CN114195862A

CN114195862A - Polypeptide and application thereof

Info

Publication number: CN114195862A
Application number: CN202111150093.2A
Authority: CN
Inventors: 叶招明; 林鹏; 薛钰铖; 滕王锶源
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-09-29
Filing date: 2021-09-29
Publication date: 2022-03-18
Anticipated expiration: 2041-09-29
Also published as: CN114195862B

Abstract

The invention discloses a polypeptide and application thereof, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO.1-SEQ ID NO. 4. The polypeptide of the invention obtains the affinity peptide of the targeted osteosarcoma HOS cell by utilizing a phage display technology, phage in-vitro cell screening and in-vivo tumor screening, and the targeting property and the specific binding of the polypeptide to the osteosarcoma HOS are verified through in-vitro fluorescence microscope observation and in-vivo experiment. The polypeptide has potential application prospect in the targeted treatment of tumors of osteosarcoma.

Description

Polypeptide and application thereof

(I) technical field

The invention belongs to the technical field of biology, relates to a polypeptide and application thereof, and particularly relates to a polypeptide targeting an osteosarcoma HOS cell line and application thereof.

(II) background of the invention

Osteosarcoma is the most common primary malignant tumor of bones in human, and the five-year survival rate of patients without metastasis is lower than 70%, and the five-year survival rate of patients with metastasis is lower than 30%. In the last thirty years, the treatment mode of osteosarcoma has not been obviously advanced, and the clinically existing main treatment modes are only surgical excision and chemotherapy, so that the long-term survival rate of osteosarcoma patients is not obviously changed in the last thirty years.

Targeted treatment of osteosarcoma is still under investigation, and most of the current research focuses on phylogenetic and somatic genetics of osteosarcoma, trying to find therapeutic targets by comparing genetic changes of osteosarcoma. Targeted treatment of osteosarcoma based on genomic examinations is however costly and still in the exploratory phase. In addition, osteosarcomas have high levels of somatic structural variation and copy number alterations, resulting in osteosarcomas with high heterogeneity with few repetitive target mutations. Therefore, there is an urgent need to develop a new targeted therapeutic strategy for osteosarcoma.

The phage display technology can be used for searching the polypeptide of the target cell and has strong application value in the aspect of identifying the affinity polypeptide of the tumor cell. Several studies currently show that the combination of short oligopeptides derived from phage display with drugs, fluorochromes, and even nanoparticles can be used for targeted therapy and accurate diagnosis of cancer.

In view of the advantages of the targeting polypeptide in targeting therapy, the screening of the polypeptide capable of targeting osteosarcoma cells has important significance in targeting therapy of osteosarcoma.

Disclosure of the invention

The invention aims to provide a polypeptide and application thereof, wherein the polypeptide is an affinity peptide of a HOS cell line of targeted human osteosarcoma by using a phage display technology and through in vitro cell screening and in vivo tumor screening, and has a wide application prospect in targeted treatment of osteosarcoma.

The technical scheme adopted by the invention is as follows:

the invention provides a polypeptide, in particular a polypeptide targeting osteosarcoma HOS cell line, wherein the amino acid sequence of the polypeptide is any one or the combination of at least two of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 from the N end to the C end, and the amino acid sequence is preferably the amino acid sequence shown in SEQ ID NO. 1;

SEQ ID NO.1：TPPRVPLLTFGS；

SEQ ID NO.2：YPAAYHTLTREP；

SEQ ID NO.3：DSPTSLVLRARS；

SEQ ID NO.4：STLLADVRIPGS。

the invention screens polypeptide sequences which can be specifically combined with a human osteosarcoma HOS cell line from a random phage polypeptide library through the construction of a phage library. And then combining the sequences with HOS cells, and screening out four amino acid sequences which are specifically combined with the HOS cells, wherein the amino acid sequence shown in SEQ ID NO.1 has the best combination effect with the HOS cells.

The invention also provides a DNA segment containing the polypeptide nucleotide sequence, a recombinant vector containing at least one copy DNA segment, and a recombinant cell containing the recombinant vector, wherein the recombinant cell is a recombinant gene engineering bacterium prepared by transforming a host bacterium; the recombinant vector is based on M13KE bacteriophage, and the recombinant genetically engineered bacterium host bacterium is preferably Escherichia coli ER 2738.

The invention also provides a pharmaceutical composition, which comprises the DNA segment, the recombinant vector or the recombinant cell, and a pharmaceutically acceptable carrier.

The invention provides an application of the polypeptide in preparing tumor localization diagnosis reagent, anti-tumor medicine or tumor cell targeting preparation.

The polypeptide is usually modified for drug attachment, and preferably, the modification is to add a Gly-Gly-Ser-Cys linker to the C-terminal of the amino acid sequence of the polypeptide.

The tumor is human osteosarcoma, or tumors which may express a receptor combined with the polypeptide, such as lung cancer, melanoma, breast cancer, colorectal cancer, ovarian cancer or liver cancer.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention selects polypeptide sequence which can be specifically combined with HOS cell of human osteosarcoma from random phage polypeptide library, and determines that the amino acid sequence shown in SEQ ID NO.1 has the best combination effect with HOS cell;

(2) the polypeptide of the invention combines with drugs, fluorescent dyes, carriers and the like to play roles of tumor diagnosis, tumor resistance and the like.

(3) The polypeptide has wide application prospect in the aspect of treating tumors with high potential expression such as osteosarcoma, lung cancer, melanoma, breast cancer, colorectal cancer, ovarian cancer or liver cancer and the like and a receptor combined with the polypeptide.

(IV) description of the drawings

FIG. 1, in vivo imaging observation of polypeptide aggregation in tumors; a is a live body imaging photo, wherein Saline is injected, CY5.5Only is injected with 200 mu M of physiological Saline solution of Cy5.5-maleimide, CY5.5-Peptide C is injected with polypeptide conjugate physiological Saline solution of sequence 2, CY5.5-Peptide S is injected with polypeptide conjugate physiological Saline solution of random sequence, and CY5.5-OTP is injected with polypeptide conjugate physiological Saline solution of sequence 1; b is a bar graph of fluorescence intensity for each group of mice.

FIG. 2, in vitro fluorescence microscopy of the affinity of the polypeptide of SEQ ID NO.1 for osteosarcoma HOS cells; a is a fluorescence micrograph, wherein Cy5.5-OTP represents a polypeptide conjugate of a sequence 1, Cy5.5-Peptide C represents a polypeptide conjugate of a sequence 2, Cy5.5-Peptide S represents a polypeptide conjugate of a random sequence Peptide S, and Cy5.5Only represents Cy5.5-maleimide; DAPI stands for DAPI staining, FITC-phaseolin stands for FITC-phaseolin staining, Cy5.5-peptide dies stands for different Cy5.5-polypeptide conjugates, MERGE stands for the combined image of the 3 staining channels; b is a bar graph of fluorescence intensity for each group.

FIG. 3, in vitro fluorescence microscopy of the specificity of binding of the polypeptide of SEQ ID NO.1 to osteosarcoma HOS cells; a is a fluorescence micrograph, in which HOS represents HOS cells, HUVECs represent control cells HUVECs, DAPI represents DAPI staining, Cy5.5-OTP represents Cy5.5-polypeptide conjugate of sequence 1, and MERGE represents the merged image of the 2 staining channels; b is a bar graph of fluorescence intensity for each group.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

the experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The reagent used in the invention comprises the following components:

LB liquid medium: 10g NaCl, 10g tryptone, 5g yeast extract, dissolved in 1L ddH₂And O. Steam sterilizing at 121 deg.C for 20 min.

LB agar medium: 10g NaCl, 10g tryptone, 5g Yeast extract, 12g Agar (Agar), dissolved in 1L ddH₂And O. Steam sterilizing at 121 deg.C for 20 min.

Bacterial culture plate: firstly, respectively preparing isopropyl-beta-D-thiogalactoside (IPTG) and galactoside (X-gal) into 20mg/ml solutions by using Dimethylformamide (DMF), and storing the solutions at the temperature of minus 20 ℃ in a dark place; then, 100. mu.L of the IPTG solution and X-gal solution were added to 100ml of LB agar medium, respectively, to prepare a bacterial culture plate.

Washing buffer solution: serum-free DMEM cell culture medium with 0.5mg/ml bovine serum albumin and 0.1% (v/v) Tween 20.

Elution buffer: 0.1M Glycine, 1mg/mL bovine serum Albumin in ddH₂O, adjusted to pH2.2 with concentrated hydrochloric acid having a mass concentration of 36%.

Cell lysis solution: 0.02mg/ml sodium deoxycholate, 10mM Tris-HCl, 2mM EDTA, in ddH₂O。

Tris-HCl buffer (Tris-HCl buffer): 121.1g Tris, 1L ddH₂And O, adjusting the pH value to 9.1 by concentrated hydrochloric acid with the mass concentration of 36%, and storing at 4 ℃.

PEG/NaCl solution: 100g polyethylene glycol (PEG), 116.9g NaCl and 475mL ddH₂O。

NAP buffer solution: 80mM NaCl, 50mM NH₄ H₂PO₄The solvent is ddH₂O, concentrated hydrochloric acid with the mass concentration of 36% is adjusted to pH 7.0.

Tris Buffer Salt (TBS): 2.42g/L Tris (Tris, hydroxymethyl aminomethane), 29.22g/L NaCl, 1L ddH₂O，Concentrated hydrochloric acid with a mass concentration of 36% was adjusted to pH 7.5.

M13KE phage library: purchased from Bio-lab Biotechnology, Inc.

The room temperature is 25-30 ℃. Human osteosarcoma HOS cells were purchased from Shanghai cell bank of Chinese academy of sciences.

The polypeptides involved in the following examples were synthesized and purified by the biotechnology limited of kingdom jinsrey, south of Jiangsu, and sequencing was performed by the biotechnology limited of wuhan baizi, Hubei.

Example 1: the human osteosarcoma HOS cell line targeting peptide is obtained by in vitro cell screening and in vivo tumor screening by adopting a phage display technology.

1. In vitro cell screening of HOS (human osteosarcoma) targeted phage

(1) Preparing Escherichia coli solution

(ii) thawing the ER2738 E.coli bacterial liquid (about 10) at room temperature¹⁰one/mL), 300. mu.L of the bacterial solution was added to 20mL of LB medium, and the mixture was shaken at 37 ℃ for 4 hours at 220rpm until the OD value became 500.

500g, centrifuging for 6min at 4 ℃ and precipitating escherichia coli; the pellet was gently resuspended in 10mL of 80mM NaCl aqueous solution;

③ shaking the resuspended solution in a shaker at 50rpm and 37 ℃ for 20min, transferring the solution into a 50mL centrifugal tube, and centrifuging the solution at 500g and 4 ℃ for 6 min; the pellet was resuspended in 1mL of 4 ℃ pre-cooled NAP buffer to obtain an e.coli solution (OD 500). Then stored in a refrigerator at 4 ℃ for use.

(2) In vitro screening of HOS cell targeting peptide of human osteosarcoma

Human osteosarcoma HOS cell (50X 10)⁶Respectively) were inoculated into 10cm cell culture bottles containing 10mL of DMEM medium containing 10% fetal bovine serum by volume, and placed in a cell culture chamber at 37 ℃ with 5% CO₂After incubation to 90% cell density, the appropriate amount of M13KE phage library (approximately 1X 10) was added to the culture broth¹⁰One), incubated at 37 ℃ for 1 h.

② abandoning the culture solution, adding 4mL of 4 ℃ precooled washing buffer solution into the culture flask, and washing for 1 minute.

③ discard the washing buffer, add 5ml PBS, then scrape the cells with cell. The scraped cells were transferred to a 15mL centrifuge tube, centrifuged at 200g at 4 ℃ for 10min, and the supernatant was discarded. Adding 200 μ L cell lysate, mixing well to obtain human osteosarcoma HOS cell affinity phage liquid.

And fourthly, adding 1mL of the escherichia coli bacterial liquid prepared in the step (1) into 1mL of human osteosarcoma cell affinity phage liquid, uniformly mixing, and culturing at room temperature for 15min to obtain phage-infected bacteria. Transferring into a 250mL conical flask, adding 20mL LB culture solution, placing in a shaking table, culturing overnight at 220rpm and 37 ℃ to obtain the amplified phage liquid.

(3) Phage purification

Centrifuging 20mL of the amplified phage liquid at 4 ℃ and 3000g for 10 min; the clear supernatant was transferred to another centrifuge bottle, 6mL of PEG/NaCl solution was added and overnight at 4 ℃ as solution A.

② solution A in 5000g centrifugal 10min, precipitation of phage, abandoning the supernatant, using 1.5mL TBS solution heavy suspension, adding 150 u L PEG/NaCl solution, and at 4 degrees C to stand overnight, recorded as solution B.

③ centrifuging the solution B for 10min at 5000g and 4 ℃; the supernatant was discarded, and then 1mL of TBS solution was added to dissolve the phage particles to obtain a purified phage solution.

2. In vivo screening of HOS targeting peptides for human osteosarcoma

Firstly, nude mouse HOS cell nodulation modeling: will be about 10⁶Individual HOS cells were injected into nude mice to form tumors subcutaneously in the left axilla.

② to about 300cm of tumor size³In this case, 100. mu.L of the phage solution purified in step 1- (3) - ③ was injected into the tail vein.

③ after 24 hours, the tumor tissue was isolated.

Cutting, grinding, adding 10ml PBS, and homogenizing the tumor tissue thoroughly by a homogenizer.

Fifthly, centrifuging homogenate for 10min at the temperature of 4 ℃ and 1000g, taking supernatant and discarding tissue precipitates.

Sixthly, adding 1mL of escherichia coli solution prepared in advance in the step 1- (1) into the supernatant, and culturing for 15min at room temperature to obtain a bacterial solution infected by the phage.

Seventhly, transferring the bacterial solution infected by the phage into LB culture solution in a 250mL conical flask, and culturing overnight on a shaking table at 37 ℃ and 220rpm to obtain the amplified phage solution.

And (b) purifying the phage in the same step 1- (3).

3. Polypeptide sequence detection

After the in vitro screening process is carried out for 4 rounds, the in-wheel screening process is carried out for 1 round. The phage liquid purified in each round is inoculated to a bacterial culture plate, cultured overnight at 37 ℃, and then a blue monoclonal colony is selected for sequencing. The sequencing result is shown in table 1, and 4 polypeptide sequences of the osteosarcoma cells with affinity are obtained by screening, wherein the polypeptide sequence 1: TPPRVPLLTFGS (SEQ ID NO.1), polypeptide sequence 2: YPAAYHTLTREP (SEQ ID NO.2), polypeptide sequence 3: DSPTSLVLRARS (SEQ ID NO.3), polypeptide sequence 4: STLLADVRIPGS (SEQ ID NO. 4).

TABLE 1 frequency of appearance of polypeptide sequences

Example 2 human osteosarcoma HOS cell line targeting and specificity verification of polypeptide sequences.

1. In vivo verification of targeting of polypeptide to osteosarcoma tissue

During the synthesis of the polypeptides (SEQ ID NO: 1 and SEQ ID NO: 2) screened in example 1, a Gly-Gly-Gly-Ser-Cys linker was added to the C-terminus of each polypeptide.

② after the polypeptide synthesis, 1mL of 10mM Cy5.5-maleimide (dissolved in DMSO) was added to 1mL of 10mM polypeptide aqueous solution and reacted at room temperature for 3 hours. Cys of the polypeptide is coupled with maleimide of Cy5.5 dye to form Cy5.5-polypeptide conjugate, wherein the polypeptide conjugate formed by the sequence 1 is marked as Cy5.5-OTP, and the polypeptide conjugate formed by the sequence 2 is marked as Cy5.5-PeptideC. Meanwhile, a random sequence Peptide S (LTPSTRLPGFPV, SEQ ID NO.5) is synthesized as a comparison and is marked as Cy5.5-Peptide S after being combined with Cy5.5-maleimide.

③ injecting 200 mu M of different Cy5.5-polypeptide conjugate physiological Saline solution (Cy5.5-OTP, Cy5.5-PeptideC, Cy5.5-PeptideS), 200 mu M of Cy5.5-maleimide physiological Saline solution (noted as Cy5.5Only) and physiological Saline (Saline) into HOS tumor-forming mice by tail vein, observing the aggregation of the polypeptide in the tumor by a mouse living body imaging system (PerkinElmer IVIS Spectrum CT) under the anesthesia state of the mice after 24 hours (A in figure 1). Wherein a stronger fluorescence signal at the tumor indicates a stronger targeting ability of the polypeptide (B in FIG. 1).

The experimental results are shown in fig. 1: the polypeptide sequence 1(TPPRVPLLTFGS) and the polypeptide sequence 2(YPAAYHTLTREP) have targeting effects on osteosarcoma, wherein the targeting effect of the polypeptide sequence 1(TPPRVPLLTFGS) is optimal, and the fluorescence signal intensity of the sequence 1 in tumors is obviously higher than that of other polypeptides, which shows that the sequence 1 has good targeting capability on the tumors.

2. Validation of affinity specificity of Polypeptides for osteosarcoma HOS cells

As the amino acid sequence shown in SEQ ID NO.1 has the best effect, the targeting property and the specificity of the amino acid sequence shown in SEQ ID NO.1 to HOS cells are verified in-vitro experiments subsequently. Cy5.5-polypeptide conjugates (marked as Cy5.5-OTP and Cy5.5-Peptide C) of the sequence 1 and the sequence 2 and polypeptide conjugates (marked as Cy5.5-Peptide S) of random sequence Peptide S are prepared according to the method of the step 1 and used as a reference, and a DMSO solution of 1mM Cy5.5-maleimide is used as a blank reference and marked as Cy5.5 Only.

(ii) HOS cells were plated at 1.0X 10 per well⁶The amount of each cell was inoculated into a 24-well plate (1ml of DMEM high-glucose medium containing 10% fetal bovine serum by volume) at 37 ℃ with 5% CO₂Cultured to 50% cell density.

② blocking with 10% BSA at 37 ℃ for 60 minutes. The blocking solution was discarded and washed 3 times with PBS buffer.

And respectively adding Cy5.5-OTP, Cy5.5-Peptide S, Cy5.5-Peptide C and Cy5.5Only with final concentration of 10 mu M and HOS cells for co-incubation for 30 minutes. After 3 subsequent washes with PBS buffer, FITC-phaseolin was stained for 1h, DAPI was stained for 5min, and observed under a fluorescent microscope at 37 ℃ with the results shown in FIG. 2: the signal of OTP in HOS cells was significantly higher than the control polypeptide, indicating that OTP has significant affinity for HOS cells.

3. Verification of binding specificity of OTP to HOS cells of osteosarcoma

HUVECs (purchased from Shanghai cell Bank of Chinese academy) as HOS cells and control cells were inoculated into 24-well plates (1.0X 10 per well), respectively⁶Cells, 1ml of DMEM high-glucose medium containing fetal bovine serum at a volume concentration of 10%), blocked with 10% BSA at 37 ℃ for 60 minutes, and then stained with cy5.5-OTP (10 μ M) prepared in step 1 for 30 minutes, respectively. After three washes, the DAPI was stained for 5min and observed under a fluorescent microscope at 37 ℃. The experimental results are shown in fig. 3: OTP accumulated significantly more in HOS cells than HUVECs cells, indicating that OTP can specifically bind to HOS cells.

In conclusion, the polypeptide and the product thereof can be combined with HOS cells of human osteosarcoma in a targeted way, so that the polypeptide has wide application prospects in the aspects of targeted treatment and diagnosis of osteosarcoma, and can have wide application prospects in the aspects of tumor targeted treatment of potential high-expression receptors combined with the polypeptide, such as lung cancer, melanoma, breast cancer, colorectal cancer, ovarian cancer or liver cancer.

The applicant states that the present invention is illustrated by the above examples of the process of the present invention, but the present invention is not limited to the above process steps, i.e. it is not meant that the present invention must rely on the above process steps to be carried out. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.

Sequence listing

<110> Zhejiang university

<120> polypeptide and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Unknown (Unknown)

<400> 1

Thr Pro Pro Arg Val Pro Leu Leu Thr Phe Gly Ser

1 5 10

<210> 2

<211> 12

<212> PRT

<213> Unknown (Unknown)

<400> 2

Tyr Pro Ala Ala Tyr His Thr Leu Thr Arg Glu Pro

1 5 10

<210> 3

<211> 12

<212> PRT

<213> Unknown (Unknown)

<400> 3

Asp Ser Pro Thr Ser Leu Val Leu Arg Ala Arg Ser

1 5 10

<210> 4

<211> 12

<212> PRT

<213> Unknown (Unknown)

<400> 4

Ser Thr Leu Leu Ala Asp Val Arg Ile Pro Gly Ser

1 5 10

Claims

1. A polypeptide is characterized in that the amino acid sequence of the polypeptide is any one or the combination of at least two of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4 from the N end to the C end;

SEQ ID NO.1：TPPRVPLLTFGS；

SEQ ID NO.2：YPAAYHTLTREP；

SEQ ID NO.3：DSPTSLVLRARS；

SEQ ID NO.4：STLLADVRIPGS。

2. a DNA fragment comprising the nucleotide sequence of the polypeptide of claim 1.

3. A recombinant vector comprising at least one copy of the DNA segment of claim 2.

4. A recombinant cell characterized in that it contains the recombinant vector according to claim 3.

5. A pharmaceutical composition comprising the polypeptide of claim 1, the DNA segment of claim 2, the recombinant vector of claim 3, or the recombinant cell of claim 4.

6. The pharmaceutical composition of claim 5, wherein said pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

7. The use of the polypeptide of claim 1 in the preparation of a tumor localization diagnostic agent.

8. The use of the polypeptide of claim 1 in the preparation of an anti-tumor medicament.

9. Use of the polypeptide of claim 1 in the preparation of a tumor cell targeting agent.

10. Use according to claim 9, wherein the tumour cells comprise cells of osteosarcoma, lung carcinoma, melanoma, breast carcinoma, colorectal carcinoma, ovarian carcinoma or liver carcinoma.