CN115260287A - Polypeptide specifically targeting human liver cancer cells and application thereof - Google Patents

Abstract

The invention belongs to the field of biological pharmacy, and particularly relates to a polypeptide specifically targeting human liver cancer cells and application thereof. Aiming at the problems that the existing polypeptide targeting liver cancer has insufficient targeting property in vivo tumor cells and the polypeptide with better targeting property in vivo needs to be developed, the invention provides a liver cancer targeting peptide F11 which is obtained by screening through a phage display technology, and the amino acid sequence is shown as SEQ ID NO. 1. The polypeptide can realize the target affinity effect on HepG2 human liver cancer cells, and in a HepG2 human liver cancer tumor-bearing mouse body, the polypeptide can realize the specific affinity on liver cancer tumor tissues to achieve the target delivery effect. The targeting polypeptide has strong specificity and high targeting property, and has good application potential in the fields of early tumor diagnosis and treatment and targeted therapy.

Description

Polypeptide specifically targeting human liver cancer cells and use thereof

technical field

The invention belongs to the field of biopharmaceuticals, and in particular relates to a polypeptide specifically targeting human liver cancer cells and its application.

Background technique

Tumor treatment technology has always been a hot and difficult point in medical research. Thanks to the development of chemotherapy drugs and the development of immunotherapy, the success rate of cancer treatment continues to increase. However, the death rate of some cancers is still at a high level. Among them, liver cancer, as one of the common malignant tumors in China, ranks second in the mortality rate of malignant tumors in my country due to the lack of obvious symptoms in the early stage and the high degree of malignancy in the middle and late stages. To this end, we need to find new targeted therapy strategies for liver cancer to improve the efficiency of liver cancer treatment and reduce drug side effects. At the same time, in view of the characteristics of early diagnosis of liver cancer patients, the invention of fluorescent dye-labeled targeting liver cancer polypeptide imaging technology is also a new way to find smaller lesions and achieve the purpose of early detection and early treatment. In summary, exploring and screening high-affinity peptides targeting liver cancer cells is an effective way to solve the difficult problem of liver cancer treatment.

At present, the methods for screening polypeptides targeting liver cancer cells mainly use phage display technology for screening. Phage display technology is a new technology that can express foreign peptides or proteins in fusion with phage surface proteins. By introducing various foreign genes into phage, a phage display library expressing various proteins on the surface can be constructed. For the screening of tumor-targeted antigens, tumor-targeting peptides with strong specificity and high affinity can be screened by specifically binding and panning the phage display peptide library with tumor cells. At present, targeting polypeptides with a certain affinity for liver cancer cells have been screened in the prior art. For example, patent CN101918433A discloses a peptide that specifically binds to HCC cells, which can specifically bind to HCC cells. Patent CN201110451767.2 discloses a polypeptide that specifically binds to the liver cancer HepG2 cell line. Although it can target the liver cancer HepG2 cell line, it does not disclose the targeting performance of the targeting polypeptide in vivo.

In the actual screening process of targeting peptides, some of them synthesize recipient cells in vitro or use tumor model mice to screen liver cancer targeting peptides, which cannot effectively simulate the tumor growth environment in patients with liver cancer, and the difference from clinical application will be relatively large. . Therefore, it is still necessary to develop some liver cancer cell-targeting polypeptides with better targeting to liver cancer cells in the human body.

Contents of the invention

The technical problem to be solved by the present invention is: the existing polypeptide targeting liver cancer has insufficient targeting ability to tumor cells in vivo, and it is necessary to develop a polypeptide with better targeting ability in vivo.

The technical solution of the present invention to solve the above-mentioned technical problems is to provide a polypeptide specifically targeting human liver cancer cells. The amino acid sequence of the polypeptide specifically targeting human liver cancer cells described in the present invention is shown in SEQ ID NO:1.

SEQ ID NO: 1 Amino acid sequence of a polypeptide specifically targeting human liver cancer cells

FYLEHPSGGLAV.

Wherein, the liver cancer cells targeted by the polypeptide specifically targeting human liver cancer cells are HepG2 cell lines.

The present invention also provides a coding nucleotide.

Wherein, the encoding nucleotide can encode the above-mentioned polypeptide specifically targeting human liver cancer cells.

Further, the coding nucleotide sequence is shown in SEQ ID NO:2.

SEQ ID NO:2 The coding nucleotide sequence of the polypeptide specifically targeting human liver cancer cells

TTTTATCTGGAACATCCGAGCGGCGGCCTGGCGGTG.

The invention also provides an expression vector.

Further, the expression vector contains the nucleotide shown in SEQ ID NO:2.

Wherein, the expression vector is a prokaryotic vector or a eukaryotic vector.

The invention also provides a host cell.

Further, the host cell contains the above-mentioned polypeptide, coding nucleotide or expression vector specifically targeting human liver cancer cells.

The present invention also provides a use of the above-mentioned polypeptide specifically targeting human liver cancer cells in targeting liver cancer cells.

Further, the liver cancer cells are HepG2 cell lines.

The present invention also provides a use of the above-mentioned polypeptide specifically targeting human liver cancer cells in the preparation of anti-liver cancer drugs or the preparation of imaging agents for diagnosis of liver cancer.

Further, in the above use, the polypeptide is prepared as a solution with a concentration of 1 mg/mL.

Further, in the above use, a buffer is also added to the polypeptide solution.

Further, the buffer is a mixture of dimethyl sulfoxide buffer and HEPES buffer.

Further, the volume ratio of the DMSO buffer to the HEPES buffer is 1:19.

The beneficial effects of the present invention are:

The present invention provides a polypeptide capable of specifically targeting human liver cancer cell line HepG2, which is named F11 polypeptide, and it has been confirmed in vivo that the polypeptide has a strong affinity for HepG2 liver cancer cells. The targeting polypeptide of the present invention has high specificity, can specifically target human liver cancer cell HepG2, but has no targeting ability to human LO2 cells and human peripheral blood cells. The targeting polypeptide of the invention has strong specificity and high targeting, and has good application potential in the fields of early diagnosis and treatment of tumors and targeted therapy.

Description of drawings

Fig. 1 shows the chemical structural formula of the F11 polypeptide of the present invention.

Figure 2 shows the verification results of the affinity of the F11 polypeptide to different cells in Experiment 1; a shows the results of fluorescence photography; b shows the results of flow cytometry.

Figure 3 shows the distribution of F11 polypeptide targeting liver cancer cells in mice.

Fig. 4 is a diagram showing the effect of immunohistochemical staining of F11 polypeptide on cancer and paracancerous tissues of liver cancer patients.

Detailed ways

The present invention uses phage display technology to screen and obtain a highly specific targeting polypeptide named F11 polypeptide for liver cancer cell HepG2. The amino acid sequence of the polypeptide is shown in SEQ ID NO:1.

The phage peptide library screened in the present invention is a randomly generated dodecapeptide library with a capacity greater than 10 ⁹ , and the F11 polypeptide is finally obtained after three rounds of screening. In the screening process of the present invention, when HepG2 is used for screening, the adsorbed cells are also selected to remove polypeptides with affinity with other cells. This makes the polypeptide F11 obtained by our screening unable to recognize LO2 and human peripheral blood cells while being able to bind to HepG2 cells.

Furthermore, the present invention also provides encoding nucleotides, vectors and host cells of the above-mentioned polypeptide specifically targeting human liver cancer cells.

Further, the present invention also provides the use of the above-mentioned polypeptide specifically targeting human liver cancer cells in targeting liver cancer cells.

The present invention also provides a use of the above-mentioned polypeptide specifically targeting human liver cancer cells in the preparation of drugs for preventing or treating liver cancer.

The present invention also provides a use of the above-mentioned polypeptide specifically targeting human liver cancer cells in the preparation of detection reagents for diagnosing liver cancer.

The polypeptide specifically targeting human liver cancer cells of the present invention is screened by the following method:

Human hepatoma cells HepG2 and LO2 cells in good growth state were inoculated in 24-well plates. Add blocking solution (4% milk+PBS), block at 37°C for 1h. Aspirate the blocking solution and wash 3 times with PBS. Mix 10 μl of phage library with 1 ml of PBS, and incubate with LO2 cells at 37°C for 1 hour. Then the supernatant was incubated with the blocked liver cancer HepG2 cells at 37° C. for 2 hours. The phages bound to the cells were collected, amplified with ER2738 host bacteria, and the amplified phages were used for the next screening of HepG2.

Repeat the above operation twice again, and the titer of the phage obtained in the third round of screening is measured on the LB/IPTG/X-Gal plate and used for identification. The 0.8 phage clone was purified and sequenced to obtain the specific polypeptide F11 capable of recognizing HepG2 of the present invention.

The following will further explain the specific implementation of the present invention through examples, but it does not mean that the protection scope of the present invention is limited to the scope described in the examples.

The preparation method of each reagent in following embodiment or test example is:

0.2M Gly-HCl PH2.2: Weigh 1.5014g glycine, dissolve it in water, adjust the pH value to 2.2 with HCl, and then dilute to 100mL with water.

1M Tris-HCl pH9.1: Weigh 121.1g Tris, dissolve it in water, adjust the pH value to 9.1 with HCl, and then dilute to 100mL with water.

20% PEG/2.5M NaCl: After dissolving 20g of polyethylene glycol and 14.6g of NaCl in water, add water to make up to 100mL.

The rest of the reagents are common commercially available products.

Example 1 HepG2 liver cancer cell screening phage polypeptide library

Human hepatoma cells HepG2 and LO2 hepatocytes (from ATCC) in good growth state were passaged separately, inoculated in 24-well cell culture plates, cultured at 37°C, saturated humidity, and 5% CO2 for 24 hours, then changed the medium, and cultured until the cells Adhere to the wall and grow in good condition. When the degree of confluence is over 90%, aspirate the medium when confluent into a monolayer of cells, gently wash twice with PBS, add serum-free medium, incubate at 37°C, 5% CO2 for 1 hour, and aspirate Remove the culture medium, add blocking solution (4% milk+PBS), and block at 37° C. for 1 hour; repeat the same operation to block the liver cancer cell HepG2 cells. After the blocking was completed, the blocking solution was aspirated, washed three times with PBS, 10 μl of phage library was mixed with 1 ml of PBS, and incubated with LO2 cells at 37°C for 1 hour for negative cell adsorption. After the adsorption is complete, absorb the supernatant, transfer to a 1.5ml sterilized centrifuge tube, centrifuge at 1000rpm for 5min, transfer the supernatant to a new centrifuge tube, and centrifuge again to remove the cells that may be contained in the supernatant. Quickly incubate the supernatant with the blocked and washed HepG2 cells at 37°C for 2 hours. After incubation, the supernatant was discarded and washed five times with PBS. Add 1ml 0.2M Gly-HCl pH2.2 for elution, then add 150μl 1MTris-HCl pH9.1 to neutralize and collect the phages adsorbed on HepG2 cells.

The collected phages were infected with 20ml of ER2738 (shake the bacteria overnight to inoculate at 1:100) bacterial solution, and infected at 37°C for 4.5h. Centrifuge at 12000 g for 10 min, take the supernatant and add 1/6 volume of 20% PEG/2.5M NaCl, extract phage at 4°C for 2 h. The phage solution was centrifuged at 12000 g at 4°C for 15 min, the supernatant was removed, and 1 mL of culture medium was added to obtain the phage solution. Repeat the above steps for the second and third rounds of screening.

Example 2 Screening to obtain the ELISA identification of phage clones

After the third round of screening, HepG2/LO2 were cultured in 96-well cell culture plates. After the cells adhered to the wall, the culture medium was removed, and the cells were washed twice with PBS. Block with 4% PBSM blocking solution at 37°C for 1 h, remove the blocking solution, and wash once with PBS. Add 50 μl 4% PBSM blocking solution and 50 μl screened phage solution to each well, and incubate at 37° C. for 1 h. After incubation, wash 5 times with PBS, add anti-P8/HRP antibody, incubate at 37°C for 1 h, wash 5 times with PBS, add 100 μl TMB substrate to each well, and stop the reaction after 15 min in the dark, and detect the absorbance with a microplate reader. A phage clone with an absorbance greater than 0.8 was selected for sequencing, the sequence was FYLEHPSGGLAV, and the polypeptide of the above sequence was named F11 polypeptide.

Example 3 Synthesis and purification of F11 polypeptide

Weigh a certain amount of 2Cl resin into the reactor, then add amino acid and alkali according to the ratio of resin: Fmoc-Cys(Trt)-OH: DIEA 1:3:6, use DCM as solvent to react for 3 hours, then add chromatography grade methanol Capped for 30min, finally washed and drained the synthetic resin. Select and accurately weigh the resin with the corresponding gauge, put it into a clean reactor, add 2 times the volume of DMF (DCM) to swell for 60-90 minutes. Remove DMF (DCM), add three times the volume of 20% piperidine solution to react for 30 minutes, then remove 20% piperidine solution, and wash with DMF for 5 times. Take a small amount of resin in the detection tube, drop two drops of ninhydrin solution (A, B, C solution), heat at 110°C for 3 minutes, if the resin is positive, it means that Fmoc has been removed. After removal, put them into the reactor in the order of 3 times AA: 6 times NMM: 2.85 times HBTU (HATU), and add DMF (so that the resin can be fully stirred). Take a small amount of resin in the detection tube, drop two drops of ninhydrin solution (A, B, C liquid) each, and heat at 110°C for 3 minutes. If the resin is negative, the reaction is complete; if the resin is positive, the reaction is incomplete. Three times the volume of 20% piperidine solution was reacted for 30 minutes and the operation was repeated. After all the peptide sequences are assembled, wash the resin with DMF*3, MeOH*2, DCM*2, and MeOH*3, and put it in a desiccator overnight to dry it. Add 8-10ml per gram of cutting solution (solution E/solution F) to the drained resin, and place it on a shaker for 2 hours. After the reaction is completed, filter with a sand core to obtain the filtrate, add glacial ether to precipitate the crude product, put it into a centrifuge for precipitation, and repeat washing with ether three times. Put the cleaned crude product into a desiccator to dry overnight to obtain the target polypeptide F11, the structure of which is shown in Figure 1.

Example 4 Synthesis and purification of FITC-F11 polypeptide

Weigh a certain amount of 2Cl resin into the reactor, then add amino acid and alkali according to the ratio of resin: Fmoc-Lys(Dde)-OH: DIEA 1:3:6, use DCM as solvent to react for 3 hours, and then add chromatography grade methanol Capped for 30min, finally washed and drained the synthetic resin. Select and accurately weigh the resin with the corresponding gauge, put it into a clean reactor, add 2 times the volume of DMF (DCM) to swell for 60-90 minutes. Remove DMF (DCM), add three times the volume of 20% piperidine solution to react for 30 minutes, then remove 20% piperidine solution, and wash with DMF for 5 times. Take a small amount of resin in the detection tube, drop two drops of ninhydrin solution (A, B, C solution), heat at 110°C for 3 minutes, if the resin is positive, it means that Fmoc has been removed. After removal, put them into the reactor in the order of 3 times AA: 6 times NMM: 2.85 times HBTU (HATU), and add DMF (so that the resin can be fully stirred). Take a small amount of resin in the detection tube, drop two drops of ninhydrin solution (A, B, C liquid) each, and heat at 110°C for 3 minutes. If the resin is negative, the reaction is complete; if the resin is positive, the reaction is incomplete. Three times the volume of 20% piperidine solution was reacted for 30 minutes and the operation was repeated. After the assembly of the linear peptide sequence is complete, remove Dde with 4% hydrazine hydrate/DMF solution, put it into the reactor in the order of 4 times NMM:2 times FITC, add DMF, take a small amount of resin in the detection tube, drop indene Two drops of triketone solutions (solutions A, B, and C) each, heated at 110°C for 3 minutes, if the resin is positive, the reaction is incomplete, repeat the previous step, if the resin is negative, the reaction is complete, wash with methanol and put it in a desiccator overnight Drain. Add 8-10ml per gram of cutting solution (solution E/solution F) to the drained resin, and place it on a shaker for 2 hours. After the reaction is completed, filter with a sand core to obtain the filtrate, add glacial ether to precipitate the crude product, put it into a centrifuge for precipitation, and repeat washing with ether three times. Put the washed crude product in a desiccator overnight to dry it up to obtain the target polypeptide FITC-F11.

Test Example 1 F11 Polypeptide Cell Targeting Affinity Verification Test

EZ细胞爬片，24小时待细胞完全贴壁后，将FITC荧光标记的F11多肽(实施例4制备得到的)加入培养孔中，5分钟后加入5μl每孔的Hoechst染细胞核。5分钟后将细胞液上清吸掉，用PBS清洗三次，再将细胞槽移掉，再盖上盖玻片。于显微镜下观察FITC-F11多肽在每种细胞上的摄取情况并拍照，如图2a所示，F11多肽大量占据在HepG2细胞表面，而在293T细胞核LO2细胞表面几乎没有F11多肽占据。之后，将每个孔的细胞收集起来测流式，结果如图2b所示，可以看出F11多肽对HepG2细胞的亲和能力明显大于293T细胞和LO2细胞。以上结果显示出F11多肽对HepG2细胞的靶向亲和能力。In order to study the targeted uptake of the liver cancer polypeptide F11 to HepG2 cells in vitro, an in vitro test was established for verification. First, HepG2 cells, LO2 cells and 293T cells were plated at a density of 5X104 per well

EZ cell slides, 24 hours after the cells were completely adhered to the wall, FITC fluorescently labeled F11 polypeptide (prepared in Example 4) was added to the culture wells, and 5 μl of Hoechst per well was added to stain the nuclei after 5 minutes. After 5 minutes, suck off the supernatant of the cell solution, wash with PBS three times, remove the cell chamber, and cover with a cover glass. The uptake of FITC-F11 polypeptide on each cell was observed under a microscope and photographed. As shown in Figure 2a, a large number of F11 polypeptides occupied the surface of HepG2 cells, while almost no F11 polypeptide occupied the surface of 293T cell nucleus LO2 cells. Afterwards, the cells in each well were collected for flow cytometry, and the results are shown in Figure 2b. It can be seen that the affinity of the F11 polypeptide to HepG2 cells is significantly greater than that of 293T cells and LO2 cells. The above results show the targeting affinity of the F11 polypeptide to HepG2 cells.

Test Example 2 F11 Polypeptide Targeted Distribution Ability Verification Test in Mice

In order to verify the targeting affinity of liver cancer polypeptide F11 to liver cancer tissue in vivo, a HepG2 subcutaneous tumor model was established in Balb/c-nude mice (4-5 weeks old, female). HepG2 cells cultured in vitro were digested with trypsin and fixed in double DmEm-free medium, and each mouse was inoculated with 1×10 ⁷ cells. After the subcutaneous tumor volume reached 1000 mm ³ , the tumor-bearing mice were taken and injected with 200 μg of FITC-F11 solution intravenously. Two hours later, the mice were sacrificed, and the subcutaneous tumors, heart, liver, spleen, lung, and kidney were removed. The tissue was cut into pieces and placed in collagenase solution, digested at 37°C for 4 hours, then the solution was filtered through a 70 μm cell mesh, the filtrate was washed three times with PBS buffer, and the flow pattern was measured. The flow cytometry data of mouse tumors and major organs are shown in Figure 3. It can be seen that the distribution of FITC-labeled polypeptide F11 in tumor tissues is significantly higher than that in other organs, which proves that the F11 polypeptide targets tumor tissues in mice Affinity.

Test 3 Verification test of F11 polypeptide on immunohistochemical staining of liver cancer patient tissue sections

In order to verify whether the F11 polypeptide may have the liver cancer targeting effect in the liver cancer patients, immunohistochemical techniques were used to stain the tissue sections of the liver cancer patients. First, slices of liver cancer patients were dried and then dewaxed and hydrated for antigen retrieval. After blocking with goat serum for 20 minutes, a 10 μg/ml FITC-F11 polypeptide solution was used as the primary antibody to cover the patient tissue chip, and incubated overnight at 4°C. After washing three times with PBS the next day, the nuclei were stained with DAPI, and then washed three times with PBS. After staining the nuclei, anti-fluorescence quencher was added dropwise, and the slides were sealed. The representative picture of the staining results is shown in Figure 4. It can be seen that the cancer tissue of the liver cancer patient is more obviously positive than the para-cancerous tissue, which proves the targeting affinity of the F11 polypeptide in the patient's body to the liver cancer tissue.

The present invention screens and obtains a polypeptide that can successfully target liver cancer cells. After research, it has a good affinity for liver cancer cells HepG2 cells, can specifically target HepG2 cells, and also shows good activity in mice. The targeting effect is suitable for preparing anti-liver cancer drugs or imaging agents for diagnosing liver cancer, and has good application prospects.

sequence listing

<110> West China Hospital of Sichuan University

<120> Polypeptides specifically targeting human liver cancer cells and uses thereof

<130> A210274K (serial)

<141> 2021-04-29

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 1

Phe Tyr Leu Glu His Pro Ser Gly Gly Leu Ala Val

1 5 10

<210> 2

<211> 36

<212>DNA

<213> Artificial Sequence

<400> 2

ttttatctgg aacatccgag cggcggcctg gcggtg 36

Claims (10)

1. A polypeptide that specifically targets human hepatoma cells, characterized in that: the amino acid sequence is shown as SEQ ID NO. 1.

2. The polypeptide specifically targeting human hepatoma cells according to claim 1, characterized in that: the liver cancer cell is a HepG2 cell line.

3. The nucleotide sequence encoding the polypeptide specifically targeting human hepatoma cells of claim 1.

4. The coding nucleotide according to claim 3, characterized in that: the sequence of the coding nucleotide is shown as SEQ ID NO. 2.

5. An expression vector comprising the polypeptide of claim 1, the coding nucleotide of claim 3 or 4.

6. The expression vector of claim 5, wherein: the expression vector is a prokaryotic vector or a eukaryotic vector.

7. A host cell comprising the polypeptide of claim 1, the coding nucleotide of claim 3 or 4, or the expression vector of claim 5 or 6.

8. Use of the polypeptide of claim 1, the coding nucleotide of claim 3 or 4, the expression vector of claim 5 or 6, or the host cell of claim 7 for targeting hepatoma cells.

9. Use of the polypeptide of claim 1, the coding nucleotide of claim 3 or 4, the expression vector of claim 5 or 6, or the host cell of claim 7 in preparing a medicament for resisting liver cancer or preparing an imaging agent for diagnosing liver cancer.

10. Use according to claim 9, characterized in that: the polypeptide is prepared into a solution for use, and the concentration is 1mg/mL.

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