CN111484547B - Anti-tumor protein AEL1, fusion protein, anti-tumor preparation and application - Google Patents

Abstract

The invention relates to an anti-tumor protein AEL1, a fusion protein, an anti-tumor preparation and application thereof, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 1. The protein has anti-tumor activity and potential of becoming an anti-tumor medicament, and provides a new treatment idea for tumor treatment.

Description

Anti-tumor protein AEL1, fusion protein, anti-tumor preparation and application

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to an antitumor protein AEL1, a fusion protein, an antitumor preparation and application.

Background

Lung cancer is the leading cause of cancer death worldwide, the mortality rate in Chinese population is high and the first of all malignant tumors, lung adenocarcinoma among them is invasive due to its biological behavior and lacks specific symptoms and signs in early stage, most patients are already in advanced stage when diagnosed and can not be resected by surgery, so that the 5-year survival rate of lung adenocarcinoma is only about 15%. With the development of molecular biotechnology, new lung cancer molecular classification is continuously discovered, and targeted drugs are designed according to lung adenocarcinoma driver protein, so that personalized treatment is successful. However, acquired resistance occurs in almost all patients with lung adenocarcinoma during treatment. Therefore, the search for novel inhibitors against lung adenocarcinoma cells is of great significance for improving the current drug resistance of lung adenocarcinoma cells and developing novel and effective lung adenocarcinoma treatment means.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an anti-tumor protein AEL1, a fusion protein, an anti-tumor preparation and application, the protein provided by the invention has anti-tumor activity, has the potential of becoming an anti-tumor drug, and provides a new choice for tumor treatment, the protein shown in SEQ ID NO.1 has the activities of inhibiting proliferation and promoting apoptosis of tumor cells, has anti-tumor activity, can be used as an active component, is used in the anti-tumor field, and provides a new choice and thought for tumor treatment, such as preparing an anti-tumor new drug or preparation.

The amino acid sequence of the anti-tumor protein AEL1 is shown in SEQ ID NO. 1.

The invention relates to an optimized codon for encoding the anti-tumor protein AEL1 to be expressed in escherichia coli, and the nucleotide sequence of the optimized codon is shown as SEQ ID NO. 2.

The invention discloses a preparation method of an antitumor protein AEL1, which comprises the following steps: the protein is prepared by using genetic engineering recombination technology.

Of course, it should be noted that in other embodiments, the protein of the present invention can be obtained by other preparation methods, such as chemical synthesis, and the like, and also fall within the scope of the present invention.

Further, in some embodiments of the invention, producing the protein using genetically engineered recombination techniques comprises: culturing recombinant cells, and separating and purifying the protein from the culture product, wherein the recombinant cells contain an expression vector for expressing the protein, and the expression vector has a gene sequence for coding the protein.

The gene sequence is shown as SEQ ID NO.2, and the base sequence shown as SEQ ID NO.2 is a sequence after codon optimization by the inventor, is suitable for expression in escherichia coli, can express the protein with higher expression efficiency, and improves the yield of the protein.

It is noted that in other embodiments of the present invention, the skilled person can use a nucleic acid sequence other than that shown in SEQ ID NO.2 to express the protein of the first aspect, which is easily realized by the skilled person without any creative work, and therefore, the present invention is also within the protection scope.

The recombinant cell is escherichia coli.

The invention discloses a preparation method of an antitumor protein AEL1, which comprises the following steps:

(1) inserting the optimized codon into an expression vector to obtain a recombinant vector;

(2) transferring the recombinant vector into a competent cell to obtain a recombinant strain;

(3) culturing the recombinant strain, separating and purifying to obtain the antitumor protein AEL 1.

The preferred mode of the above preparation method is as follows:

the step (1) is specifically as follows: the nucleotide sequence of the optimized codon was inserted into the pCreat-SII vector.

The recombinant strain in the step (2) is DE3-AEL 1.

A recombinant large intestine expression plasmid is obtained by inserting the nucleotide sequence of the optimized codon into a pCreat-SII vector according to the sequence shown in SEQ ID NO. 2.

The invention provides a fusion protein comprising an active domain of the amino acid sequence of the material.

The fusion protein is one or more of the anti-tumor protein fusion tag protein, the fluorescent marker protein and the antibody of claim 1.

Other proteins, such as tag proteins, are fused on the protein AEL1 in the invention, so that the protein can be separated and purified in the process of preparing the protein based on engineering technology; for example, the protein is fused with a fluorescence labeling protein so as to observe the distribution, metabolism and the like of the protein in an experimental animal body when the protein is used as a medicament, and for example, the protein is fused with an antibody which can specifically target tumor tissues, so that the protein can be better enriched in tumors through the antibody, and the anti-tumor performance can be better exerted. Therefore, based on the disclosed proteins, the fusion proteins are within the scope of the present invention, regardless of the proteins or other analogs to which they are fused.

The protein provided by the invention is used in the field of pharmacy, such as preparing anti-tumor medicines or preparations, and provides a new choice for the current medicines for treating tumors.

The tumor is any one selected from lung cancer, breast cancer, colon cancer, kidney cancer, liver cancer, esophagus cancer, bladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, skin cancer, head and neck cancer and prostate cancer.

Further, the lung cancer is non-small cell cancer.

Further, the lung cancer is lung adenocarcinoma.

The research of the embodiment of the invention shows that the protein provided by the invention has the activity of inhibiting the proliferation and promoting the apoptosis of tumor cells such as lung adenocarcinoma cells.

The invention provides an anti-tumor preparation, which contains an effective amount of the anti-tumor protein AEL1 or the fusion protein.

The invention provides application of the anti-tumor preparation in preparing medicaments for preventing and treating lung cancer, breast cancer, colon cancer, kidney cancer, liver cancer, esophagus cancer, bladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, skin cancer, head and neck cancer and prostate cancer.

The invention provides application of the anti-tumor preparation in preparing a medicament for preventing and treating non-small cell cancer

The invention provides application of the anti-tumor preparation in preparing a medicament for preventing and treating lung adenocarcinoma.

Advantageous effects

The amino acid sequence of the protein disclosed by the invention is shown in SEQ ID NO.1, and the protein has anti-tumor activity and potential of becoming an anti-tumor medicament, and provides a new treatment idea for tumor treatment.

The molecular weight of the recombinant protein is about 12.877kDa, AEL1 is efficiently expressed in escherichia coli, the expression amount is 640mg/L, and the purity reaches more than 90%.

The active protein AEL1 of the invention presents a good concentration dependence on the growth inhibition effect of Spca-1 cells. The newly found active protein AEL1 has good inhibition effect, and the inhibition effect is better when the concentration is 16 mu g/mL.

The active protein AEL1 has the function of remarkably promoting apoptosis of lung adenocarcinoma cells Spca-1.

Drawings

FIG. 1: identifying the expression of active protein AEL 1; in the figure, M is Protein Marker; 1, sample before induction of AEL 1; 2: AEL1 post-induction sample I; 3: sample II after AEL1 induction; 4: AEL1 post-induction sample III; 5: AEL1 post-induction sample IV; 6: AEL1 induced post-sample V.

FIG. 2: optimizing the heterologous expression condition of the recombinant active protein AEL 1; in the figure, M is Protein Marker; 1: AEL1 non-induced sample; 2-4: 1.0mM IPTG induced sample at 37 ℃; 5-7: 37 ℃ of: 0.2mM IPTG post-induction samples; 8-10: 1.0mM IPTG post-induction samples at 15 ℃; 11-13: post-induction samples were 0.2mM IPTG at 15 ℃.

FIG. 3: the solubility of the recombinant active protein AEL1 is analyzed; in the figure, M is Protein Marker; 1: AEL1 uninduced sample; 2, AEL1 induced samples; 3, precipitating the sample after induction by 1.0mM IPTG at 37 ℃; 4: 1.0mM IPTG induced supernatant sample at 37 ℃; 5, precipitating the sample after induction by 0.2mM IPTG at 37 ℃; 6: a supernatant sample after induction with 0.2mM IPTG at 37 ℃; 7:15 ℃ 1.0mM IPTG induced precipitation of the sample; 8: supernatant samples after 1.0mM IPTG induction at 15 ℃; precipitating the sample after induction with 0.2mM IPTG at the temperature of 9:15 ℃; supernatant samples after induction with 0.2mM IPTG at 10:15 ℃.

FIG. 4: affinity purification analysis of recombinant active protein AEL 1; in the figure, M is Protein Marker; 1: precipitating after crushing; 2: crushed supernatant 3: effluent after Ni-NTA incubation; 4: buffer B elution sample; 5: buffer C eluted sample; 6: buffer D eluted.

FIG. 5 is a schematic view of: western Blot assay of purified active protein AEL 1; m is Pre-stabilized Protein Marker; sample: post-induction samples.

FIG. 6: SDS-PAGE electrophoretic detection of the eluate with the label removed; in the figure: m: protein Marker; 1: sample before enzyme digestion reaction; 2: performing enzyme digestion reaction on a sample; 3: flowing out the sample; 4: buffer E eluted sample; 5: buffer F eluted sample; 6: buffer G eluted sample; 7: buffer H eluted.

FIG. 7: the active protein AEL1 was isolated and purified for unlabeled SDS-PAGE analysis.

FIG. 8: effect of active protein AEL1 on the proliferation of lung adenocarcinoma Spca-1 cells.

FIG. 9: flow cytometry test that the active protein AEL1 promotes apoptosis of lung adenocarcinoma Spca-1 cells; in the figure: NC negative control. FIG. 10: map of expression plasmid pCreat-SII-AEL 1.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Test materials

1. Cell lines: BL21(DE3) was competently purchased from general biosystems (Anhui) Inc. (CP 01010); the lung adenocarcinoma Spca-1 cell strain is purchased from the cell resource center of Shanghai Life sciences research institute of Chinese academy of sciences.

2. Main reagent consumables and instrument:

reagent: restriction enzyme (TaKaRa, Japan), T4 DNA ligase (TaKaRa, Japan), plasmid extraction kit (TIANGEN), agarose gel DNA recovery kit (TIANGEN), SUMO Protease (biosystems, Anhui, Inc.), Annexin V-FITC/PI apoptosis detection kit (Katy Biotech development, Inc., Nanjing)

The instrument comprises the following steps: electrophoresis tank (American Bio-Rad Company), PCR apparatus (Applied Biosystems Company), low-temperature centrifuge (Eppendorf Company), constant temperature shaker (Duke (Shanghai) Automation Equipment Co., Ltd.), ultrapure water apparatus (American Millipore Company), ice maker (Japan SANYO Company), electrophoresis apparatus (American Bio-Rad Company), cell disrupter (Ningbo New Biotech Co., Ltd.), microplate reader (BIO-RAD), incubator (Yiheng (Shanghai) Instrument Co., Ltd.), vortex oscillator (German IKA Company), flow cytometer (American Becton Dickinson), centrifuge (Seifel).

Example 1

The amino acid sequence of the protein with anti-tumor activity provided by the embodiment is shown in SEQ ID NO.1, the active protein is named as AEL1, and the coded nucleic acid sequence is shown in SEQ ID NO. 2.

This example provides the following preparation method for preparing the active protein:

firstly, constructing a carrier containing optimized codons of active protein AEL 1:

the coding sequence of the active protein AEL1 (SEQ ID NO.2) was inserted between the BamHI and XhoI restriction sites on the pCreat-SII vector, resulting in the recombinant large intestine expression plasmid pCreat-SII-AEL1 (shown in FIG. 10).

The method specifically comprises the following steps: the amino acid sequence (SEQ ID NO.1) of the protein AEL1 is determined by molecular docking screening, virtual amino acid mutation, bioinformatics means such as protein stability evaluation and the like. According to the AEL1 amino acid sequence (SEQ ID NO.1), carrying out codon optimization of an escherichia coli expression system, entrusting the optimized codon sequence (SEQ ID NO.2) to general biological system (Anhui) limited for gene synthesis, then placing the obtained product in 5mL of LB liquid culture medium for shaking at 37 ℃ overnight, and placing the obtained product in 5mL of LB liquid culture medium for shaking at 37 ℃ overnight by using the vector pCeat-SII. The plasmid was extracted, digested with BamHI and XhoI, and ligated with T4 ligase to give recombinant large intestine expression plasmid pCreat-SII-AEL1 (FIG. 10).

Secondly, the plasmid pCreat-SII-AEL1 is transformed into BL21(DE3) competent cells to obtain a recombinant strain DE3-AEL1, and expression identification is carried out:

1. adding 2 μ L of plasmid into 100 μ L of competent bacteria, and placing on ice for 30 min;

heat shock at 2.42 deg.C for 90s, rapidly placing in ice for 5min, and adding 500 μ L LB culture solution;

after centrifugation, all the cells were spread on resistant LB plates at 3.37 ℃ for 1h with shaking at 220rpm and cultured in an inverted state at 37 ℃ overnight.

4. Selecting 5 plates, and inoculating the single clones in a test tube containing 4mL of LB culture solution with proper resistance;

shaking at 5.37 deg.C and 220rpm to OD ₆₀₀ 0.6-0.8;

6. taking out 1mL of culture, centrifuging at 12000g for 5min at room temperature, discarding the supernatant, resuspending the thallus precipitate with 80. mu.L of 1 XPBS Buffer solution, and adding 20. mu.L of 5 XPoading Buffer;

7. adding IPTG to the rest culture to final concentration of 0.5mM, shaking at 37 deg.C and 220rpm for 4h, inducing fusion protein expression;

8. 0.5mL of the culture was removed, 12000g was centrifuged at room temperature for 5min, the supernatant was discarded, the pellet was resuspended in 80. mu.L of 1 XPBS Buffer and 20. mu.L of 5 XPoading Buffer was added.

9. SDS-PAGE analysis showed that the correct plasmid was transformed into E.coli competent cells and the fusion protein AEL1 was normally expressed, as shown in FIG. 1.

Thirdly, the heterologous expression condition of the recombinant active protein AEL1 is optimized:

1. selecting a single clone on the streak plate, and inoculating the single clone into 13 test tubes containing 4mL of LB culture solution with proper resistance;

shaking at 2.37 deg.C and 220rpm to OD ₆₀₀ 0.6-0.8;

3. taking out 0.4mL of culture, centrifuging at 12000g for 5min at room temperature, discarding the supernatant, resuspending the bacterial pellet with 80. mu.L of 1 XPBS Buffer solution, and adding 20. mu.L of 5 XPoading Buffer;

4. adding IPTG to the remaining culture to final concentrations of 0.2mM and 1mM respectively, shaking at 37 deg.C and 15 deg.C and 220rpm for 4h and 16h respectively, and inducing expression of the fusion protein;

5. 0.2mL of the culture was removed, centrifuged at 12000g for 5min at room temperature, the supernatant was discarded, and the cell pellet was resuspended in 80. mu.L of 1 XPBS Buffer and 20. mu.L of 5 XPoading Buffer was added.

6. SDS-PAGE analysis shows that the fusion protein AEL1 is obviously expressed under each optimized condition, wherein 0.2Mm IPTG is the optimal expression condition at 37 ℃ as shown in figure 2.

Fourthly, the solubility analysis of the recombinant active protein AEL 1:

centrifuging 2mL of the induced bacterial liquid at 37 ℃ and 2mL of the induced bacterial liquid at 15 ℃ for 5min at the room temperature of 12000g, discarding the supernatant, re-suspending and centrifuging the supernatant by using 1mL of Buffer A (20mM Tris, 300mM NaCl, pH8.0), precipitating the supernatant after centrifugation, carrying out ultrasonic disruption (phi 3, 15 percent, 3s/6s and 5min), respectively sampling the supernatant precipitate, and carrying out SDS-PAGE analysis. As a result, as shown in FIG. 3, it was confirmed that the fusion protein of interest AEL1 was a soluble protein.

Fifthly, amplifying expression and affinity purification of the recombinant active protein AEL 1:

1. inoculating the optimal clone strain to 1L LB culture medium containing appropriate antibiotics, and shaking at 37 deg.C and 220rpm to OD ₆₀₀ And (5) amplifying and expressing under the optimal condition of 0.6-0.8.

2. Centrifuging at 8000rpm for 10min, discarding supernatant, and collecting all thallus.

3. The cells were resuspended using Buffer A (20mM Tris, 300mM NaCl, pH8.0) and sonicated (. PHI.10, 15%, 3s/6s, 30 min). 16000rpm for 10min, and collecting the supernatant.

4. Adding 3mLNi-NTA into the supernatant, mixing uniformly, and incubating for 1h at 4 ℃.

5. The incubation was added to an empty column and the effluent collected.

6. The filler was washed with Buffer B (20mM Tris, 300mM NaCl, 20mM Imidazole, pH8.0) and Buffer C (20mM Tris, 300mM NaCl, 40mM Imidazole, pH8.0), respectively, and the washing solutions were collected.

7. The eluate was collected by elution with Buffer D (20mM Tris, 300mM NaCl, 250mM Imidazole, pH 8.0).

SDS-PAGE electrophoretic detection. The results are shown in FIG. 4, and the fusion protein AEL1 can be purified by Ni-NTA affinity chromatography after the optimal conditions are selected for amplification of expression.

Sixthly, Western Blot test of the purified active protein AEL 1:

SDS-PAGE electrophoretic samples: pre-stabilized Protein Marker, IPTG induced samples. Electrophoresis conditions: 200V for 50 min;

2. semi-dry type electric transfer printing: during the electrophoresis, the PVDF membrane is cut out to have the same size as the gel, a small angle is cut off to help the judgment of the direction, and the PVDF membrane is placed in methanol for soaking for 30s and then transferred to an electrotransformation solution. The cut areas of the thin filter paper and the thick filter paper are slightly larger than the gel, and the two pieces of the thin filter paper and the thick filter paper are transferred to the buffer solution for soaking. After electrophoresis, the gel concentrate was cut off and placed in an electrotransfer solution. The thick filter paper, the thin filter paper, the NC membrane, the glue, the thin filter paper and the thick filter paper are placed on the plane of the electric rotating machine in the order named. The air bubbles between each layer are all removed. One end can be lightly pressed by hand, then a 50mL centrifuge tube is used for gently moving the upper layer to one side to remove air bubbles, one end is replaced by hand, and the centrifuge tube is used for removing air bubbles from the other end. Linking the electrotransformation instrument to the instrument for 30mins with the protein of less than 50KDa and the protein of 30V; the protein is larger than 50KDa, and 45mins is used.

3. Blocking of membranes and antibody incubation and final treatment: washing the transfer printing film: rinse 3 times with 1 XPBST at room temperature for 5min, not add to the membrane at the time of any solution addition, add slowly along the corner of the box to avoid washing away the membrane bound material, and rotate the shaker at 40 rpm. PBST was poured off, placed in a 5% skim milk powder confining liquid, the shaker turned to the lowest speed, and confined at room temperature for 2 h. Rinse with 1 × PBST for 5min at room temperature for 3 times. Adding antibody (1: 2000 to 5% skimmed milk powder), incubating for 1h, and recovering to-20 deg.C after use. Rinse with 1 XPBST for 5min at room temperature for 3 times. Slowly adding a small amount of ECL solution to cover the membrane, standing for 2min, and taking a picture. The results are shown in FIG. 5, where the fusion protein AEL1 was successfully purified.

Seventhly, removing the purified active protein AEL1 label:

1. the eluted sample was dialyzed into Buffer E (1 × PBS, ph 7.4).

2. After dialysis, a proper amount of SUMOProtease was added to the fusion protein, mixed well, and left to react overnight at 4 ℃.

3. And (3) adding the product after the reaction is finished into a Ni-NTA purification column, slowly loading the sample, and collecting effluent liquid.

4. The purification column was washed with Buffer E (1 × PBS, ph 7.4).

5. Elution was performed using Buffer F (1 × PBS, 20mM Imidazole, ph 7.4).

6. Elution was performed using Buffer G (1 × PBS, 40mM Imidazole, ph 7.4).

7. Elution was performed using Buffer H (1 × PBS, 250mM Imidazole, ph 7.4).

SDS-PAGE electrophoretic detection. The results are shown in FIG. 6, and the active protein AEL1 was successfully separated from the tag by the enzyme digestion reaction.

9. And (4) subpackaging the effluent sample or the elution sample to Buffer E, and then subpackaging for subsequent activity verification. SDS-PAGE shows (as shown in figure 7) that the molecular weight of the recombinant protein is about 12.877kDa, AEL1 is efficiently expressed in escherichia coli, the expression amount is 640mg/L, and the purity reaches more than 90%.

Example 2

The active protein AEL1 inhibits the activity detection of lung adenocarcinoma Spca-1 cells:

succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit in cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its absorbance at 570nm and 630nm can be measured by enzyme linked immunosorbent assay (ELISA) to indirectly reflect the number of living cells. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number.

1. Collecting cells in logarithmic phase, digesting, centrifuging and collecting when the cells grow to the density of about 80-90%, removing supernatant, adding 2ml culture medium, and mixing well;

2. adjusting the cell suspension concentration (typically 5-10X 10 cell concentration) ⁴ One/ml), add 100 μ l per well (32 wells at the edge of 96 well plate are filled with sterile PBS because water in the wells at the edge evaporates quickly and the drug is easily concentrated, which has a large impact on the experiment);

3.5％CO ₂ incubate at 37 ℃ for about 24 hours until the cell monolayer is confluent at the bottom of the well (96-well flat bottom plate);

4. firstly, absorbing culture solution in the hole, and then adding the medicine with concentration gradient: triplicate settings for each concentration of 0. mu.g, 1. mu.g, 2. mu.g, 4. mu.g, 8. mu.g, 16. mu.g, 5% CO ₂ Incubating at 37 ℃ for about 24 hours, and observing under an inverted microscope; (proteins are filter sterilized before loading the protein);

5. adding 10ul MTT solution (5mg/ml, namely 0.5% MTT) into each well, and continuing culturing for 4 h;

6. terminating the culture, and carefully sucking out the culture solution in the holes;

7. adding 150ul MTT into each well, and placing on a shaking table to shake at low speed for 3min to fully dissolve the crystals;

8. the absorbance of each well was measured at enzyme linked immunosorbent assays OD570nm and OD630nm, as follows:

the growth condition of Spca-1 cells after the treatment of different concentrations of the newly found active protein AEL1 for 24 hours is detected by an MTT method. As shown in FIG. 8, the active protein AEL1 showed a good concentration-dependent relationship with respect to the growth inhibitory effect on Spca-1 cells. When the mass concentration of the protein AEL1 is 1 mug/mL, 2 mug/mL, 4 mug/mL, 8 mug/mL and 16 mug/mL respectively, the survival rate of Spca-1 cells is 97.75%, 94.09%, 91.64%, 90.46% and 73.08% respectively. The inhibition effect is strongest when the protein AEL1 is at the concentration of 16 mu g/mL, and the anti-lung adenocarcinoma effect is very obvious compared with a blank control group (P < 0.05).

Example 3

The active protein AEL1 promotes the apoptosis detection of lung adenocarcinoma Spca-1:

in normal cells, Phosphatidylserine (PS) is distributed only inside the lipid bilayer of the cell membrane, whereas in early apoptosis, the Phosphatidylserine (PS) in the cell membrane is turned outside from inside the lipid membrane. Annexin V is Ca with molecular weight of about 35KD ²⁺ The dependent phospholipid binding protein has high affinity with phosphatidylserine, so that the phospholipid binding protein can be bound to the cell membrane of cells in early apoptosis through the phosphatidylserine exposed outside the cells. Therefore, Annexin V is used as one of indexes for detecting early apoptosis of cells. Annexin V is labeled by fluorescein FITC, and the labeled Annexin V is used as a fluorescent probe, and the occurrence of apoptosis can be detected by using a flow cytometer.

Propidium Iodide (PI) is a nucleic acid dye that cannot penetrate the intact cell membrane, but can penetrate the membrane to stain red nuclei in cells in the middle and late stages of apoptosis, and dead cells. Therefore, cells are often stained with Annexin V matched to PI to differentiate between cells at different apoptotic stages. The invention utilizes flow cytometry to detect lung adenocarcinoma Spca-1 apoptosis, and the specific steps are as follows:

1. digesting the tumor cells in logarithmic growth phase to prepare single cell suspension;

2. adjusting the cell concentration to a final concentration of 5-10 × 10 ⁵ Per ml; adding 1ml of tumor cell suspension in 5% CO into 6-well culture plate ₂ Culturing in an incubator at 37 ℃ for 24 hours;

3. adding active protein with final concentration of 16 μ g/ml, and using culture medium as negativeA sexual control; at 5% CO ₂ Culturing in 37 deg.C incubator for 24-36 hr;

4. digesting and collecting adherent cells by using pancreatin without EDTA;

5. washing the cells twice with PBS (centrifugation at 2000rpm for 5min) to collect 1-5X 10 ⁵ A cell;

6. adding 500 mu l of Binding Buffer suspension cells;

7. adding 5 mul annexin V-FITC, mixing, adding 5 mul propdium Iodide, and mixing; keeping the mixture away from light at room temperature, and reacting for 5-15 min; flow cytometry observations and measurements were performed over 1 h.

The invention detects the influence of the active protein AEL1 on the apoptosis of Spca-1 cells after the cells are treated for 36 hours. As shown in figure 9, when the active protein AEL1 acts on Spca-1 cells, the apoptosis rate of the negative control is 3.9%, the apoptosis rate of the active protein AEL1 promoting Spca-1 cells is 13.3%, and the results show that the active protein AEL1 has the effect of remarkably promoting the apoptosis of lung adenocarcinoma cells Spca-1.

In conclusion, the active protein AEL1 provided by the invention has activities of inhibiting the proliferation and promoting the apoptosis of lung adenocarcinoma cells, and has strong anti-tumor activity, and the active protein AEL1 has good application prospect, can be used for preparing anti-tumor protein medicines, and provides a new idea for the treatment of the current tumor.

SEQUENCE LISTING

<110> Shanghai city academy of agricultural sciences

2, 1

<120> anti-tumor protein AEL1, fusion protein, anti-tumor preparation and application

<130> 1

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 113

<212> PRT

<213> Artificial sequence

<400> 1

Met Ser Thr Pro Ser Ser Leu Pro Ile Phe Leu Ala Ala Thr Leu Arg

1 5 10 15

Lys Leu His Phe Asp Tyr Thr Pro Lys Trp Gly Arg Gly Thr Pro Asn

20 25 30

Thr Phe Val Asp Asn Val Thr Phe Pro Val Val Leu Thr Asp Lys Ala

35 40 45

Tyr Thr Tyr Arg Ile Val Val Asp Asp Asp Thr Asp Leu Gly Ile Lys

50 55 60

Ser Gly Tyr Ala Val Gln Ala Asp Gly Ser Gln Arg Leu Asn Phe Leu

65 70 75 80

Glu Trp Asn Lys Gly Tyr Gly Ile Glu Gln Gly Arg Arg Ile Arg Val

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Tyr Val Val Glu Pro Asp Thr Gly Asn Gln Tyr Leu Ile Ala Gln Trp

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Lys

<210> 2

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<212> DNA

<213> Artificial sequence

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ccggttgttc tgaccgataa agcatatacc tatcgtattg ttgtggatga tgataccgat 180

ctgggtatta agagtggtta tgcagtgcag gccgatggta gccagcgcct gaattttctg 240

gaatggaata agggttatgg tattgaacag ggccgccgta ttcgtgtgta tgtggtggaa 300

ccggataccg gtaatcagta tctgattgcc cagtggaaat aa 342

Claims (7)

1. An anti-tumor protein AEL1, which is characterized in that the amino acid sequence of the protein is shown in SEQ ID NO. 1.

2. A gene for coding the expression of the antitumor protein AEL1 in Escherichia coli as claimed in claim 1, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

3. A method for preparing the antitumor protein AEL1 of claim 1, which comprises the following steps:

(1) inserting the gene of claim 2 into an expression vector to obtain a recombinant vector;

(2) transferring the recombinant vector into a competent cell to obtain a recombinant strain;

(3) culturing the recombinant strain, separating and purifying to obtain the antitumor protein AEL 1.

4. A recombinant large intestine expression plasmid obtained by inserting the nucleotide sequence of the gene of claim 2 into a pCreat-SII vector as shown in SEQ ID NO. 2.

5. A fusion protein, which is characterized in that the fusion protein is one or more of the anti-tumor protein further fusion tag protein, the fluorescent marker protein and the antibody of claim 1.

6. An anti-tumor agent comprising the anti-tumor protein AEL1 according to claim 1 or the fusion protein according to claim 5.

7. Use of the anti-tumor agent of claim 6 in the preparation of a medicament for inhibiting lung adenocarcinoma cell proliferation and promoting lung adenocarcinoma cell apoptosis.

Images