CN112500470A

CN112500470A - Polypeptide with tumor cell proliferation inhibition function and application thereof

Info

Publication number: CN112500470A
Application number: CN202011494425.4A
Authority: CN
Inventors: 贾静; 杨新燕; 晏沙沙; 王屹喆
Original assignee: Hangzhou Medical College
Current assignee: Hangzhou Medical College
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2021-03-16
Anticipated expiration: 2040-12-17
Also published as: CN112500470B

Abstract

The invention discloses a polypeptide with a function of inhibiting tumor cell proliferation and application thereof. The polypeptide is a Ku70 protein truncation, and the amino acid sequence is shown in SEQ ID No. 1. The invention separates polypeptide Ku70-C which can effectively inhibit tumor proliferation from Ku70 protein by using a mutation PCR method. In vitro anti-tumor activity detection shows that the modified protein shows good anti-tumor proliferation property, can be used for optimizing anti-tumor polypeptide and provides a model for developing anti-tumor drugs. The polypeptide Ku70-C only has 74 amino acid residues, and has the advantages of small molecular weight, low immunogenicity and good tissue permeability. Has certain application prospect in the field of medicine.

Description

Polypeptide with tumor cell proliferation inhibition function and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a polypeptide with a function of inhibiting tumor cell proliferation and application thereof.

Background

The traditional tumor treatment method mostly adopts operation, ray or chemical drug treatment means, has great side effect and easy relapse, and can not overcome the defects of great side effect, easy relapse and the like of tumor treatment drugs even in the emerging tumor immunotherapy. The search for safe and effective tumor-targeted therapeutic drugs is a problem to be solved urgently in the field of tumor therapy at present.

The polypeptide medicine has low immunogenicity, easy synthesis and modification and good tissue permeability, and has good application prospect in the treatment of tumor, cardiovascular diseases, immune diseases and other diseases. At present, more than 80 polypeptide drugs are on the market globally, and nearly 200 polypeptide drugs are developing preclinical tests.

Polypeptide drugs are generally composed of several to several tens of natural or unnatural amino acids, and can be obtained by natural product extraction, genetic recombination, chemical synthesis, and the like. In addition to direct application to disease treatment, polypeptide drugs also have other broad application fields, such as drug carrier delivery, molecular probe detection of disease, or as small molecule drug models, providing structural basis for the development of targeted small molecule drugs.

The Ku70 protein is formed by transcription and translation of XRCC6(X-ray repair complementary defective repair in Chinese hamse hamster cells 6) gene, and is closely related to the generation, the infiltration and the metastasis of tumor. Research shows that the expression of Ku70 in the junctional and malignant ovarian serous tissues is obviously higher than that of normal ovarian tissues and ovarian cystadenomas, the expression in the rectal cancer tissues is obviously higher than that of rectal mucosa, the expression of Ku70 in the cervical cancer, pancreatic cancer and other tissues is obviously higher than that of normal tissues, and the expression condition of the Ku70 is also related to the prognosis of lung cancer, prostate cancer and other tumors. The research of tumor molecular genetics provides a large amount of evidence that Ku70 is related to tumor susceptibility such as gastric cancer, liver cancer, childhood leukemia and the like, and shows that Ku70 may be involved in the tumorigenesis process.

The Ku70 protein has been studied as the most widely biological function, and is involved in the repair of DNA double strand breaks by constituting an heterodimer with the Ku80 protein. In addition to NHEJ-dependent biological functions, Ku70 was also found to have NHEJ-independent biological functions: ku70 can induce epithelial cell-mesenchymal transition (EMT) or promote tumor cell proliferation, and participate in tumor development; ku70 also binds to Bax protein, preventing Bax from inducing apoptosis.

The invention with publication number CN111333712A discloses a Ku70 protein mutant with the function of inhibiting tumor cell proliferation, a gene and application thereof. The amino acid sequence of the Ku70 protein mutant is shown in SEQ ID NO. 1. The invention constructs the variant Ku70 protein Mu4 which can effectively inhibit tumor proliferation by utilizing the influence of the Ku70 protein on the tumor cell proliferation and through artificial modification. In-vitro antitumor activity detection and animal experiments show that the modified protein shows good antitumor proliferation characteristics, can be used for researching the action mechanism of Ku70 participating in tumor cell proliferation, and provides a theoretical basis and a model for developing antitumor drugs. The mutant Ku70 protein Mu4 has obvious proliferation inhibition effect on lung cancer or cervical cancer cell lines, and has no obvious inhibition effect on liver cancer, colon cancer cell lines and non-cancer cell lines. However, in this application, Mu4 has a total of 572 amino acids, and the overall molecular weight is large and needs further improvement.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention separates a section of polypeptide from Ku70 protein, constructs an expression plasmid for expressing the polypeptide, can over-express the polypeptide in tumor cells in a liposome cell transfection way, and inhibits the proliferation of the tumor cells, thereby indicating that the polypeptide has potential drug development value.

A polypeptide with tumor cell proliferation inhibiting function is a Ku70 protein truncation, and has an amino acid sequence shown in SEQ ID No. 1. The polypeptide is a C-terminal truncated body of Ku70 protein, and comprises 74 amino acids at the C terminal.

The invention also provides the application of the polypeptide in preparing anti-tumor drugs. Preferably, wherein the tumor is lung cancer or cervical cancer.

The invention also provides a gene encoding the polypeptide. Preferably, the nucleotide sequence of the gene is shown as SEQ ID No. 2.

The invention also provides application of the gene in preparation of antitumor drugs. Preferably, wherein the tumor is lung cancer or cervical cancer. Preferably, the application is carried out by introducing the gene into cancer cells to express the polypeptide.

The invention separates polypeptide Ku70-C which can effectively inhibit tumor proliferation from Ku70 protein by using a mutation PCR method. In vitro anti-tumor activity detection shows that the modified protein shows good anti-tumor proliferation property, can be used for optimizing anti-tumor polypeptide and provides a model for developing anti-tumor drugs. The polypeptide Ku70-C only has 74 amino acid residues, and has the advantages of small molecular weight, low immunogenicity and good tissue permeability. Has certain application prospect in the field of medicine.

Drawings

FIG. 1 is a plasmid map of pBICEP-CMV-3.

FIG. 2 shows that wild-type Ku70 protein (pCMV-N-Flag-Ku70), Ku 70C-terminal polypeptide (pCMV-N-Flag-Ku70-C) and variant Ku70 protein (pCMV-N-Flag-Ku70-Mu-C) lacking the C-terminal were overexpressed in NCI-H1975, A549, HeLa and HCT-116 cells by lipofection, and after transfection for 48 hours, the proliferation rate of each group of cells was measured by the MTT method. In NCI-H1975, A549 and HeLa, the proliferation rate of the cell over-expressing Ku70-C polypeptide is obviously lower than that of the cell transfected with other two plasmids (FIG. 2.A-C), and in HCT-116 cell, the proliferation rate of the cell transfected with three different plasmids is not obviously different (FIG. 2. D).

Detailed Description

Example 1: ku70-C polypeptide expression vector pBICEP-N-Flag-Ku70-C clone construction targeting Skp2

(1) preparation of pBICEP-N-Flag-Ku70-C, pBICEP-N-Flag-Ku70-Mu-C expression vector

The expression vector pBICEP-N-Flag-Ku70-C for expressing the Ku70-C polypeptide (the amino acid sequence is shown as SEQ ID No.1, and the coding gene sequence is shown as SEQ ID No. 2) is obtained by amplifying a wild-type full-length Ku70 expression vector pBICEP-N-Flag-Ku70 (the full-sequence GenBank number of the gene of the invention application Ku70 protein with the publication number of CN111333712A is NM-001288977) by a mutation PCR method, and the expression vector pBICEP-N-Flag-Ku70-Mu-C and the comparison vector pBICEP-N-Flag-Ku70-Mu-C are used for expressing the variant Ku70 protein lacking the C terminal (the variant Ku70 protein lacking the C terminal is shown as Ku70-Mu-C, the amino acid sequence is shown as SEQ ID No.3, and the coding gene sequence is shown as SEQ ID No. 4).

The specific method for preparing the pBICEP-N-Flag-Ku70-C, pBICEP-N-Flag-Ku70-Mu-C expression vector comprises the following steps: synthesizing a mutation PCR primer, using pBICEP-N-Flag-Ku70 plasmid as a template by a mutation PCR method, amplifying to obtain DNA of a pBICEP-N-Flag-Ku70-C, pBICEP-N-Flag-Ku70-Mu-C expression vector, and obtaining a complete plasmid of the pBICEP-N-Flag-Ku70-C, pBICEP-N-Flag-Ku70-Mu-C expression vector for later use by methods of transforming escherichia coli, selecting positive clone and sequencing verification.

Mutation PCR primer:

Ku70-C：

an upstream primer:

5'-AATCGATATCTGCAGAATTCACGCGTCATGCCTGAAGGGAAAGTTACCAAGAGAAAACAC-3'；

a downstream primer:

5'-GTGTTTTCTCTTGGTAACTTTCCCTTCAGGCATGACGCGTGAATTCTGCAGATATCGATT-3'。

Ku70-Mu-C：

an upstream primer:

5'-AAGGAGCTTGTTTACCCACCAGATTACAATTGAGGATCCTCTAGATCTGTTAACTCCGGG-3'；

a downstream primer:

5'-CCCGGAGTTAACAGATCTAGAGGATCCTCAATTGTAATCTGGTGGGTAAACAAGCTCCTT-3'。

TABLE 1 mutant PCR reaction System

pBICEP-N-Flag-Ku70 vector	500ng
		Upstream primer (10mM)	1μl
DownstreamPrimer (10mM)	1μl
		Pfu DNA Polymerase	1μl
Pfu DNA Polymerase Reaction Buffer(10×)	5μl
		dNTP mix (10mM each)	1μl
Sterile Milli-Q Water	Water was added to 50. mu.l.

Mutation PCR reaction conditions:

pre-denaturation at 95 ℃ for 5 min;

denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 7min for 20 cycles;

extending for 10min at 72 ℃;

storing at 4 ℃.

PCR product purification (using Biomega PCR product purification kit, following kit instructions):

1. and adding 2 times volume of Buffer GC into the PCR reaction solution, uniformly mixing, performing instantaneous centrifugation, and collecting the solution to the bottom. Fragments of less than 200bp were purified and 5 volumes of Buffer GC were added to 1 volume of PCR reaction.

2. Transferring the mixed solution to an adsorption column with a collecting pipe, centrifuging at 13000g for 1 minute at room temperature, pouring the waste liquid in the collecting pipe, and returning the adsorption column to the collecting pipe. And (3) repeating the step (2) until the rest of the mixed solution passes through the adsorption column.

3. Add 650. mu.l of DNA Wash Buffer to the adsorption column, centrifuge at 13000g for 30s at room temperature, discard the collection tube, place the adsorption column back into the collection tube, repeat step "3".

4. 13000g at room temperature, the column was uncapped and centrifuged for 2 minutes to remove residual ethanol.

5. Transfer the adsorption column to a 1.5ml collection tube and add 30. mu.l ddH preheated at 60 ℃₂O is put on the center of the adsorption column membrane at room temperature for 1 minute, and centrifuged at 13000g for 1 minute.

TABLE 2 Dpn I digestion System

pBICEP-N-Flag-Ku70-C (or pBICEP-N-Flag-Ku70-Mu-C) vector	1μg
		Dpn I enzyme	1μl
Cut smart Buffer(10×)	5μl
		Sterile Milli-Q Water	Water was added to 50. mu.l.

The enzyme digestion system is subjected to enzyme digestion in a water bath kettle at 37 ℃ overnight and then inactivated at 65 ℃ for 10 min.

And (3) transforming escherichia coli: the Ku70-C polypeptide expression vector pBICEP-N-Flag-Ku70-C and the control vector pBICEP-N-Flag-Ku70-Mu-C were transferred into competent cells DH5 α and were preserved and propagated in E.coli DH5 α.

The method comprises the following steps:

1. taking 2 competent cell suspensions from a refrigerator at-70 deg.C, and thawing on ice

2. 1ul of each of the expression vector pBICEP-N-Flag-Ku70-C and the control vector pBICEP-N-Flag-Ku70-Mu-C was added, gently shaken, and left on ice for 30 minutes.

The mixture was heated in a water bath at 3.42 ℃ for 90 seconds and then rapidly placed on ice for 2 min.

4. 800ul of LB liquid medium was added to the tube, mixed well and cultured with shaking (225rpm) at 37 ℃ for 1 hour.

5. And shaking the bacterial liquid uniformly, coating the bacterial liquid on an LB agar plate containing Amp, placing the bacterial liquid for half an hour with the front side upward, and performing inverted culture at 37 ℃ for 12-16 hours after the bacterial liquid is completely absorbed by a culture medium.

6. The positive colonies growing on the plates were picked, cultured overnight in LB liquid medium (shaking culture at 37 ℃), and the cells were collected and sent to Protektionaceae sequencing for sequencing verification. And selecting a colony with a correct sequencing result, storing and extracting a plasmid for later use.

Example 2: detection of proliferation inhibition activity of Ku70-C polypeptide on lung cancer cell lines A549, NCI-H1975 and cervical cancer cell HeLa

(1) Culturing lung cancer cell line A549, NCI-H1975, cervical cancer cell HeLa and colorectal cancer cell HCT-116;

the cell culture medium is complete medium including RPMI-1640 medium (A549, NCI-H1975, HCT-116) and complete MDEM medium (HeLa). The culture conditions are as follows: the seeding density of each cell was 1X 10 per well⁵And inoculating the cells into a 96-well plate, culturing at 37 ℃ until the cells are 50-60% confluent.

(2) The liposome transfection method is respectively transferred into plasmids of pBICEP-N-Flag-Ku70-C, pBICEP-N-Flag-Ku70-Mu-C and pBICEP-N-Flag-Ku70 by a lung cancer cell line A549, NCI-H1975, a cervical cancer cell HeLa and a colorectal cancer cell HCT-116;

a549, NCI-H1975, HeLa and HCT-116 cells were lipofected when cultured to 50% -60% confluence in 96-well plates. Cell proliferation viability was measured 48 hours after transfection using the MTT method.

Liposome transfection was performed using Lipofectamin3000 from Invitrogen.

The transfection method is as follows:

1. dilution of

3000 reagent:

1 sterile 1.5ml of EP tube was selected and diluted to 0.2. mu.L using 5. mu.L of Opti-MEM medium

3000 reagent, fully and uniformly mixing the components,

2. dilution of the plasmid to be transfected:

a sterile 1.5ml EP tube was selected, and 0.1. mu.g of plasmid pBICEP-N-Flag-Ku70-C, pBICEP-N-Flag-Ku70-Mu-C and pBICEP-N-Flag-Ku70 were diluted with 5. mu.L of Opti-MEM medium to prepare DNA premixes, and 0.2. mu. L P3000 was added to each of the premixes^TMAnd (5) fully and uniformly mixing the reagents.

3. Preparation of DNA-liposome complexes:

diluted in each tube

The diluted DNA was added to the 3000 reagent at a ratio of 1: 1.

4. Transfection of cells:

the DNA-liposome complex was incubated at room temperature for 10-15 minutes. mu.L of the DNA-liposome complex was added to each well of the cells, and the cells were cultured in a carbon dioxide incubator at 37 ℃.

(3) After 48 hours of transfection, the MTT method examined the proliferation inhibition of each cell line.

MTT experiments were performed 48 hours after transfection: adding 20 mu L of MTT (5mg/ml) into each well, and continuing to incubate for 4 hours; the supernatant was aspirated, 150. mu.L of dimethyl sulfoxide was added thereto, the mixture was shaken for 5min, and the absorbance (OD value) was measured at a wavelength of 490nm using a microplate reader.

The cell proliferation condition is detected through an MTT experiment, an MTT substrate can be reduced into a bluish purple product by succinate dehydrogenase of mitochondria of living cells, and dead cells do not have the function; the more the living cells are, the stronger the proliferation activity rate is, and the darker the solution color is; the proliferation activity of the cells was deduced from this.

As shown in Table 3, the cell proliferation activities of A549, NCI-H1975 and HeLa cells transfected with pBICEP-N-Flag-Ku70-C expressing the Ku70-C polypeptide were significantly less than those of cells transfected with pBICEP-N-Flag-Ku70-Mu-C and pBICEP-N-Flag-Ku70 plasmids, and the cell proliferation inhibition rates were calculated as follows:

inhibition ratio 1 ═ OD490(Ku70) -OD490(Ku70-C))/OD490(Ku70) × 100%),

inhibition ratio 2 ═ ((OD490(Ku70-Mu-C) -OD490(Ku70-C))/OD490(Ku70-Mu-C) × 100%

The experimental operation of example 2 was repeated on the lung cancer cell lines A549, NCI-H1975, cervical cancer cell HeLa and colon cancer cell line HCT-116 to verify the inhibitory effect of the Ku70-C polypeptide on the proliferation of other tumor cells. The results are shown in Table 3.

TABLE 3 proliferation inhibition of A549, NCI-H1975, HeLa, HCT-116 cells by Ku70-C polypeptide

As shown in Table 3, the cells A549, NCI-H1975, HeLa and HCT-116 were transfected into pBICEP-N-Flag-Ku70-C, pBICEP-N-Flag-Ku70-Mu-C and pBICEP-N-Flag-Ku70 respectively, and the MTT detection results after 48H showed that the cell viability of the cells transfected with different plasmids was significantly different. The average value of OD490 of A549 cells over-expressing Ku70-C polypeptide is 1.18, which is smaller than that of A549 cells over-expressing Ku70 protein (the average value of OD490 is 1.51), the difference is significant (P <0.01), and the difference is not significant with that of A549 cells over-expressing Ku70-Mu-C polypeptide (the average value of OD490 is 1.34); after 48 hours of transfection, the proliferation inhibition rate of the Ku70-C polypeptide on a549 cells was 22% relative to a549 cells overexpressing Ku 70. NCI-H1975 cells overexpressing the Ku70-C polypeptide, which had an OD490 average of 0.67, were less significant (P <0.01) than NCI-H1975 cells overexpressing the Ku70 protein (OD490 average of 0.85), but did not differ significantly from NCI-H1975 cells overexpressing the Ku70-Mu-C polypeptide (OD490 average of 0.86); 48 hours after transfection, the Ku70-C polypeptide inhibited cell proliferation by 21% relative to NCI-H1975 cells which overexpress Ku 70. The average OD490 of the HeLa cell over-expressing the Ku70-C polypeptide is 1.08, which is smaller than that of the HeLa cell over-expressing the Ku70-C polypeptide (the average OD490 is 1.53), the difference is significant (P <0.01), and the difference is not significant with the HeLa cell over-expressing the Ku70-Mu-C polypeptide (the average OD490 is 1.24); 48 hours after transfection, the Ku70-C polypeptide inhibited cell proliferation by 29% relative to HeLa cells overexpressing Ku 70. The Ku70-C polypeptide has obvious proliferation inhibition on lung cancer and cervical cancer cells.

And the results of MTT detection after 48h of transfection of colon cancer HCT-116 cells transfected with pBICEP-N-Flag-Ku70-C, pBICEP-N-Flag-Ku70-Mu-C and pBICEP-N-Flag-Ku70 plasmid show that the cell viability of the cells transfected with different plasmids is not obviously different. It is shown that the cell proliferation level of colon cancer cells is probably not obviously inhibited by over-expressing Ku70-C polypeptide.

In conclusion, the Ku70-C polypeptide expression vector pBICEP-N-Flag-Ku70-C constructed by the inventor has obvious effect of inhibiting the proliferation of cervical cancer and lung cancer cell lines, and has no obvious effect of inhibiting the proliferation of colon cancer cell lines HCT-116 cells, so that the Ku70-C polypeptide has tumor specificity on the inhibition of the proliferation of tumor cells, and the inhibition of the lung cancer and the cervical cancer is found to be obvious at present.

Sequence listing

<110> Hangzhou college of medicine

<120> polypeptide with tumor cell proliferation inhibiting function and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 74

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Pro Glu Gly Lys Val Thr Lys Arg Lys His Asp Asn Glu Gly Ser Gly

1 5 10 15

Ser Lys Arg Pro Lys Val Glu Tyr Ser Glu Glu Glu Leu Lys Thr His

20 25 30

Ile Ser Lys Gly Thr Leu Gly Lys Phe Thr Val Pro Met Leu Lys Glu

35 40 45

Ala Cys Arg Ala Tyr Gly Leu Lys Ser Gly Leu Lys Lys Gln Glu Leu

50 55 60

Leu Glu Ala Leu Thr Lys His Phe Gln Asp

65 70

<210> 2

<211> 225

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cctgaaggga aagttaccaa gagaaaacac gataatgaag gttctggaag caaaaggccc 60

aaggtggagt attcagaaga ggagctgaag acccacatca gcaagggtac gctgggcaag 120

ttcactgtgc ccatgctgaa agaggcctgc cgggcttacg ggctgaagag tgggctgaag 180

aagcaggagc tgctggaagc cctcaccaag cacttccagg actga 225

<210> 3

<211> 535

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Ser Gly Trp Glu Ser Tyr Tyr Lys Thr Glu Gly Asp Glu Glu Ala

1 5 10 15

Glu Glu Glu Gln Glu Glu Asn Leu Glu Ala Ser Gly Asp Tyr Lys Tyr

20 25 30

Ser Gly Arg Asp Ser Leu Ile Phe Leu Val Asp Ala Ser Lys Ala Met

35 40 45

Phe Glu Ser Gln Ser Glu Asp Glu Leu Thr Pro Phe Asp Met Ser Ile

50 55 60

Gln Cys Ile Gln Ser Val Tyr Ile Ser Lys Ile Ile Ser Ser Asp Arg

65 70 75 80

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

85 90 95

Val Asn Phe Lys Asn Ile Tyr Val Leu Gln Glu Leu Asp Asn Pro Gly

100 105 110

Ala Lys Arg Ile Leu Glu Leu Asp Gln Phe Lys Gly Gln Gln Gly Gln

115 120 125

Lys Arg Phe Gln Asp Met Met Gly His Gly Ser Asp Tyr Ser Leu Ser

130 135 140

Glu Val Leu Trp Val Cys Ala Asn Leu Phe Ser Asp Val Gln Phe Lys

145 150 155 160

Met Ser His Lys Arg Ile Met Leu Phe Thr Asn Glu Asp Asn Pro His

165 170 175

Gly Asn Asp Ser Ala Lys Ala Ser Arg Ala Arg Thr Lys Ala Gly Asp

180 185 190

Leu Arg Asp Thr Gly Ile Phe Leu Asp Leu Met His Leu Lys Lys Pro

195 200 205

Gly Gly Phe Asp Ile Ser Leu Phe Tyr Arg Asp Ile Ile Ser Ile Ala

210 215 220

Glu Asp Glu Asp Leu Arg Val His Phe Glu Glu Ser Ser Lys Leu Glu

225 230 235 240

Asp Leu Leu Arg Lys Val Arg Ala Lys Glu Thr Arg Lys Arg Ala Leu

245 250 255

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

260 265 270

Ile Tyr Asn Leu Val Gln Lys Ala Leu Lys Pro Pro Pro Ile Lys Leu

275 280 285

Tyr Arg Glu Thr Asn Glu Pro Val Lys Thr Lys Thr Arg Thr Phe Asn

290 295 300

Thr Ser Thr Gly Gly Leu Leu Leu Pro Ser Asp Thr Lys Arg Ser Gln

305 310 315 320

Ile Tyr Gly Ser Arg Gln Ile Ile Leu Glu Lys Glu Glu Thr Glu Glu

325 330 335

Leu Lys Arg Phe Asp Asp Pro Gly Leu Met Leu Met Gly Phe Lys Pro

340 345 350

Leu Val Leu Leu Lys Lys His His Tyr Leu Arg Pro Ser Leu Phe Val

355 360 365

Tyr Pro Glu Glu Ser Leu Val Ile Gly Ser Ser Thr Leu Phe Ser Ala

370 375 380

Leu Leu Ile Lys Cys Leu Glu Lys Glu Val Ala Ala Leu Cys Arg Tyr

385 390 395 400

Thr Pro Arg Arg Asn Ile Pro Pro Tyr Phe Val Ala Leu Val Pro Gln

405 410 415

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

420 425 430

Gln Leu Val Phe Leu Pro Phe Ala Asp Asp Lys Arg Lys Met Pro Phe

435 440 445

Thr Glu Lys Ile Met Ala Thr Pro Glu Gln Val Gly Lys Met Lys Ala

450 455 460

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

465 470 475 480

Pro Val Leu Gln Gln His Phe Arg Asn Leu Glu Ala Leu Ala Leu Asp

485 490 495

Leu Met Glu Pro Glu Gln Ala Val Asp Leu Thr Leu Pro Lys Val Glu

500 505 510

Ala Met Asn Lys Arg Leu Gly Ser Leu Val Asp Glu Phe Lys Glu Leu

515 520 525

Val Tyr Pro Pro Asp Tyr Asn

530 535

<210> 4

<211> 1605

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgtcagggt gggagtcata ttacaaaacc gagggcgatg aagaagcaga ggaagaacaa 60

gaagagaacc ttgaagcaag tggagactat aaatattcag gaagagatag tttgattttt 120

ttggttgatg cctccaaggc tatgtttgaa tctcagagtg aagatgagtt gacacctttt 180

gacatgagca tccagtgtat ccaaagtgtg tacatcagta agatcataag cagtgatcga 240

gatctcttgg ctgtggtgtt ctatggtacc gagaaagaca aaaattcagt gaattttaaa 300

aatatttacg tcttacagga gctggataat ccaggtgcaa aacgaattct agagcttgac 360

cagtttaagg ggcagcaggg acaaaaacgt ttccaagaca tgatgggcca cggatctgac 420

tactcactca gtgaagtgct gtgggtctgt gccaacctct ttagtgatgt ccaattcaag 480

atgagtcata agaggatcat gctgttcacc aatgaagaca acccccatgg caatgacagt 540

gccaaagcca gccgggccag gaccaaagcc ggtgatctcc gagatacagg catcttcctt 600

gacttgatgc acctgaagaa acctgggggc tttgacatat ccttgttcta cagagatatc 660

atcagcatag cagaggatga ggacctcagg gttcactttg aggaatccag caagctagaa 720

gacctgttgc ggaaggttcg cgccaaggag accaggaagc gagcactcag caggttaaag 780

ctgaagctca acaaagatat agtgatctct gtgggcattt ataatctggt ccagaaggct 840

ctcaagcctc ctccaataaa gctctatcgg gaaacaaatg aaccagtgaa aaccaagacc 900

cggaccttta atacaagtac aggcggtttg cttctgccta gcgataccaa gaggtctcag 960

atctatggga gtcgtcagat tatactggag aaagaggaaa cagaagagct aaaacggttt 1020

gatgatccag gtttgatgct catgggtttc aagccgttgg tactgctgaa gaaacaccat 1080

tacctgaggc cctccctgtt cgtgtaccca gaggagtcgc tggtgattgg gagctcaacc 1140

ctgttcagtg ctctgctcat caagtgtctg gagaaggagg ttgcagcatt gtgcagatac 1200

acaccccgca ggaacatccc tccttatttt gtggctttgg tgccacagga agaagagttg 1260

gatgaccaga aaattcaggt gactcctcca ggcttccagc tggtcttttt accctttgct 1320

gatgataaaa ggaagatgcc ctttactgaa aaaatcatgg caactccaga gcaggtgggc 1380

aagatgaagg ctatcgttga gaagcttcgc ttcacataca gaagtgacag ctttgagaac 1440

cccgtgctgc agcagcactt caggaacctg gaggccttgg ccttggattt gatggagccg 1500

gaacaagcag tggacctgac attgcccaag gttgaagcaa tgaataaaag actgggctcc 1560

ttggtggatg agtttaagga gcttgtttac ccaccagatt acaat 1605

<210> 5

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aatcgatatc tgcagaattc acgcgtcatg cctgaaggga aagttaccaa gagaaaacac 60

<210> 6

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gtgttttctc ttggtaactt tcccttcagg catgacgcgt gaattctgca gatatcgatt 60

<210> 7

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aaggagcttg tttacccacc agattacaat tgaggatcct ctagatctgt taactccggg 60

<210> 8

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cccggagtta acagatctag aggatcctca attgtaatct ggtgggtaaa caagctcctt 60

Claims

1. A polypeptide with the function of inhibiting tumor cell proliferation is characterized by being a Ku70 protein truncated body, and the amino acid sequence is shown as SEQ ID No. 1.

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

3. The use of claim 2, wherein the neoplasm is lung cancer or cervical cancer.

4. A gene encoding the polypeptide of claim 1.

5. The gene of claim 4, wherein the nucleotide sequence is as shown in SEQ ID No. 2.

6. Use of the gene according to claim 4 or 5 for the preparation of an antitumor medicament.

7. The use of claim 6, wherein the neoplasm is lung cancer or cervical cancer.

8. The use of claim 6, wherein said polypeptide is expressed by introducing said gene into a cancer cell.