CN113527430B

CN113527430B - Novel polypeptide for tumor cell specific targeting and application thereof

Info

Publication number: CN113527430B
Application number: CN202010293449.7A
Authority: CN
Inventors: 魏丽杰
Original assignee: Shenyang Yijian Life Technology Co ltd
Current assignee: Shenyang Yijian Life Technology Co ltd
Priority date: 2020-04-15
Filing date: 2020-04-15
Publication date: 2024-04-26
Anticipated expiration: 2040-04-15
Also published as: CN113527430A

Abstract

The invention belongs to the biomedical field, in particular to a novel polypeptide specifically targeted and combined with human liver cancer cells and application thereof. The invention also relates to application of the polypeptide and the bioactive fragments and derivatives thereof as molecular imaging probes and drug targets in tumor diagnosis and treatment, wherein the amino acid sequence of the polypeptide is ：SEQ ID NO.1：QTNYTSVHMGSS;SEQ ID NO.2：NSQHPSLYVWAS;SEQ ID NO.3：DPQHSSLYVSSA;SEQ ID NO.4：QTNYTSVHMASS;SEQ ID NO.5：HTASYYSAVGSA;SEQ ID NO.6：YPSAHHSLMRPA;SEQ ID NO.7：MPSMGHAAIAPN;SEQ ID NO.8：VSAVWYSALSTG;SEQ ID NO.9：DPNHAAVHMGAA. respectively. The polypeptide has specific targeting binding capacity to liver cancer cells and strong selectivity, and lays a foundation for early diagnosis of clinical tumors and research and development of targeted drugs.

Description

Novel polypeptide for tumor cell specific targeting and application thereof

Technical Field

The invention belongs to the biomedical field, in particular to a polypeptide capable of specifically binding with human liver cancer cells; derivatives comprising the polypeptide; nucleic acids encoding the above polypeptides and derivatives thereof; and the use of the polypeptide or derivative, especially for the prevention, diagnosis and treatment of tumors.

Background

The current urgent situation is that in modern times of such developed medical technology, malignant tumors remain the peak of being difficult to overcome worldwide. Although the three traditional treatment methods of operation, chemotherapy and radiotherapy can treat or delay the development of malignant tumor, the three traditional treatment methods still cause great pains to patients due to serious defects of large wounds, strong toxic and side effects and the like. So the efficient and low-toxicity treatment means aiming at malignant tumors become the pursuit of cumin tiredness of modern medical researchers. In the field of chemotherapy drug research, the killing effect of anti-malignant tumor drugs on non-tumor tissues has been the most obvious and difficult problem to overcome by the drugs. Therefore, it is important to find out a high-specificity targeting molecule capable of binding with tumor cells and tissues and develop a liver cancer early diagnosis reagent and a targeting drug system with more sensitivity by taking the targeting molecule as a break.

At present, phage Display Technology (PDT) has been widely applied to screening tumor targeting molecules, and the basic principle is that a foreign gene library with a certain rule is inserted into phage genome, polypeptide or protein molecules encoded by the foreign gene are fused on phage envelope proteins, so that the established phage expression library can be used for affinity screening of polypeptide or protein capable of specifically binding with target molecules, and cell protein components and functions thereof can be analyzed in a high throughput manner. The technology is also applied to a plurality of fields such as affinity screening of known (or unknown) receptor/antibody, identification of new receptor or natural ligand, mapping and simulation of receptor/antibody epitope, research and development of vaccine and new medicine.

Phage display peptide libraries, which are an important branch of phage display technology, are widely used to identify and develop tumor homing peptides (THP _S). The tumor homing peptide can specifically bind with tumor cells or tumor microenvironment components, has no or low affinity with normal cells, and plays a role in guiding through specifically recognizing receptors expressed by tumor cells or tumor blood vessels; in addition, part of tumor homing peptides have the characteristic of penetrating membranes, so that medicines can enter tumor cells more effectively, thereby improving the curative effect of therapeutic medicines in cancer treatment and reducing adverse reactions. The tumor homing peptide mediated targeting drug delivery system is expected to be a new strategy and a new means for molecular imaging diagnosis and targeted drug development.

The inventor uses phage display 12 peptide library to screen and obtain positive phage clone combined with tumor cell in targeting mode, and obtains the sequence and structure of polypeptide interacted with human tumor cell through DNA structure analysis and triple code translation. The invention has the advantages that (1) the polypeptide can be combined with the targeting of human tumor cells, namely has tumor targeting property and can be used as a tool for diagnosis and treatment. (2) The polypeptide fragment has higher water solubility, relatively stable property and difficult inactivation, and greatly widens the application range of the targeting peptide.

Disclosure of Invention

The invention aims to provide a novel polypeptide with high affinity and specificity for binding tumor cells and application thereof, wherein the novel targeting polypeptide or a derivative product thereof is preferably used for preparing an antitumor drug or an imaging preparation for targeting tumors by specifically targeting the tumor cells without affecting normal human cells.

The principle of the invention is as follows: the method comprises the steps of taking a normal human liver epithelial cell HL7702 as a control, adopting a human liver cancer cell HepG2 to carry out subtractive screening on a phage display peptide library, selecting positive phage clones capable of specifically binding with the human liver cancer cell by a blue-white screening test, and verifying the binding specificity of phage and the human liver cancer cell by an ELISA test. Then taking escherichia coli as a carrier, extracting DNA (deoxyribonucleic acid) of the phage after amplification and purification, sequencing to obtain a polypeptide coding sequence capable of specifically combining with human liver cancer cells, artificially synthesizing fluorescent-labeled positive polypeptide, and verifying the combining effect of fluorescent-labeled polypeptide and human liver cancer cells, thereby providing an experimental basis for early diagnosis and targeted treatment of liver cancer.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

The primary purpose of the present invention is to provide polypeptides capable of specifically targeted binding to human tumor cells, which have the amino acid sequences of ：SEQ ID NO.1：QTNYTSVHMGSS;SEQ ID NO.2：NSQHPSLYVWAS;SEQ ID NO.3：DPQHSSLYVSSA;SEQ ID NO.4：QTNYTSVHMASS;SEQ ID NO.5：HTASYYSAVGSA;SEQ ID NO.6：YPSAHHSLMRPA;SEQ ID NO.7：MPSMGHAAIAPN;SEQ ID NO.8：VSAVWYSALSTG;SEQ ID NO.9：DPNHAAVHMGAA.

The polypeptide has targeting effect on tumor cells and can be specifically combined with the tumor cells.

The tumor cells are human liver cancer cells.

Amino acid residues represented by single letter symbols in the above polypeptide chains are shown in the amino acid abbreviations:

TABLE 1 amino acid abbreviations

Single letter	Three letters	English name	Chinese name
				A	ALA	Alanine	Alanine (Ala)
C	CYS	Cysteine	Cysteine (S)
				D	ASP	Aspartic Acid	Aspartic acid
E	GLU	Glutamic Acid	Glutamic acid
				F	PHE	Phenylalanine	Phenylalanine (Phe)
G	GLY	Glycine	Glycine (Gly)
				H	HIS	Histidine	Histidine
I	ILE	Isoleucine	Isoleucine (Ile)
				K	LYS	Lysine	Lysine
L	LEU	Leucine	Leucine (leucine)
				M	MET	Methionine	Methionine
N	ASN	Asparagine	Asparagine derivatives
				P	PRO	Proline	Proline (proline)
Q	GLN	Glutamine	Glutamine
				R	ARG	Arginine	Arginine (Arg)
S	SER	Serine	Serine (serine)
				T	THR	Threonine	Threonine (Thr)
V	VAL	Valine	Valine (valine)
				W	TRP	Tyrptophan	Tryptophan
Y	TYR	Tyrosine	Tyrosine

The invention provides a bioactive fragment or derivative, which is characterized by comprising any polypeptide sequence as a core sequence, wherein the core sequence comprises a mixture of covalently linked compounds and a polymer consisting of the core sequence.

Preferably, the biologically active fragment or derivative has the same biological function as the polypeptide of claim 1, i.e. also has the effect of specifically targeting human liver cancer.

It is an object of the present invention to provide a polynucleotide sequence which codes for a polypeptide sequence comprising any one of SEQ ID NO.1-SEQ ID NO.9 as defined in claim 1 and an active fragment of a polypeptide as defined in claim 2 and derivatives thereof.

The invention further provides a polypeptide molecular probe for tumor diagnosis, which comprises polypeptides with the amino acid sequences of SEQ ID NO.1-SEQ ID NO. 9.

The invention further provides an imaging agent which comprises the polypeptide and the imaging agent or the radionuclide and is used for imaging and diagnosing clinical tumors.

The invention also provides a pharmaceutical composition, which comprises the polypeptide with the amino acid sequence of SEQ ID NO.1-SEQ ID NO.9 as a targeting peptide, and can be used as a medicament for tumor treatment, a target head increasing medicament or a medicament-carrying carrier such as a nano material, a liposome and the like.

The pharmaceutical composition provided by the invention also comprises a carrier which is conjugated or mixed with the polypeptide and can prepare a targeting drug.

Further, the carrier is natural high molecular material and synthetic molecular polymer and their mixture widely used in pharmacy at present.

Preferably, the preparation is any one of alkylating agent, antimetabolite, natural antineoplastic medicine, hormone, antitumor antibiotic, metal complex or tumor target.

More preferably, the preparation is any one of chemical drugs, biological drugs, nano-drugs, radiopharmaceuticals, photodynamic therapy or photothermal therapy drugs or carriers for encapsulating the drugs, which can kill tumor cells.

The invention also provides any pharmaceutically acceptable dosage and dosage form of the above pharmaceutical composition.

The invention also provides application of the polypeptide with the amino acid sequence of SEQ ID NO.1-SEQ ID NO.9 and bioactive fragments or derivatives thereof in preparing medicaments or imaging preparations for preventing, treating or diagnosing tumors.

The applicable tumor range is any one of liver cancer, lung cancer, colorectal cancer, pancreatic cancer, breast cancer and ovarian cancer, wherein the liver cancer is the first choice.

The invention utilizes phage display technology to screen polypeptide specifically combined with human liver cancer, identifies specificity and affinity thereof, and lays foundation for developing clinical liver cancer diagnosis reagent and targeted therapeutic drug.

The invention has the advantages that:

(1) The small molecular polypeptide medicine is easier to permeate into tissues, and has the advantages of less dosage, stronger selectivity, better specificity, better effect and smaller side effect.

(2) The chemical synthesis technology of the small molecule polypeptide drugs is mature, the operation flow is simple and convenient, mass production is easier to realize, and the production cost is low.

(3) The polypeptide has the effect of specifically targeting tumor cells, can be widely applied to the fields of drug screening, vaccine and research and development of diagnostic reagents, and has a great potential clinical application prospect.

Drawings

FIG. 1 is a graph showing the results of three rounds of differential screening experiments of phage display technology on liver cancer cell-specific binding positive phage clones: a is the titration number of the eluent after three rounds of phage panning; b is a three-cycle phage panning eluate drop-size blue-white plate.

FIG. 2 is a graph showing the results of ELISA to identify the OD ₄₀₅ of the No. 1-30 positive phage clone with the affinity of the human hepatoma cell HepG 2.

FIG. 3 shows the DNA sequencing results of 30 positive phage clones. A is a positive phage clone DNA sequencing statistical table; b is a DNA sequencing map of the positive phage clone with the highest repetition rate.

FIG. 4 is a graph showing the result of fluorescence labeled polypeptide FITC-QS in targeted binding with liver normal epithelial cell Hl-7702 and liver cancer cell HepG 2. A is a confocal result diagram of the combination of FITC-QS and liver normal epithelial cells Hl-7702 and liver cancer cells HepG 2; b is a statistical histogram of relative fluorescence intensity.

Detailed Description

The technical scheme of the invention is further described by the following specific examples.

Example 1 three rounds of differential screening of liver cancer cell-specific binding positive phage clones Using phage display technology

1.1 Recovery and cultivation of host E.coli ER2738

Preparing an escherichia coli flat plate, taking an LB-TET culture plate, preheating the LB-TET culture plate in a incubator at 37 ℃ for 1 hour, dipping a small amount of bacterial liquid in an inoculating loop after E.coli ER2738 bacterial liquid is melted, uniformly spreading the bacterial liquid on the culture plate, and then pouring the bacterial liquid into a constant-temperature incubator at 37 ℃ for overnight culture. A host bacterial solution is prepared, a single colony selected from a culture plate with good growth is placed into LB bacterial culture solution containing tetracycline, the culture is carried out at 37 ℃ and 180rpm for overnight shaking, and the shaking is stopped when the bacteria are in the logarithmic phase. Preparing LB-Tet plate containing Escherichia coli, storing in refrigerator at 4deg.C, and storing host bacterial liquid in refrigerator at-80deg.C.

1.2 In vitro subtractive screening of phage display dodecapeptide libraries

Human liver cancer cell HepG2 is used as target cell, and human normal liver epithelial cell Hl-7702 is used as adsorption cell. In vitro screening was performed 3-round in vitro using phage display technology in vitro screening for Ph.D. -12 ^TM PHAGE DISPLAY PEPTIDE Library from Neurolim Biotechnology Co., ltd (NEW ENGLAND Biolabs). And ensuring the same total input amount of phages in each round as much as possible, increasing the screening pressure in each round, and finally obtaining highly enriched phages. The specific screening steps are as follows:

1.2.1 preparation of cells for screening

Selecting a well-grown human liver cancer cell HepG2 and a human normal liver epithelial cell Hl-7702. Trypsin is added into a culture dish to digest cells respectively, pancreatic enzymes are sucked off when the cells are round and lighten under a microscope, a cell culture medium containing serum is added to stop digestion, and a gun is used for lightly blowing the cells to suspend the adherent cells. The two cell suspensions are respectively added into a prepared culture dish treated by polylysine, the culture dish is put into a cell incubator, and the culture is carried out until the cells are attached to the wall, and the screening is carried out when the cell growth state is good.

1.2.2 Preparation of bacterial liquid

The host bacterial liquid cultured overnight on the day of screening was added to the LB medium. Shaking at 180rpm at 37℃for about 3 hours. The bacterial liquid used for screening titer measurement and amplification was used as described above.

1.2.3 Blocking cells

The liquid in the dish was sucked up with a suction tube and the liquid in the dish was thrown off on a sterile filter paper. Blocking with 0.5% BSA and standing at 37℃for 1 hour.

1.2.4 Peptide library binding

The blocking solution in the dish was discarded. Add Ph.D. -12 ^TM original phage display dodecapeptide library diluted 100-fold with TBST and slightly shake at 120rpm at room temperature for 1 hour.

1.2.5 Washing

The first round of unbound phage solution was aspirated, and the dish was placed on sterile filter paper and vigorously patted dry. The dishes were washed 10 times with 0.1% TBST for about 30 seconds each time, the bottom and the edges of the dishes were rinsed, the wash was poured off, and the dishes were patted dry on sterile filter paper (note that a new piece of clean filter paper was replaced after each wash to prevent cross-contamination).

1.2.6 Elution

Eluent (0.2M Glycine-HCl, pH 2.2) was added to the dish and the dish was gently shaken at 80rpm for 15min at normal temperature. After the elution is finished, the eluent is gently blown and sucked out, and then added into an EP tube containing the neutralization buffer solution, and the mixture is uniformly mixed.

1.3 Determination of phage titers

Shaking bacteria in advance, adding TET to LB, 37 deg.180 rpm, and after 3 hr, it reaches logarithmic growth phase (OD value is about 0.6). LB/IPTG/Xgal plates were pre-heated at 37℃for more than 1 hour prior to titer determination. Agarose gel was prepared and heated to melt with microwaves. The phage was diluted with LB medium fold by taking the post-adsorption neutralization eluate (or post-amplification phage supernatant). The dilution range is: diluting the neutralization eluent by 10 ²-10⁵ after adsorption; phage supernatant was diluted 10 ⁹-10¹² after amplification. Each EP tube was filled with the prepared bacterial solution, and 10. Mu.l of phage solution at different dilution factors was added to each tube. And (5) oscillating for 5min on an oscillator for uniform mixing. Phage solution after infection was added to room temperature thawing agar, and the suspension was immediately added to the pre-warmed LB/IPTG/Xgal plates and spread evenly with cooled coated glass rods (one plate per dilution and labeled). The coated plates were placed in an incubator at 37℃overnight for incubation. The phage blue spot growth on the plates was checked the following day and counted.

1.4 Phage amplification purification

The remaining adsorbed eluate was subjected to amplification purification for subsequent screening. Taking a sterile centrifuge tube, and inoculating the prepared overnight host bacteria into an LB culture medium to form a pre-logarithmic host bacteria liquid. All the eluate after adsorption was added to the pre-log host bacterial solution and amplified by rapid shaking at 200rpm at 37℃for 5 hours. The amplified phage solution was centrifuged at 12000rpm at 4℃for 10min, and the supernatant was transferred to another new centrifuge tube and centrifuged again under the same conditions. Carefully taking 80% of supernatant in the upper part of the centrifuge tube into a new centrifuge tube, adding one sixth of the volume of PEG/NaCl into the centrifuge tube, and standing at 4 ℃ for overnight precipitation. The next day, 50ml tubes from the previous day were centrifuged at 12000rpm at 4℃for 15min, the supernatant was discarded, and the remaining supernatant was discarded after centrifugation at the same conditions for 2 min. The pellet was resuspended in 1ml TBS and transferred to a sterile EP tube and centrifuged at 14000rpm at 4℃for 5min. The supernatant was transferred to a fresh EP tube, and one sixth of the volume of PEG/NaCl was added again and precipitated on ice for 1 hour. Centrifuging at 14000rpm at 4deg.C for 10min, discarding supernatant, and retaining precipitate. The pellet was resuspended with 200. Mu.l of TBS, centrifuged at 1000rpm for 1min at 4℃and the supernatant was kept in a fresh EP tube (this is the phage solution after amplification) and stored in a refrigerator at 20 ℃.

1.5 Second to third round screening

The basic steps of screening are the same as in the first round. In each next round of screening, phage liquid amplified in the previous round is selected as a secondary peptide library, and phage input amount basically consistent with that in the first round is kept as much as possible in each round, and three rounds of subtractive screening are performed in total. The phage titer was measured for each round of the eluate after screening, and the phage recovery was calculated.

The results are shown in figure 1, and figure 1 is a diagram of the results of three rounds of subtractive screening experiments for specifically binding positive polypeptides to liver cancer cells by using phage display technology; wherein A is phage titer of each round of eluent for three rounds of screening; b is a plate diagram of the liquid droplet size of the elution liquid of three rounds of phage panning, and the results show that the titration numbers of positive phage eluents capable of specifically binding to liver cancer cells after each round of panning are respectively as follows: 4.6X10 ⁵pfu/ml、9.8*10⁶pfu/ml、1.2*10⁸ pfu/ml, it was seen that positive phage clones capable of specifically binding to liver cancer cells after three rounds of panning were significantly enriched.

Example 2 ELISA detection of affinity of positive phage clones to hepatoma cells

2.1 Purification of positive phage clones

2.1.1 Amplification of positive phages: 20ml of LB/Tet liquid culture medium is added into a conical flask, then coliform bacteria liquid and phage to be amplified are added according to a ratio of 1:100, and the mixture is placed at 37 ℃ and vigorously oscillated for 4.5 hours in a constant-temperature oscillator, so that phage amplification liquid is obtained.

2.1.2 Purification of positive phages: centrifuging phage amplification liquid obtained through the steps at 4 ℃ for 10min at 12000r/min, adding 1/6 volume of PEG-NaCl to precipitate overnight after taking supernatant, centrifuging for 15min at 12000r/min, discarding supernatant, dissolving precipitate with TBS buffer, adding 1/6 volume of PEG-NaCl again, and incubating on ice for 1h. Centrifuging at 4 ℃ for 15min at 14000r/min, discarding supernatant, dissolving the obtained precipitate with TBS-NaN3, and storing in a 4 ℃ refrigerator.

2.2 ELISA (enzyme-linked immunosorbent assay) for detecting affinity

2.2.1 Preparation of cell 96-well plates, plating rules: two columns of 16 wells at the edge of the 96-well plate were each filled with 100 μl×pbs as a blank; then, each of the 1, 2, 3 and 4 rows of small holes is paved with 100 mu human normal liver epithelial cell Hl-7702 suspension according to snakelike shape, each of the 5, 6, 7 and 8 rows of small holes is paved with 100 mu human liver cancer cell HepG2 suspension according to snakelike shape, and the paved cell plate is placed in a 37 ℃ cell constant temperature incubator with 5% CO2 for overnight, so that ELISA experiments can be carried out.

2.2.2 Fixation: the 96-well plate with the cells spread overnight was removed, and after the liquid in the well was dried, washed 3 times with PBS, and then fixed with 4% paraformaldehyde for 20min.

2.2.3 Blocking: taking out the fixed 96-well plate, taking out the liquid in the well, washing with PBS for 3 times, adding 3% hydrogen peroxide, and sealing in a 37 ℃ cell constant temperature incubator for 30min to block the activity of endogenous peroxidase.

2.2.4 Blocking: taking out the blocked 96-well plate, taking out the liquid in the well, washing 3 times by using PBS, adding 3% BSA/PBS into a 37 ℃ cell constant temperature incubator, and sealing for 1h.

2.2.5 Phage samples: taking out the closed 96-well plate, beating the liquid in the well, adding 20 positive phages obtained by purification, and reacting for 1h in a 37 ℃ cell constant temperature incubator.

2.2.6 Adding primary antibody: taking out the reacted 96-well plate, beating the liquid in the well, washing 3 times with PBS, adding 1:4000M 13 antibody, and standing at 4 ℃ overnight.

2.2.7: Taking out the reacted 96-well plate, taking out the liquid in the well, washing 3 times by using PBS, adding secondary antibody, and reacting for 30min in a 37 ℃ cell constant temperature incubator.

2.2.8 Addition of substrate TMB: adding TMB display agent into the 96-well plate washed for 3 times by PBS under the light-proof condition, and placing the 96-well plate in a 37 ℃ cell constant temperature incubator for 15min in a light-proof mode.

2.2.9 Termination: the 96-well plate after the reaction was removed, and 2M sulfuric acid was added to terminate the reaction.

2.2.10 Determination of the results: the 96-well plate with all the reactions completed was placed in an enzyme-labeled instrument, its OD was measured at 405nm, and the results were saved and analyzed.

As shown in FIG. 2, the average absorbance value OD ₄₀₅ of the liver cancer cells HepG2 of the experimental group is obviously enhanced compared with the average absorbance value OD ₄₀₅ of the normal liver epithelial cells Hl-7702 of the control group. The positive phage clone can be specifically combined with liver cancer cell HepG2, and has weaker combination effect with liver normal epithelial cell Hl-7702.

EXAMPLE 3 assay of the DNA sequence of positive phage clones

3.1. Positive monoclonal phage selection

The phage solution obtained after the third round of screening was subjected to titer measurement. LB plates were prepared, and 30 well-grown blue spots were randomly picked on plates with less than 100 growing spots at 5mm intervals. The 30 randomly picked blue spots were added to 1ml of the pre-logarithmic host bacterial liquid (with phage amplification), and the mixture was rapidly shaken at 37℃and 200rpm for 4.5 hours for amplification.

3.2 Extraction of Single-stranded DNA from Positive monoclonal phages

The amplified monoclonal phage liquid is respectively centrifugated at 4 ℃ and 14000rpm for 30 seconds, the supernatant is transferred to a new tube, the supernatant is centrifugated at 4 ℃ and 1000rpm for 30 seconds, 80 percent of the supernatant is transferred to a new nuclease-free centrifuge tube, 300ul of the bacterial liquid is added with 300ul of glycerol according to the ratio of 1:1, and the mixture is frozen and stored in a refrigerator at-20 ℃, thus obtaining the amplified monoclonal phage liquid. Adding 200ul PEG into the remaining 500ul bacterial liquid, uniformly mixing, standing at room temperature for 20min, centrifuging at 4 ℃ for 14000r and 10min, discarding the supernatant, centrifuging at 4 ℃ for 3min at 14000rpm, discarding the supernatant, adding 100ul NaI, uniformly mixing, adding 250ul absolute ethanol, standing for 10min, centrifuging at 4 ℃ for 10min at 10000rpm, discarding the supernatant, slightly washing with precooled 70% ethanol for 3 times, airing for 30min, centrifuging at 10000rpm for 5min, discarding the supernatant, and adding 60ul TE.

3.3 DNA purification

Taking the 60ul TE pipe in the last step, adding 40ul TE to complement to 100ul. 500ul Buffer B3 is added into the centrifuge tube and fully and uniformly mixed. The mixture was transferred to an adsorption column, 8000g was centrifuged at room temperature for 30 seconds, and the filtrate was again added to the adsorption column and passed through the column again. The liquid in the collection tube is poured out, and the adsorption column is put back into the collection tube. 500 mu l WashSolution g, 9000g was added to the column and centrifuged for 30 seconds. The liquid in the collection tube is poured out, and the adsorption column is placed in the collection tube again. The above procedure was repeated, and the empty adsorption column and collection tube were put into a centrifuge, 9000g, and centrifuged for 1min. 40. Mu.l of an adsorption Buffer was added to the center of the adsorption film, and the mixture was allowed to stand at room temperature for 2 minutes. 9000g was centrifuged for 1min, and the resulting DNA solution was sequenced.

3.4 Determination of DNA sequence and analysis

The extracted single-stranded DNA is sequenced and the corresponding amino acid sequence is translated according to the triplet codon principle. Translating the polypeptide into a short peptide sequence by DNAstar software, analyzing the structure, and applying NCBI Blast to compare homology with a known protein polypeptide sequence; the GeneBank, swiss-prot protein database performs a similarity analysis on nucleotide sequences.

The results are shown in FIG. 3, wherein A is the 9 sequences and the repetition times screened from the DNA sequencing results, 30 positive monoclonal phages are totally detected, wherein SEQ ID NO.1 with the highest repetition times is 7 times, other sequences are respectively 5 times of SEQ ID NO.2, 4 times of SEQ ID NO.3, 4 times of SEQ ID NO.4, 3 times of SEQ ID NO.5, 3 times of SEQ ID NO.6, 2 times of SEQ ID NO.7, 1 time of SEQ ID NO.8, 1 time of SEQ ID NO.9, and the 9 sequences are compared with known protein polypeptide sequences to have no homology with the known protein polypeptide sequences and have no similarity with nucleotide sequences. B is a DNA sequencing waveform of the positive phage clone with the highest repetition rate.

Example 4 confocal detection of FITC-Positive polypeptide fragment FITC-QS specific targeting binding Capacity to liver cancer cells

According to the determined amino acid sequence, preparing fluorescent labeled polypeptide FITC-QS by adopting a solid phase synthesis method, purifying by adopting a C18 reverse phase preparation column, and freeze-drying to obtain white solid, namely a product. HepG2 and Hl-7702 cells were plated in 24 well plates with slides, and the cells were incubated in incubator to adhere the cells and confluent with monolayers. After 24 hours, the medium was discarded and washed 3 times with 5min each with PBS. 4% paraformaldehyde was fixed for 10min. PBS was washed 3 times, each for 5min. Incubation was performed with PBS containing 0.5% Triton X-100 for 10min. PBS was washed 3 times, each for 5min. Blocking with 3% BSA, and standing at room temperature for 15min. 5. Mu.M FITC-QS was added thereto, and the mixture was allowed to stand at 37℃for 15 minutes. PBS was washed 3 times, each for 5min. Adding DAB dye liquor, standing at 37 ℃ for 15min. And taking out the glass slide in the 24-hole plate, reversely buckling the glass slide with the anti-fluorescence quenching sealing tablet, positioning the position of the FITC-QS on the cells by using a confocal laser microscope, and identifying the specific targeting effect of the liver cancer cells.

The results are shown in FIG. 4, which is a graph of the result of the cell immunofluorescence of FITC-QS in targeted binding with liver normal epithelial cells Hl-7702 and liver cancer cells HepG 2. Wherein A is a graph of copolymerization Jiao Shice, and as a result, it was found that a strong fluorescent signal was observed on hepatoma cell HepG2, while almost no fluorescent signal was detected on hepatonormal epithelial cell Hl-7702. B is a relative fluorescence intensity histogram, and compared with normal epithelial cells, the binding capacity of the polypeptide FITC-QS and liver cancer cells is obviously enhanced, and the polypeptide FITC-QS has statistical significance, wherein P is less than 0.001.

Sequence listing

<110> Liaoning medical science and technology research and development center Limited

<120> A novel polypeptide specifically targeted to tumor cells and use thereof

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Artificial sequence

<400> 1

Gln Thr Asn Tyr Thr Ser Val His Met Gly Ser Ser

1 5 10

<210> 2

<211> 12

<212> PRT

<213> Artificial sequence

<400> 2

Asn Ser Gln His Pro Ser Leu Tyr Val Trp Ala Ser

1 5 10

<210> 3

<211> 12

<212> PRT

<213> Artificial sequence

<400> 3

Asp Pro Gln His Ser Ser Leu Tyr Val Ser Ser Ala

1 5 10

<210> 4

<211> 12

<212> PRT

<213> Artificial sequence

<400> 4

Gln Thr Asn Tyr Thr Ser Val His Met Ala Ser Ser

1 5 10

<210> 5

<211> 12

<212> PRT

<213> Artificial sequence

<400> 5

His Thr Ala Ser Tyr Tyr Ser Ala Val Gly Ser Ala

1 5 10

<210> 6

<211> 12

<212> PRT

<213> Artificial sequence

<400> 6

Tyr Pro Ser Ala His His Ser Leu Met Arg Pro Ala

1 5 10

<210> 7

<211> 12

<212> PRT

<213> Artificial sequence

<400> 7

Met Pro Ser Met Gly His Ala Ala Ile Ala Pro Asn

1 5 10

<210> 8

<211> 12

<212> PRT

<213> Artificial sequence

<400> 8

Val Ser Ala Val Trp Tyr Ser Ala Leu Ser Thr Gly

1 5 10

<210> 9

<211> 12

<212> PRT

<213> Artificial sequence

<400> 9

Asp Pro Asn His Ala Ala Val His Met Gly Ala Ala

1 5 10

Claims

1. A targeting polypeptide specifically binding to human hepatoma cells, wherein the amino acid sequences are:

SEQ ID NO.1：QTNYTSVHMGSS；

SEQ ID NO.2：NSQHPSLYVWAS；

SEQ ID NO.3：DPQHSSLYVSSA；

SEQ ID NO.4：QTNYTSVHMASS；

SEQ ID NO.5：HTASYYSAVGSA；

SEQ ID NO.6：YPSAHHSLMRPA；

SEQ ID NO.7：MPSMGHAAIAPN；

SEQ ID NO.8：VSAVWYSALSTG；

SEQ ID NO.9：DPNHAAVHMGAA。