CN111499696B - Anti-tumor protein ILAP1, and preparation and application thereof - Google Patents

Abstract

The invention relates to an anti-tumor protein ILAP1, and a preparation method and an application thereof, wherein the amino acid sequence of the anti-tumor protein ILAP1 is shown as SEQ ID NO. 1. The preparation method disclosed by the invention can be used for preparing the protein with the anti-tumor activity, the preparation method is simple to operate and high in efficiency, and in addition, the anti-tumor protein provided by the invention has obvious anti-tumor activity, can inhibit the proliferation of tumor cells and promote the apoptosis of the tumor cells, and can be used for preparing anti-tumor drugs.

Description

Anti-tumor protein ILAP1, and preparation and application thereof

Technical Field

The invention belongs to the field of antitumor proteins, and particularly relates to an antitumor protein ILAP1, and a preparation method and an application thereof.

Background

Malignant tumor is one of the main diseases endangering human health at present, and the death rate of the malignant tumor is second to cardiovascular and cerebrovascular diseases. Among them, lung cancer is one of the most common malignant tumors in the world today, and the incidence and mortality of lung cancer are on the rising trend year by year, and the lung cancer has become the main cause of cancer death in human beings. Lung cancer is classified by histopathology into non-small cell lung cancer and small cell lung cancer, of which non-small cell lung cancer (NSCLC) is the most common pathological type of lung cancer, accounting for about 70% -80% of primary lung cancer, and having an overall 5-year survival rate of 8% -11%.

Lung adenocarcinoma (lung adenocarinoma) is a non-small cell lung cancer, which is a kind of lung cancer, and is easy to occur in women and patients without smoking.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an anti-tumor protein ILAP1 and preparation and application thereof, the protein shown in SEQ ID NO.1 has activities of inhibiting proliferation and promoting apoptosis of tumor cells, and the protein can be quickly and conveniently prepared in large quantity by a genetic engineering technical means, so that the time for obtaining the protein is shortened.

The invention provides an anti-tumor protein ILAP1, wherein the amino acid sequence of the anti-tumor protein ILAP1 is shown in SEQ ID NO. 1.

The invention provides a nucleotide sequence for coding the anti-tumor protein ILAP1, which is shown in SEQ ID NO. 2.

The invention relates to a recombinant large intestine expression plasmid, which contains the nucleotide sequence.

The recombinant expression plasmid is inserted between BamHI and XhoI restriction sites on a pCreat-SII vector by a nucleic acid sequence shown as SEQ ID NO.2, and the recombinant expression plasmid pCreat-SII-ILAP1 is obtained.

A recombinant cell obtained by transferring said recombinant large intestine expression plasmid into a competent cell.

The protein has anti-tumor activity, the amino acid sequence of the protein is shown as SEQ ID NO.1, and the recombinant cell contains a nucleic acid sequence for coding the protein.

The nucleic acid sequence shown in SEQ ID NO.2 is subjected to codon optimization, is suitable for expression in common escherichia coli, can express the protein with higher expression efficiency, and improves the yield of the protein.

The recombinant cell is escherichia coli.

The preparation method of the anti-tumor protein ILAP1 comprises the following steps: culturing the recombinant cells, and separating and purifying the culture product to obtain the protein.

Further specifically:

the invention provides a preparation method of an antitumor protein ILAP1, which comprises the following steps:

(1) constructing the recombinant expression vector;

(2) constructing a recombinant cell containing the recombinant expression vector in the step (1);

(3) culturing the recombinant cell, and inducing the expression of the fusion protein ILAP 1;

(4) separating and purifying to obtain the antitumor protein ILAP 1.

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

the skeleton of the recombinant expression vector in the step (1) is a pCreat-SII vector, and the nucleic acid sequence is positioned between BamHI and XhoI restriction enzyme cutting sites of the recombinant expression vector.

The selection of a suitable expression vector is also a means for increasing the efficiency of protein expression, and in some embodiments of the present invention, a pCreat-SII vector is selected as the expression vector, which can increase the expression efficiency and thus increase the protein content in the final culture product.

The conditions for culturing the recombinant cells were as follows: the temperature is 36-38 ℃, and the rotation speed is 200-240 rpm.

Suitable culture conditions are favorable for growth of the recombinant cells and increase of protein yield, and in some embodiments of the invention, the recombinant cells are cultured at the temperature of 36-38 ℃ and the rotation speed of 200-240rpm, so that the expression level of the protein can be increased.

The step (3) is specifically as follows: when the OD600 of the culture medium of the recombinant cells is 0.6-0.8, IPTG is added into the culture medium to induce and express proteins; wherein IPTG is added to the medium at a final concentration of 0.18-022 mM.

When the IPTG is used for inducing the expression of the protein, the expression amount can be improved, the protein product is more stable, the adding time of the inducer IPTG is also an important influence factor on the expression amount of the product, and the ideal induction effect can not be achieved easily when the inducer IPTG is added too early or too late. The research of the invention finds that when the OD600 is 0.6-0.8, IPTG is added, the expression of the protein can be well induced, and the expression quantity of the protein is improved.

The final concentration of IPTG added in the medium was 0.18-022 mM.

The addition amount of IPTG is also a factor influencing the expression efficiency of the protein, too much or too low is not beneficial to the expression of the protein, and the addition amount in what concentration range is the optimal induction use amount cannot be reasonably expected by a person skilled in the art. The research of the invention shows that the expression level of the protein can be improved by controlling the concentration of IPTG in the culture medium to be in the range of 0.18-022 mM.

The invention provides an application of the anti-tumor protein ILAP1 in preparation of a medicine for treating or preventing lung cancer.

The invention provides application of the anti-tumor protein ILAP1 in preparation of a medicine for treating or preventing non-small cell lung cancer.

The invention provides application of the anti-tumor protein ILAP1 in preparation of a medicine for treating or preventing lung adenocarcinoma.

As used herein, the terms "comprises," comprising, "" has, "" having, "" contains, "" containing, "and" includes "and variations thereof, as used herein, mean" including but not limited to. While various compositions and methods are described in terms of "comprising" various components or steps (interpreted as meaning "including, but not limited to"), the compositions, methods, and devices can also "consist essentially of" or "consist of" the various components and steps, and such terms should be interpreted as defining an essentially closed group of members.

"expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector may comprise sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or by an in vitro expression system. Expression vectors include all expression vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), which contain recombinant polynucleotides.

Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns, and to some extent the nucleotide sequence encoding the protein may contain introns in some versions. In certain embodiments, the nucleotide sequence does not comprise an intron, comprising only the coding sequence.

Advantageous effects

The preparation method disclosed by the invention can be used for preparing the protein with anti-tumor activity, and is simple to operate and high in efficiency.

The molecular weight of the protein is about 12.35kDa, the ILAP1 is efficiently expressed in escherichia coli, the expression amount can reach 100mg/L, the expression efficiency is high, and the purity of the protein reaches more than 90 percent.

The protein ILAP1 of the invention presents a good concentration dependence on the growth inhibition effect of Spca-1 cells. The newly found protein ILAP1 has good inhibition effect, has obvious cancer cell proliferation inhibition capacity at a lower concentration (2 mu g/mL), and has a sharp increase when the concentration is more than 4 mu g/mL.

The protein ILAP1 of the invention has the function of remarkably promoting the apoptosis of lung adenocarcinoma cells Spca-1, and the apoptosis rate can reach 36.0%.

The invention provides a novel anti-tumor protein, which has good activities of inhibiting proliferation and promoting apoptosis on tumor cells, can be used in the field of anti-tumor treatment, provides a novel drug selection for tumor treatment, and also provides a novel treatment idea for tumor treatment.

Drawings

FIG. 1: identifying the expression of the recombinant protein ILAP 1; in the figure, M is Protein Marker; 1 sample before ILAP1 induction; 2: ILAP1 post-induction sample I; 3: ILAP1 post-induction sample II; 4: ILAP1 post-induction sample III; 5: ILAP1 post-induction sample IV; 6: ILAP1 post-induction sample V.

FIG. 2: optimizing the heterologous expression condition of the recombinant protein ILAP 1; in the figure, M is Protein Marker; 1: ILAP1 uninduced sample; 2-4 samples after 1.0mM IPTG induction 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 with 0.2mM IPTG at 15 ℃.

FIG. 3: solubility analysis of the recombinant protein ILAP 1; in the figure, M is Protein Marker; 1: ILAP1 uninduced sample; 2, ILAP1 post-induction 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: 1.0mM IPTG induced supernatant sample 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 the recombinant protein ILAP 1; in the figure, M is Protein Marker; 1: crushing and precipitating; 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: western Blot to examine the purified protein ILAP 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 the 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 protein ILAP1 was isolated and purified for unlabeled SDS-PAGE analysis.

FIG. 8: effect of the protein ILAP1 on the proliferation of lung adenocarcinoma Spca-1 cells.

FIG. 9: flow cytometry test of promoting lung adenocarcinoma Spca-1 apoptosis by using the protein ILAP 1; in the figure: NC negative control.

FIG. 10: plasmid map of plasmid vector pCreat-SII-ILAP 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 (Bio-Rad, USA), PCR instrument (Applied Biosystems Company), low temperature centrifuge (Eppendorf Company), constant temperature shaker (Duke (Shanghai) Automation equipment Co., Ltd.), ultrapure water equipment (Millipore, USA), ice maker (SANYO, Japan), electrophoresis instrument (Bio-Rad, USA), cell disrupter (Ningbo New Biotech, Ltd.), enzyme reader (BIO-RAD), incubator (Heng (Shanghai) Instrument Co., Ltd.), vortex oscillator (Germany IKA), flow cytometer (Becton Dickinson, USA), centrifuge (Seifenesiel).

Example 1

The amino acid sequence of the anti-tumor protein provided by the embodiment is shown in SEQ ID No.1, and the nucleotide sequence coded by the anti-tumor protein is shown in SEQ ID No. 2. In the examples, this antitumor protein was designated ILAP 1.

The preparation method of the anti-tumor protein provided by the embodiment is as follows:

firstly, constructing a vector containing optimized codons of protein ILAP 1:

the coding sequence of the protein ILAP1 (SEQ ID NO.2) was inserted between the BamHI and XhoI restriction sites on the pCreat-SII vector to give a recombinant large intestine expression plasmid pCreat-SII-ILAP1 (shown in FIG. 10).

Specifically, the method comprises the following steps: the amino acid sequence (SEQ ID NO.1) of the protein ILAP1 is determined by molecular docking screening, virtual amino acid mutation, bioinformatics means such as protein stability evaluation and the like. According to the amino acid sequence of ILAP1 (SEQ ID NO.1), codon optimization of an escherichia coli expression system is carried out, the optimized codon sequence (SEQ ID NO.2) is entrusted to general biological system (Anhui) limited for gene synthesis, and then the obtained product is placed in 5mL of LB liquid culture medium and shaken at 37 ℃ overnight, and the vector pCeat-SII is placed in 5mL of LB liquid culture medium and shaken at 37 ℃ overnight. The plasmid was extracted, digested with BamHI and XhoI and ligated with T4 ligase to give recombinant large intestine expression plasmid pCreat-SII-ILAP1 (FIG. 10).

Secondly, the plasmid pCreat-SII-ILAP1 is transformed into BL21(DE3) competent cells to obtain a recombinant strain DE3-ILAP1, 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, the whole was spread on a resistant LB plate at 3.37 ℃ for 1h with shaking at 220rpm, and cultured overnight in an inverted state at 37 ℃.

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 bacterial pellet with 80. mu.L of 1 XPBS Buffer solution, and adding 20. mu.L of 5 Xloading Buffer;

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

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 ILAP1 was normally expressed, as shown in FIG. 1.

Thirdly, the heterologous expression condition of the recombinant protein ILAP1 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 rest culture to final concentration of 0.2mM and 1mM respectively, shaking at 37 deg.C and 15 deg.C and 220rpm for 4h and 16h respectively, inducing expression of 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 ILAP1 is obviously expressed under each optimized condition, wherein 37 ℃ and 0.2Mm IPTG are optimal expression conditions, as shown in figure 2.

Fourthly, carrying out solubility analysis on the recombinant protein ILAP 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, the obtained fusion protein ILAP1 of interest was a soluble protein.

Fifthly, amplifying expression and affinity purification of recombinant protein ILAP 1:

1. inoculating the optimal clone strain into 1L LB culture medium containing appropriate antibiotics, and shaking at 37 deg.C and 220rpm to OD ₆₀₀ The expression was amplified under the above-mentioned optimum conditions (37 ℃ C., 0.2Mm IPTG) at 0.6 to 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. 3mL of Ni-NTA was added to the supernatant, mixed well and incubated at 4 ℃ for 1 h.

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 electrophoresis detection shows that the result is shown in FIG. 4, and after the optimal condition is selected for amplification expression, the fusion protein ILAP1 can be purified by Ni-NTA affinity chromatography.

Sixthly, Western Blot test of purified protein ILAP 1:

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

2. semi-dry type electric transfer printing: during the running of electrophoresis, the PVDF membrane is cut out to have the same size as the gel, a small angle is cut off to help the direction judgment, and the PVDF membrane is placed in methanol for soaking for 30s and then transferred to an electrotransformation liquid. The cut areas of the thin filter paper and the thick filter paper are slightly larger than the gel, and two pieces of the thin filter paper and two pieces of the thick filter paper are soaked in the transfer buffer solution. 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 for 5min at room temperature, without adding any solution to the membrane, slowly along the corner of the box to avoid washing away the bound material on the membrane, 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 × PBST 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 figure 5 of the drawings,

the fusion protein ILAP1 was successfully purified.

Seventhly, removing the purified protein ILAP1 label:

1. all eluted samples were dialyzed into Buffer E (1 × PBS, ph 7.4).

2. After dialysis, appropriate amount of SUMO Protease was added to the fusion protein, mixed and left to react overnight at 4 ℃.

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

4. The 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 with Buffer H (1 × PBS, 250mM Imidazole, pH7.4)

SDS-PAGE electrophoretic detection. As shown in FIG. 6, the active protein ILAP1 was successfully separated from the tag by the enzyme cleavage 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 protein is about 12.35kDa, the ILAP1 is efficiently expressed in escherichia coli, the expression level is 100mg/L, the expression efficiency is high, and the purity of the protein reaches more than 90 percent.

Example 2

Detection of activity of protein ILAP1 in inhibiting 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 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. the culture solution in the holes is firstly sucked and thenAdding a concentration gradient of the drug: 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 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 treated by the newly found protein ILAP1 at different concentrations for 24h is detected by an MTT method. As shown in FIG. 8, the growth inhibitory effect of protein ILAP1 on Spca-1 cells showed a good concentration-dependent relationship. When the mass concentration of the protein ILAP1 is 1 mu g/mL, 2 mu g/mL, 4 mu g/mL, 8 mu g/mL and 16 mu g/mL respectively, the survival rates of Spca-1 cells are 98.80%, 92.79%, 86.76%, 64.85% and 37.38% respectively. Half inhibitory concentration IC of protein ILAP1 on lung adenocarcinoma Spca-1 cells ₅₀ 16 mu g/mL which is less than the concentration standard of 30 mu g/mL in modern tumor treatment pharmacology, and proves that the protein ILAP1 has strong lung adenocarcinoma resisting effect and has the potential of being used as a medicament.

Example 3

The protein ILAP1 promotes 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, Phosphatidylserine (PS) in the cell membrane turns from inside to outside of the lipid bilayer. Annexin V is Ca with molecular weight of 35KD or so ²⁺ The phospholipid-dependent binding protein has high affinity with phosphatidylserine, and is therefore useful for treating diseasesPhosphatidylserine exposed outside the cell binds to the cell membrane of early apoptotic 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). times.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 protein with final concentration of 16 mug/ml, and taking the culture medium as negative 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 protein ILAP1 on the apoptosis of Spca-1 cells after the Spca-1 cells are treated for 36 hours. As shown in FIG. 9, when the recombinant human lung adenocarcinoma cell line was applied to Spca-1 cells, the apoptosis rate of the negative control was 3.9%, and the apoptosis rate of the protein ILAP1 promoting Spca-1 cells was 36.0%, and it can be seen from the results that the protein ILAP1 has a significant effect of promoting apoptosis of lung adenocarcinoma cells Spca-1.

SEQUENCE LISTING

<110> Shanghai city academy of agricultural sciences

2, 1

<120> anti-tumor protein ILAP1, and preparation and application thereof

<130> 1

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 114

<212> PRT

<213> Artificial sequence

<400> 1

Met Ser Leu Thr Gly Val Asn Glu Gly Leu Val Phe Leu Leu Val Ala

1 5 10 15

Gln Val Lys Lys Ile Asp Phe Asp Tyr Ala Pro Ala Tyr Tyr Arg Pro

20 25 30

Thr Ser Gly Tyr Thr Asp Ala Val Thr Phe Pro Ala Val Leu Ala Asn

35 40 45

Lys Ala Tyr Lys Tyr Gln Val Val Val Asp Gly Val Ser Lys Gly Ile

50 55 60

Arg Arg Asp His Ala Val Ala Pro Asp Gly Ser Ala Lys Ile Asn Phe

65 70 75 80

Leu Asp Tyr Asn Ala Gly Tyr Gly Ile Pro Asn Lys Ser Ser Thr Gln

85 90 95

Val Tyr Ala Val Asp Pro Asp Thr Gly Ile Ala Tyr Tyr Ile Leu Thr

100 105 110

Val Ser

<210> 2

<211> 342

<212> DNA

<213> Artificial sequence

<400> 2

atgagcctga ccggcgtgaa tgaaggcctg gtttttctgc tggttgcaca ggtgaaaaag 60

attgattttg attacgcacc ggcatattat cgcccgacca gcggctatac cgatgcagtt 120

acctttccgg cagttctggc caataaggca tataaatatc aggtggtggt ggatggcgtg 180

agcaaaggta ttcgccgtga tcatgccgtg gcaccggatg gtagcgcaaa aattaatttt 240

ctggattata acgccggcta tggcattccg aataagagca gcacccaggt gtatgcagtg 300

gaccctgata ccggtattgc atattatatt ctgaccgtta gc 342

Claims (10)

1. An anti-tumor protein ILAP1, which is characterized in that the amino acid sequence of the anti-tumor protein ILAP1 is shown in SEQ ID NO. 1.

2. A nucleotide sequence encoding the antitumor protein ILAP1 of claim 1, wherein the nucleotide sequence is set forth in SEQ ID No. 2.

3. A recombinant large intestine expression plasmid comprising the nucleotide sequence of claim 2.

4. The recombinant expression plasmid of claim 3, wherein the recombinant expression plasmid is inserted between the BamHI and XhoI restriction sites on the pCreat-SII vector by the nucleic acid sequence shown in SEQ ID No.2, resulting in the recombinant expression plasmid pCreat-SII-ILAP 1.

5. A recombinant cell obtained by transferring the recombinant large intestine expression plasmid of claim 3 into a competent cell.

6. A preparation method of an antitumor protein ILAP1 comprises the following steps:

culturing the recombinant cell of claim 5, inducing expression of fusion protein ILAP1, and separating and purifying to obtain antitumor protein ILAP 1.

7. The preparation method according to claim 6, wherein the step (3) is specifically: when the OD600 of the culture medium of the recombinant cells is 0.6-0.8, IPTG is added into the culture medium to induce and express proteins; wherein IPTG is added to the medium at a final concentration of about 0.18-022 mM.

8. The use of the anti-tumor protein ILAP1 of claim 1 in the preparation of a medicament for the treatment of lung cancer.

9. The use of the anti-tumor protein ILAP1 of claim 1 in the preparation of a medicament for the treatment of non-small cell lung cancer.

10. The use of the anti-tumor protein ILAP1 of claim 1 in the preparation of a medicament for the treatment of lung adenocarcinoma.

Images