CN105311617B

CN105311617B - Application of bullacta oligopeptide in lung cancer resistance

Info

Publication number: CN105311617B
Application number: CN201410279283.8A
Authority: CN
Inventors: 马剑茵; 李云涛; 卜天
Original assignee: Zhejiang Ocean University ZJOU
Current assignee: Zhejiang Ocean University ZJOU
Priority date: 2014-06-20
Filing date: 2014-06-20
Publication date: 2020-11-24
Anticipated expiration: 2034-06-20
Also published as: CN105311617A

Abstract

The invention relates to an application of a bullacta oligopeptide in lung cancer resistance, in particular to an application of the bullacta oligopeptide in a lung cancer H1299 tumor cell resisting medicine. The amino acid sequence of the mud snail oligopeptide is Gln-Pro-Pro-Gly-Leu, mud snails are used as raw materials, the mud snail tissues are subjected to enzymolysis through trypsin, and the mud snail tissues are separated and purified by combining gel chromatography and high performance liquid chromatography. The bullacta exarata oligosaccharide can obviously inhibit lung cancer H1299 tumor cells, wherein the proliferation inhibition rate of the H1299 cells can reach 21.3-74.3%, the influence rates on early apoptosis and late apoptosis of the lung cancer H1299 cells are 15-19.74% and 33-36.43%, and the bullacta exarata oligosaccharide has obvious anti-tumor activity. The bullacta oligopeptide has good development prospect, and the research on the anti-lung cancer H1299 tumor cells of the bullacta oligosaccharide can provide theoretical support for the development and research of anti-lung cancer drugs.

Description

Application of bullacta oligopeptide in lung cancer resistance

Technical Field

The invention relates to application of mud snail enzymolysis oligopeptides, in particular to application of mud snail oligopeptides in anti-lung cancer drugs.

Background

Mud snails (Bullacta exarata), commonly known as "iron spitting", "yellow mud snails", "plum snails", and the like, belong to the phylum mollusca, class gastropoda, class epigillidae, order tenotenocephalia, family ardisiaceae, genus mud snails, are specialty species of seawater and brackish water in the western pacific, are composed of shells and soft bodies, contain rich components such as protein, calcium, phosphorus, iron, vitamins and the like, have rich oligopeptide substances, are widely distributed in the Yangtze river on both sides of the east sea and the yellow sea in China, and are common aquatic products.

Malignant tumor is a disease which seriously threatens human health and life, the incidence rate is higher and higher, and the existing chemotherapy drugs have certain curative effect on most tumors, but the problems of poor selectivity, large toxic and side effects, drug resistance and the like are still very obvious. Therefore, the search for highly effective, low-toxicity and specific antitumor drugs is still imperative. The oligopeptide has small molecular weight, no immunogenicity, high activity and small side effect, and particularly has good inhibitory activity on multi-drug resistant tumor cell lines. Marine oligopeptides are an important area of research in marine bioactive substances, and some bioactive oligopeptides are often present in inactive state in proteins which are hydrolyzed by protease to be hidden in the proteinsThe active peptide in (1) is released, and more biological activity than protein is possibly exerted. For example: the phycocyanin enzymolysis oligopeptide has obvious inhibition effect on the in-vitro growth of the HeLa cell strain of the human cervical carcinoma as found in Dunwei and the like; mytilus edulis oligopeptides obtained by carrying out enzymolysis on Mytilus edulis such as Yangyongfang and the like have specific proliferation inhibition on prostate cancer PC-3 cells and DU-145 cells, and the inhibition on the DU-145 cells is stronger than the proliferation inhibition on the PC-3 cells; the oligopeptide content in tegillarca granosa in the research of Yao Yongyong is 0.25-1.0 g.L^-1Within the range, the oligopeptide can obviously inhibit the proliferation of lung cancer cells A549 and Ketr-3 cells, can also inhibit the synthesis of cell proteins, and has obvious dose dependence.

Lung cancer is one of the most serious malignant tumors threatening the health and life of human beings, and in recent years, the incidence and the mortality of lung cancer are both remarkably increased in many countries, the incidence and the mortality of lung cancer in men account for the first of all malignant tumors, the incidence of lung cancer in women accounts for the second and the mortality accounts for the second. The research on the inhibition effect of the bullacta exarata enzymolysis oligopeptide on lung cancer H1299 tumor cells has important significance on the research on the application of the bullacta exarata oligopeptide in anti-lung cancer drugs.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of the bullacta oligopeptide in the anti-lung cancer medicine, in particular the anti-lung cancer H1299 tumor cells, on the basis of obtaining the bullacta oligopeptide by enzymolysis of the bullacta tissue.

The technical scheme adopted by the invention for solving the technical problems is as follows: an application of mud snail oligopeptide in anti-lung cancer H1299 cell medicines, wherein the specific implementation form of the medicines can be tablets, capsules, granules, pills and the like.

The amino acid sequence of the mud snail oligopeptide is shown in SEQ ID NO. 1.

The mud snail oligopeptide is prepared by the following steps: cleaning and shelling fresh bullacta exarata, taking a bullacta exarata tissue, mashing, adding distilled water for homogenate, wherein the material-liquid ratio is 1 (3-4), adjusting the pH value of homogenate to 8-9 by using a hydrochloric acid solution and a sodium hydroxide solution, adding trypsin with the volume of 0.4-0.5% of the homogenate, carrying out heat preservation and hydrolysis at 40-50 ℃ for 7-9 h, carrying out enzyme inactivation at 95-100 ℃ for 10-15 min after the hydrolysis is finished, centrifuging the hydrolysate at 4 ℃ at 9500-10000 r/min for 15-20 min, taking supernatant, carrying out ultrafiltration by a 3kd ultrafiltration membrane to obtain a first component with the molecular weight of less than 3kd, carrying out gel chromatography elution on the first component to obtain a lowest peak to obtain a second component, and finally carrying out reverse phase high performance liquid chromatography on the second component to obtain the bullacta oligopeptide.

Preferably, the gel chromatography conditions are as follows: the concentration of the first component is 48-50 mg/mL, the sample loading amount is 3-4 mL, the speed is 2.5-3.5 mL/min, the mobile phase is ultrapure water, and 280nm ultraviolet detection is carried out.

Preferably, the reverse phase high performance liquid phase conditions are as follows: zorbax SB-C18(4.6 × 250, 5um) is selected; the column temperature is 20 ℃; mobile phase was 1% TFA and acetonitrile; gradient elution: from the beginning to the end of 30min, the acetonitrile concentration is changed from 0 to 40 percent, and the elution speed is 1.0 mL/min; the sample injection volume is 50ul, and the ultraviolet detection wavelength is 214nm and 280nm respectively.

Furthermore, after the 6-14 mg/ml mud snail oligopeptide acts on the H1299 cells for 24-36H, the proliferation inhibition rate of the lung cancer H1299 cells is 21.3-74.3%, and the proliferation inhibition rate is in positive correlation with the concentration of the mud snail oligopeptide.

Furthermore, after the 6-14 mg/ml mud snail oligopeptide acts on the H1299 cells for 24 hours, the influence rates on the early apoptosis and the late apoptosis of the lung cancer H1299 cells are 15-19.74% and 33-36.43%, respectively, and the proliferation inhibition rate is positively correlated with the concentration of the mud snail oligopeptide

Compared with the prior art, the invention has the advantages that: the method utilizes the bullacta exarata as a raw material to extract the bullacta exarata oligopeptide through enzymolysis, the bullacta exarata is a low-value shellfish and is widely distributed in China, the source is wide, the cost is low, and the method for extracting the bullacta exarata oligopeptide is mild and easy to control, the process is simple, and the operation is convenient. The bullacta exarata oligosaccharide can obviously inhibit lung cancer H1299 tumor cells, wherein the proliferation inhibition rate of the H1299 cells can reach 21.3-74.3%, the influence rates on early apoptosis and late apoptosis of the lung cancer H1299 cells are 15-19.74% and 33-36.43%, and the bullacta exarata oligosaccharide has obvious anti-tumor activity. The bullacta oligopeptide has good development prospect, and the research on the anti-lung cancer H1299 tumor cells of the bullacta oligosaccharide can provide theoretical support for the development and research of anti-lung cancer drugs.

Drawings

FIG. 1 is a gel chromatogram of the first component of example 1;

FIG. 2 is a high performance liquid chromatogram of the second component of example 1;

FIG. 3 is a line graph showing the relationship between the proliferation inhibition effect of the bullacta oligopeptide on lung cancer H1299 tumor cells and the action time and action concentration in example 4;

FIG. 4 is a bar graph of FIG. 3;

FIG. 5 is a graph of HE staining of H1299 cells after the mudsnail oligopeptides of example 4 are acted on, wherein A is 6mg/ml, B is 10mg/ml, C is 14 mg/ml;

FIG. 6 is a fluorescence image of AO/EB double staining of H1299 cells after the mudsnail oligopeptides with different concentrations in example 4 act, wherein A is a control group, B is 6mg/ml, C is 10mg/ml, and D is 14 mg/ml;

FIG. 7 is a graph showing the results of Annexin V-FITC/PI staining after various concentrations of bullacta oligopeptides in example 4 act on H1299 cells, A-1: control group, A-2:6mg/ml, A-3:10mg/ml, A-4:14 mg/ml.

Detailed Description

The invention is described in further detail below with reference to the accompanying examples.

Example 1: preparation of mud snail oligopeptide

(1.1) raw Material treatment

Taking fresh mud snails, removing shells of the mud snails, draining, and homogenizing for later use.

(1.2) enzymolysis process of mud snail

Mashing and homogenizing bullacta exarata tissues by using a high-speed tissue mashing machine, precisely weighing homogenate, adjusting the pH value by using 0.1mol/L hydrochloric acid solution and 0.1mol/L NaOH solution, adding trypsin for enzymolysis for hours, wherein the enzymolysis condition is that the enzymolysis temperature is 45 ℃, the pH value is 8.7, the material-liquid ratio is 1:4, the enzymolysis time is 8 hours, and the enzyme adding amount is 0.48%. Inactivating enzyme at 100 deg.C for 15min, centrifuging at 4 deg.C for 15min (10000r/min), and concentrating the supernatant.

(1.3) separation and purification of mud snail polypeptide

Firstly, selecting ultrafiltration methodSeparating mud snail polypeptide enzymatic hydrolysate, selecting 10kd, 5kd and 3kd by ultrafiltration membranes, respectively intercepting three components with molecular weights of 10-5kd, 5-3kd and less than 3kd, respectively preparing the components into concentrations of 20mg/mL after freeze drying, and determining the inhibition rate of each component on lung cancer H1299 by an MTT method. Selecting the first component with highest activity (molecular weight less than 3kd), performing Sephadex G-25 gel chromatography, loading into column, balancing with deionized water at concentration of 50mg/mL, loading sample of 4mL and speed of 3mL/min, detecting with mobile phase ultrapure water at 280nm ultraviolet, collecting the highest elution peak, freeze drying to obtain powder, detecting proliferation inhibition rate of H1299 cells, and drawing curve to obtain IC₅₀And collecting a large amount of the second component with the highest activity, freeze-drying and carrying out high performance liquid chromatography analysis to obtain the mud snail oligopeptide.

The lyophilized second component was dissolved in 0.06% TFA water in a 0.6ml centrifuge tube and centrifuged at 12000rpm for 10min to obtain the supernatant. High performance liquid phase conditions: zorbax SB-C18 (4.6X 250, 5 um); the column temperature is 20 ℃; mobile phase was 1% TFA and acetonitrile; gradient elution: from the beginning to the end of 30min, the acetonitrile concentration is changed from 0 to 40 percent, and the elution speed is 1.0 mL/min; the sample injection volume is 50ul, and the ultraviolet detection wavelength is 214nm and 280nm respectively. And (3) respectively collecting elution peaks A by using a high performance liquid chromatogram as shown in figure 2 to obtain the bullacta oligopeptide.

Example 2: determination of amino acid sequence of mud snail oligopeptide

The amino acid sequence analysis of the target polypeptide is determined by adopting an N-terminal amino acid degradation detection method. Establishing a standard amino acid map: and (3) operating under a conventional condition by using a mixed amino acid standard product (PTH-AA) to generate a standard product chromatogram, correcting the retention time of the mixed amino acid standard product, and generating a standard method file. Sample pretreatment: centrifuging the pure sample, and taking a supernatant for later use; 15ul of Polybrene (Polybrene) was applied to a Glass Fiber membrane (Glass Fiber Disk) and dried with nitrogen; and (3) pretreating the glass fiber membrane by a machine, namely, performing 5 cycles to add sufficient pure sample points on the pretreated glass fiber membrane, and drying by nitrogen. And (3) computer detection: and sealing the glass fiber membrane added with the sample in a reactor of a protein sequencer PPSQ-31A by using a PTFE filter membrane, and setting the number of detected amino acids and other parameters.

In this example, the mudsnail oligopeptide separated by high performance liquid chromatography was collected, and the amino acid sequence of the peptide was shown in SEQ ID NO.1 by detection.

Example 3: culture and passage of H1299 cells

(3.1) cell recovery

Taking out the stored H1299 cell strain from the liquid nitrogen tank, quickly putting the H1299 cell strain into a constant-temperature water bath kettle at 37 ℃ for thawing, putting the H1299 cell strain into a sterile working room for operation after thawing, sucking the cell strain into a centrifuge tube by using a sterilized straw, adding 2mL of F12 nutrient solution or RPMI1640 nutrient solution, centrifuging for 10min at 1000rpm, removing supernatant, adding 4mL of nutrient solution, and repeatedly blowing and beating the cell to form a single cell. Then, the cells were uniformly aspirated into 2 25mL culture flasks with a pipette, and 5% CO was added₂Culturing in a constant-temperature incubator at 37 ℃, and pouring off dead cells which are not attached to the wall by changing the culture solution the next day.

(3.2) cell culture

Human lung cancer cell H1299 was inoculated into F12 and 1640 nutrient solutions containing 10% fetal bovine serum (volume fraction) FBS and diabodies (penicillin G100IU/mL, streptomycin 100IU/mL), respectively, and placed at 37 ℃ in 5% CO₂Culturing in a constant-temperature incubator, carrying out adherent growth of cells, changing the liquid every 1 day, and carrying out passage when the cells grow to be about 80% of the culture bottle. The cells were passaged at a ratio of 1: 2, and cells in the logarithmic growth phase were collected for experiments.

(3.3) cell passaging

The culture flask full of cells is taken out from the constant temperature incubator and put on a sterile operation table. During passage, firstly pouring the nutrient solution in the bottle, removing dead cells growing without adherence, and carrying out mixed digestion by using 0.25% trypsin/0.02% EDTA digestive juice, wherein the digestion time of different cells is different, and the general digestion time is 3-5 min; when the cells are observed under a microscope and the cell gaps are obviously enlarged and the cells become round and bright, the cells are completely digested, and the digestive enzyme solution is removed. Adding about 2.5mL of nutrient solution into a culture bottle, repeatedly blowing and beating the digested adherent cells to form single cells, generally transferring one bottle of cells into 2 bottles, placing the passaged cells at 37 deg.C、5％CO₂The incubator of (2) for cultivation.

(3.4) cryopreservation of cells

Observing under a microscope, when the cells grow to about 80% of the culture bottle, selecting the cells with good shapes for cryopreservation, pouring out the culture solution, adding the digestive juice to digest the cells, pouring out the digestive enzyme when the intercellular space is observed to be obvious under the microscope, adding about 3mL of nutrient solution into the culture bottle, repeatedly blowing to make the cells become single cells, sucking the single cells into a centrifugal tube, centrifuging for 10min at 1000rpm, carefully sucking to remove the culture solution, adding 1mL of fetal calf serum containing 10% DMSO, repeatedly blowing the cells uniformly by using a suction tube, and sucking the cells into a sterile cryopreservation tube. Putting into a refrigerator at 4 deg.C for 30min, putting into a liquid nitrogen bottle mouth for 2h, and freezing.

Example 4: bullacta oligopeptide anti-lung cancer H1299 cell activity test

(4.1) Effect on inhibition of H1299 cell proliferation

Preparing suspension from cells in logarithmic growth phase, inoculating to 96-well plate with 200 μ L per well, setting 5 parallel wells in 5% CO₂And adhering the mud snail to the wall for 16-48h at 37 ℃, observing under an inverted microscope, discarding the culture solution, and simultaneously respectively dissolving the mud snail enzymolysis solution into the culture solution at different concentrations. Then, each well was filled with 5% CO, and a control group without the sample was placed in each well₂Incubation was carried out for 36h at 37 ℃ in an incubator, washed 2 times with PBS, and the culture was continued for 4h by adding a nutrient solution containing MTT. The culture was terminated and the culture medium in the wells was carefully aspirated. Adding dimethyl Acer Truncatum Bunge, shaking on a shaker at low speed for 10min, and measuring absorbance (OD value) at 490nm with enzyme-linked immunosorbent assay. The Inhibition Index (IR) of cell proliferation, i.e., the Inhibition rate of proliferation, was calculated according to the following formula:

the bullacta oligopeptides prepared in example 1 were set to 5 concentration groups of 6mg/mL, 8mg/mL, 10mg/mL, 12mg/mL and 14mg/mL, respectively. After 24 and 36 hours of action, MTT detection is carried out on the inhibition rate of H1299 cell proliferation. The results show that: after 36H of action, the proliferation inhibition rate of the bullacta oligopeptide on H1299 cells reaches 74.3 percent as shown in Table 1, and the proliferation inhibition rate on the H1299 cells is positively correlated with the concentration and the action time of the bullacta oligopeptide respectively as shown in figures 3 and 4.

TABLE 1 inhibition of H1299 cell proliferation by bullacta oligopeptide

)(n＝3)

Note: ﹡ P <0.05 compared to the minimal cytostatic rate of each fraction

(4.2) Effect on apoptosis of H1299 cells

(4.2.1) HE staining

Acidifying cover glass with polylysine for 24h, washing with tap water for 20 times, soaking in distilled water for 24h, drying, autoclaving, spreading in six-hole culture plate, digesting digestive juice 0.25% of cancer cells in culture bottle, sucking off digestive juice when cell gap is obvious and becomes round and bright, adding 10% fetal calf serum into nutrient solution, repeatedly blowing with dropper to reach single cell, adjusting cell concentration to 1 × 10 with culture solution⁵·mL^-1Left and right. Carefully inoculating the cells on a cover glass, dripping about 2mL of the cells into each hole, then putting the cover glass into an incubator for culture, and adding a drug group and a control group with different concentrations prepared by nutrient solution for culture when the cells on the cover glass grow to be about 80%.

After cell slide climbing is finished, the cover glass is carefully taken out, and is gently washed with PBS for 2 times, about 1min each time, then fixed with 95% alcohol for 15min, and gently washed with PBS for 3 times, 3min each time. Adding hematoxylin, dyeing for 8min, soaking in tap water for 30s, differentiating with hydrochloric acid and ethanol, and soaking in tap water. When the cell nucleus turns blue, the cell nucleus is re-stained with eosin stain for 30s, and then washed with tap water. Gradient-dehydrating with 50%, 75%, 95% and anhydrous ethanol, respectively, and removing xylene for 3 times (5 min each time). The stained slide is mounted with neutral resin, and the side with cells must be attached to the slide. The morphology was observed under an optical microscope and photographed.

As shown in FIG. 5, the lung cancer H1299 cells in the control group can observe regular cell morphology, uniform size, uniform and fine chromatin, and uniform nucleus size. After the bullacta exarata oligopeptide acts on H1299 cells for 24 hours, the B picture is the shape under the concentration of 6mg/mL, and is characterized in that the appearance of the cells is changed, the cell membranes become irregular, the cell nucleus is stained more densely, and the cytoplasm is slightly concentrated. The C picture is the cell under the drug concentration of 10mg/mL, the cell nucleus is obviously solidified and condensed, vacuole appears, the cell membrane becomes irregular further than the B picture, the cytoplasm is stained into a clot shape, and the cell has the small body status of apoptosis; the D picture is the cell morphology under the drug concentration of 14mg/mL, and the cell has obvious apoptosis characteristics, irregular cell, cell membrane shedding and the like.

(4.2.2) AO/EB fluorescent staining

Taking lung cancer H1299 tumor cells in logarithmic growth phase, adding a proper amount of pancreatin for digestion, preparing suspension by using culture solution containing 10% fetal calf serum, tiling a cover glass on a 6-hole cell culture plate, adding 2mL of cell suspension into each hole, placing the cell plate at 37 ℃, and placing the cell plate at 5% CO₂Culturing in the incubator until the cells grow to about 80% of the glass slide, and taking out. Adding the liquid medicine into each hole, putting the cell plate into an incubator for culture, and taking out the cell culture plate after 24 hours. Taking out the glass slide, adding 1 drop of mixed fluorescent staining solution: 100 mu g/mL Acridine Orange (AO) and 100 mu g/mL olfactory Ethidine (EB) are mixed uniformly, dropped on a glass slide, pressed on a cover glass full of cells, and the shapes of apoptotic cells are observed under a fluorescence microscope.

The fluorescence staining method is to stain the mixed dye of Acridine Orange (AO) and Ethidium Bromide (EB) into different colors according to different nuclear structures and cell membrane integrality so as to distinguish whether the cells are apoptotic. FIG. 6 shows that A is normal H1299 cells without obvious apoptosis under a fluorescence microscope, and the cells are uniform in size and distribution. The B picture shows that after the medicine concentration is 6mg/mL and acts for 24 hours, the membrane structure of early apoptotic cells is relatively complete, the chromatin is green, but the nuclei begin to be condensed, and the C picture shows that the cell nucleus is AO stained and shows yellow green fluorescence, condensed and granular, is positioned at one side of the cell, the number of early apoptotic cells is increased, the cell nucleus is AO stained and shows yellow green fluorescence, and late apoptotic cells are also increased; the D picture shows that the chromatin of the high dose group (14mg/mL) fluoresces orange-red and is in a solid state.

(4.2.3) FCM analysis of apoptosis

The cultured H1299 cells are digested by using a mixed solution of 0.25% pancreatin and 0.02% EDTA, and the digested cells are washed for 1-2 times by using PBS, wherein the pancreatin digestion time is not too long so as to avoid causing false positive. Sucking the digested cells into a centrifuge tube, centrifuging at 4-6 deg.C (1200r/min) for 5min, removing supernatant, adding 400ul of binding solution, and gently blowing repeatedly to make the cells into single cells with cell concentration of 1 × 10⁶About cells/mL, adding 5ul of FITC staining solution into the cell suspension, mixing uniformly, incubating for 15min at 4-6 ℃ in the dark, then adding 10ul of PI staining solution, mixing lightly, incubating for 5min at 4-6 ℃, and immediately placing the treated cells into FACS for detection.

Flow Cytometry (FCM) can separate normal, necrotic and apoptotic cells, etc. in a sample. As shown in FIG. 7, the cells in each of the maps A-1, A-2, A-3, and A-4 were divided into four regions: the upper left quadrant represents mechanically damaged cells; the lower left quadrant represents normal cells; the upper right quadrant is late apoptotic or necrotic cells; the lower right quadrant represents early apoptotic cells. It can be seen that the early apoptosis and the late apoptosis of the H1299 cells are gradually increased along with the increase of the polypeptide concentration, and when the concentration of the bullacta oligopeptide is 14mg/mL, the early apoptosis is 19.74 percent, and the late apoptosis is 36.43 percent.

The main instruments and materials used in the above examples are as follows:

main instrument

Superclean ZHJM-C12090, Shanghai Zhicheng Analyzer manufacturing, Inc.;

forma3111 cell culture box, bio-technology ltd, hangzhou baocheng;

enzyme-linked immunosorbent assay (BIO-RAD, USA); hangzhou Baocheng Biotechnology Co., Ltd;

FACS Calbur flow cytometer, BD company;

OLYMPUS CKX41 inverted phase contrast microscope, OLYMPUS corporation, japan;

berle 680 plate reader (usa), bio-technology ltd, baocheng, hangzhou;

OLYMPUS microscope 500 ten thousand pixels Pro-MicroScan CCD, OLYMPUS corporation, Japan;

reagent

Bovine serum (Hangzhou Sijiqing bioengineering, ltd);

MTT (SIGMA corporation, usa);

f12 powder medium (SIGMA, usa);

RPMI1640 medium, Gibco;

DMSO; (SIGMA corporation, USA)

AO/EB dyeing Material Hao Tian Biotechnology Ltd

Annexin V-FITC/PI apoptosis kit; bebo biological Co Ltd

Penicillin, streptomycin; lukang pharmaceutical Co., Ltd

Cell line

Human lung cancer cell H-1299 was purchased from Shanghai cell biology cell Bank, Chinese academy of sciences.

Claims

1. The application of the bullacta oligopeptide in preparing the anti-lung cancer H1299 cell medicine is characterized in that: the amino acid sequence of the mud snail oligopeptide is shown in SEQ ID NO. 1.

2. The application of the bullacta oligopeptide of claim 1 in preparing anti-lung cancer H1299 cell medicaments, which is characterized in that: after 6-14 mg/ml of the bullacta oligopeptide acts on H1299 cells for 24-36H, the proliferation inhibition rate of the H1299 cells of lung cancer is 21.3-74.3%, and the proliferation inhibition rate is in positive correlation with the concentration of the bullacta oligopeptide.

3. The application of the bullacta oligopeptide of claim 1 in preparing anti-lung cancer H1299 cell medicaments, which is characterized in that: after 14mg/ml of the bullacta oligopeptide acts on H1299 cells for 24 hours, the influence rate on early apoptosis and late apoptosis of lung cancer H1299 cells is 19.74 percent and 36.43 percent respectively.