molecular weight is the molecular weight of the target polypeptide.

[0105] Example 11

[0106] Surface plasmon resonance (SPR): detecting the affinity constant of the amino acid sequences prepared in Examples 1-10 (SEQ. ID NO. 1-10) to CLDN6 protein, respectively.

[0107] The human recombinant protein CLDN6 was coupled to the CM 5 chip, and the analyte was diluted to a concentration gradient (50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.0390625, 0.1953125, 0.09965625, 0nM). The appropriate regeneration conditions (such as Glycine 2.5) were used, the flow rate was set to 30μL / min, the injection time of each cycle was set to 90s, and the dissociation time was also 90s. According to the Biacore instrument procedure, multi-cycle kinetic test was carried out, and finally the binding and dissociation curves were analyzed by Biacore T200 Evaluation Software to calculate the affinity related parameters, and the results are shown in Table 1 below;

[0108] Table 1: Affinity constant (KD) summary

[0109]

[0110]

[0111] As can be seen from Table 1, the best affinity reaches <10 -8 , reaching the level of 10nM.

[0112] Example 12

[0113] CCK-8 cell killing experiment: detecting the toxicity of the amino acid sequences prepared in Examples 1-10 (SEQ. ID NO. 1-10) to the constructed CLDN6 high expression ovarian cancer stable cell line at different concentrations.

[0114] Select the well-grown logarithmic growth phase cells to make cell suspension, and count, 5*103 per hole in 96-hole plate inoculation cell suspension (100 μL / hole): set 5 duplicate holes in each group, after the cell overnight iron wall, add different concentrations (1 uM, 10 uM, 100 uM) of SEQ.ID NO.1-22 polypeptide, then add CCK-8 reagent after 24 hours of culture, use the enzyme-labeled instrument to detect, detection wavelength 450-490nm, reference wavelength 600-650nm.Use Graphpad to calculate the toxicity of polypeptide, the results are shown in Figure 11 , Figure 11 The test results of SEQ.ID NO.1-10 are shown in

[0115] Example 13

[0116] Specifically, the ovarian cancer cell line OV90 is lentivirus transfected to overexpress CLDN6 protein, the above-mentioned 10 peptide segments are connected with FITC, the cells are incubated with anti-CLDN6 protein antibody, FITC-labeled polypeptide and DAPI respectively, and the drug internalization and co-localization with target protein are detected by fluorescence microscope, and the results are shown in Figure 12 (a) and (b) of FIG. 1-10, which reflect the immunofluorescence staining images of different time points after the polypeptide sequences in examples 1-10 are connected with FITC fluorescent group and co-incubated with tumor cells, it can be seen from the figure that each peptide segment is well internalized and well co-localized with the target protein.

[0117] Specifically, the above-mentioned polypeptide is applied in drugs, and the original structure of the polypeptide is not modified or modified by using a cleavable or non-cleavable linker (modification includes but is not limited to the following types: cyclic peptide, peptidomimetic, small molecule, using D-type amino acid, changing single or multiple amino acids in the polypeptide, modifying both ends of the polypeptide, modifying the polypeptide by biological macromolecules, physically wrapping the polypeptide, and using a combination of multiple modifications, etc.), and the modification after application includes but is not limited to the following types: polypeptide conjugated drugs, radionuclide conjugated drugs, small molecule conjugated drugs, aptamer drug conjugates, polypeptide oligonucleotide conjugates, immunostimulatory polypeptide conjugated drugs, virus-like drug conjugates, polypeptide-siRNA conjugates, bicyclic peptide radioconjugated drugs, antibody biopolymer conjugates, degrading agent-antibody conjugates, polypeptide-siRNA drugs.

[0118] In summary, the polypeptide of the present application can effectively target CLDN6 positive cells, and has no toxicity itself, and can be used as an excellent targeting peptide, thus having the potential as a drug component or drug carrier for the treatment of ovarian cancer or other tumors with high expression of CLDN6 (such as endometrial tumors, prostate tumors).

[0119] In the description of the specification, the description of the terms "one embodiment", "an example", "a specific example" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate way in one or more embodiments or examples.

[0120] The basic principles, main features and advantages of the present application are shown and described above. Those skilled in the art should understand that the present application is not limited by the above embodiments, and the above embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application.

Claims

1. A polypeptide targeting CLDN6 protein, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. The use of the polypeptide targeting CLDN6 protein as described in claim 1 in the preparation of a drug carrier for treating ovarian tumors.

3. A pharmaceutical carrier, characterized in that, Includes a polypeptide targeting the CLDN6 protein as described in claim 1.

4. The drug carrier of claim 3, wherein The drug carrier is a polypeptide hydrogel formed by coupling a polypeptide with any amino acid that is charged or has different hydrophilicity or hydrophobicity.

5. The method of claim 1, wherein the method of producing a polypeptide targeting CLDN6 protein is characterized by, Includes the following steps: S1, weigh Fmoc-Rink amide-MBHA Resin, swell it with DCM, filter off the DCM, swell it with NMP for 30 min, and finally wash it with NMP, DCM and NMP respectively. S2, the swollen resin is placed in the reactor, and a piperidine / NMP solution containing HOBT is added to react. After the reaction is completed, the solution is filtered off and washed with NMP. S3, Fmoc-Arg(Pbf)-OH, HBTU, HOBT and DIPEA are dissolved in NMP, and this solution is added to the depiperidine-treated resin for reaction. After the reaction is completed, the reaction solution is filtered off and washed with DCM and NMP. S4. The coupling efficiency of the resin is qualitatively tested using the ninhydrin method or the bromophenol blue method. If the color reaction is negative, the next coupling cycle can begin. S5, following the sequence of the polypeptide, repeat S2 and S3 to sequentially attach the corresponding amino acids to obtain a resin with the polypeptide sequence attached. S6, a cutting agent is added to perform resin cutting to obtain the polypeptide.

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