CN111499696A - Anti-tumor protein I L AP1, and preparation and application thereof - Google Patents

Abstract

The invention relates to an antitumor protein I L AP1, and a preparation method and an application thereof, wherein the amino acid sequence of the antitumor protein I L AP1 is shown in SEQ ID NO. 1.

Description

Anti-tumor protein I L AP1, and preparation and application thereof

Technical Field

The invention belongs to the field of antitumor proteins, and particularly relates to an antitumor protein I L AP1, and preparation and application thereof.

Background

The lung cancer is divided into non-small cell lung cancer and small cell lung cancer according to histopathology, wherein the non-small cell lung cancer (NSC L C) is one of the most common pathological types of the lung cancer, accounts for about 70% -80% of the primary lung cancer, and has a total 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 invention aims to solve the technical problem of providing an anti-tumor protein I L AP1 and preparation and application thereof, the protein shown as SEQ ID NO.1 has the activities of inhibiting the proliferation and promoting the 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 antitumor protein I L AP1, wherein the amino acid sequence of the antitumor protein I L AP1 is shown in SEQ ID NO. 1.

The invention provides a nucleotide sequence for coding the anti-tumor protein I L AP1, which is shown as 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 enzyme sites on a pCreat-SII vector by a nucleic acid sequence shown as SEQ ID NO.2, and the recombinant expression plasmid pCreat-SII-I L AP1 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 I L AP1 comprises the steps of culturing recombinant cells, and separating and purifying the cultured product to obtain the protein.

Further specifically:

the invention provides a preparation method of an antitumor protein I L AP1, 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 I L AP 1;

(4) separating and purifying to obtain the antitumor protein I L AP 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 I L AP1 in preparation of a medicine for treating or preventing lung cancer.

The invention provides application of the anti-tumor protein I L AP1 in preparation of a medicine for treating or preventing non-small cell lung cancer.

The invention provides application of the anti-tumor protein I L AP1 in preparation of a medicament 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 the anti-tumor activity, and is simple to operate and high in efficiency.

The molecular weight of the protein is about 12.35kDa, the I L AP1 is efficiently expressed in escherichia coli, the expression amount can reach 100 mg/L, the expression efficiency is high, and the purity of the protein reaches more than 90%.

The protein I L AP1 of the invention presents a good concentration dependence on the growth inhibition effect of Spca-1 cells, the newly found protein I L AP1 has a good inhibition effect, has obvious cancer cell proliferation inhibition capacity at a lower concentration (2 mu g/m L), and has a sharp increase of the inhibition effect when the concentration is more than 4 mu g/m L.

The protein I L AP1 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 shows the expression identification of recombinant Protein I L AP1, in which M is Protein Marker, 1 is sample before induction of I L AP1, 2 is sample I after induction of I L AP1, 3 is sample II after induction of I L AP1, 4 is sample III after induction of I L AP1, 5 is sample IV after induction of I L AP1, and 6 is sample V after induction of I L AP 1.

FIG. 2 shows the optimization of heterologous expression conditions of recombinant Protein I L AP1, in which M is Protein Marker, 1 is I L AP1 uninduced sample, 2-4 is sample induced at 37 deg.C by 1.0mM IPTG, 5-7 is sample induced at 37 deg.C by 0.2mM IPTG, 8-10 is sample induced at 15 deg.C by 1.0mM IPTG, and 11-13 is sample induced at 15 deg.C by 0.2mM IPTG.

FIG. 3 shows solubility analysis of recombinant Protein I L AP1, in which M is Protein Marker, 1: I L AP1 non-induced sample, 2: I L AP1 induced sample, 3: 1.0mM IPTG induced precipitation sample at 37 deg.C, 4: 1.0mM IPTG induced supernatant sample at 37 deg.C, 5: 0.2mM IPTG induced precipitation sample at 37 deg.C, 6: 0.2mM IPTG induced supernatant sample at 37 deg.C, 7: 1.0mM IPTG induced precipitation sample at 15 deg.C, 8: 1.0mM IPTG induced supernatant sample at 15 deg.C, 9:15 deg.C 0.2mM IPTG induced precipitation sample at 10:15 deg.C, and 0.2mM IPTG induced supernatant sample at 10:15 deg.C.

FIG. 4 shows affinity purification analysis of recombinant Protein I L AP1, wherein M is Protein Marker, 1 is precipitation after disruption, 2 is supernatant after disruption, 3 is effluent after Ni-NTA incubation, 4 is Buffer B elution, 5 is Buffer C elution, and 6 is Buffer D elution.

FIG. 5 Western Blot assay purified Protein I L AP1, M Pre-stabilized Protein Marker, Sample after induction.

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: BufferG eluted sample; 7: buffer H eluted.

FIG. 7 SDS-PAGE analysis of the separation and purification of protein I L AP 1.

FIG. 8 Effect of protein I L AP1 on Spca-1 cell proliferation of lung adenocarcinoma.

FIG. 9 is a flow cytometry test that protein I L AP1 promotes lung adenocarcinoma Spca-1 apoptosis, and NC is a negative control.

FIG. 10 plasmid map of plasmid vector pCreat-SII-I L AP 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 line B L21 (DE3) was obtained from general biosystems (Anhui) Inc. (CP01010) and lung adenocarcinoma Spca-1 cell line was obtained from the cell resource center of Shanghai institute of Life sciences, 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 anti-tumor protein provided by the embodiment is shown as SEQ ID NO.1, and the coded nucleotide sequence is shown as SEQ ID NO. 2.

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

firstly, constructing a vector containing protein I L AP1 optimized codons:

the coding sequence for protein I L AP1 (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-I L AP1 (shown in FIG. 10).

Specifically, a protein I L AP1 amino acid sequence (SEQ ID NO.1) is determined through molecular docking screening, virtual amino acid mutation, bioinformatics means such as protein stability evaluation and the like, codon optimization of an escherichia coli expression system is carried out according to an I L AP1 amino acid sequence (SEQ ID NO.1), the optimized codon sequence (SEQ ID NO.2) is entrusted to a general biological system (Anhui) limited company for gene synthesis, then the obtained product is placed in a 5m L L B liquid culture medium and shaken at 37 ℃ overnight, a vector pCreat-SII is shaken at 37 ℃ in a 5m L L B liquid culture medium and shaken at overnight, plasmids are extracted, and the obtained product is subjected to enzyme digestion by BamHI and XhoI and then connected by T4 ligase, so that a recombinant large intestine expression plasmid pCreat-SII-I L AP1 is obtained (figure 10).

Secondly, the plasmid pCreat-SII-I L AP1 is transformed into B L21 (DE3) competent cells to obtain a recombinant strain DE3-I L AP1, and expression identification is carried out:

1. plasmid 2 μ L was added to 100 μ L competent bacteria and placed 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 L B culture solution;

after centrifugation at 220rpm for 1h at 3.37 ℃ all were plated on resistant L B plates and incubated overnight at 37 ℃ in an inverted position.

4. Selecting 5 plates, and inoculating the single clones in test tubes containing proper amount of resistant 4m L L B culture solution;

shaking at 5.37 deg.C and 220rpm to OD₆₀₀0.6-0.8;

6. taking out 1m L culture, centrifuging at 12000g room temperature for 5min, discarding supernatant, resuspending the thallus precipitate with 80 μ L1 × PBS Buffer solution, and adding 20 μ L5 ×L loading 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. 0.5m L culture was removed, centrifuged at 12000g for 5min at room temperature, the supernatant discarded, the pellet resuspended in 80. mu. L1 × PBS Buffer and 20. mu. L of 5 ×L loading Buffer was added.

9. SDS-PAGE analysis showed that the correct plasmid was transformed into E.coli competent cells and normally expressed fusion protein I L AP1, as shown in FIG. 1.

Thirdly, the heterologous expression condition of the recombinant protein I L AP1 is optimized:

1. selecting a single clone on a streak plate, and inoculating the single clone into 13 test tubes containing a proper amount of resistant 4m L L B culture solution;

shaking at 2.37 deg.C and 220rpm to OD₆₀₀0.6-0.8;

3. 0.4m L culture was removed, centrifuged at 12000g for 5min at room temperature, the supernatant was discarded, the pellet was resuspended in 80. mu. L1 × PBS Buffer and 20. mu. L of 5 ×L loading Buffer was added;

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. 0.2m L culture was removed, centrifuged at 12000g for 5min at room temperature, the supernatant discarded, and the pellet resuspended in 80. mu. L1 × PBS Buffer and 20. mu. L of 5 ×L loading Buffer was added.

6. SDS-PAGE analysis shows that the fusion protein I L AP1 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 of the recombinant protein I L AP1 is analyzed:

the induced bacterial liquid at 37 ℃ and the induced bacterial liquid at 15 ℃ are taken, centrifuged for 5min at room temperature of 2m L and 12000g, the supernatant is discarded, 1m L of Buffer A (20mM Tris, 300mM NaCl, pH8.0) is used for resuspension and centrifugation, the precipitate is crushed by ultrasonic waves ( phi 3, 15 percent, 3s/6s and 5min), the supernatant precipitate is respectively sampled and analyzed by SDS-PAGE, and the result is shown in figure 3, and the obtained target fusion protein I L AP1 is soluble protein.

Fifthly, amplifying expression and affinity purification of the recombinant protein I L AP 1:

1. inoculating the optimal clone strain into 1L L B culture medium containing appropriate antibiotics, and shaking at 37 deg.C and 220rpm to OD₆₀₀The 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. 3m L 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, the result is shown in figure 4, after selecting the optimal condition to enlarge the expression, the fusion protein I L AP1 can be purified by Ni-NTA affinity chromatography.

Sixthly, Western Blot test of purified protein I L AP 1:

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

2. semi-dry type electrotransfer, cutting a PVDF membrane into a size same as that of gel, cutting a small angle to help to judge the direction, soaking the membrane in methanol for 30s, transferring the membrane into electrotransfer liquid, cutting thin filter paper and thick filter paper into two pieces with the area slightly larger than that of the gel, placing the two pieces of thin filter paper and thick filter paper in a transfer buffer solution for soaking, cutting concentrated gel of the gel after the electrophoresis is finished, placing the gel in the electrotransfer liquid, placing the thick filter paper, the thin filter paper, an NC membrane, the gel, the thin filter paper and the thick filter paper on the plane of an electrotransfer in sequence, removing all bubbles between each layer, slightly pressing one end by hand, slightly moving the upper layer to one side by using a centrifugal tube of 50m L to remove the bubbles, changing one end by hand, removing the bubbles from the other end by using the centrifugal tube, linking the electrotransfer to the instrument, setting of 30V, protein less than 50KDa, using 30mins, protein more than 50KDa, and using 45 mins.

3. Sealing the membrane, incubating the membrane and finally processing the membrane, namely washing the transfer membrane, namely rinsing the transfer membrane for 3 times by 1 × PBST at room temperature for 5min, adding the solution slowly along the corner of the box to avoid flushing away substances bound on the membrane, pouring the PBST at the rotating speed of a shaking table of 40rpm, putting the PBST into 5% skimmed milk powder sealing solution, adjusting the shaking table to the lowest rotating speed, sealing the transfer membrane for 2h at room temperature, rinsing the transfer membrane for 5min by 1 × PBST at the room temperature, adding the antibody (1: 2000 to 5% skimmed milk powder) for 3 times, incubating for 1h, recovering the transfer membrane to-20 ℃ after use, rinsing the transfer membrane for 5min by 1 × PBST at the room temperature, slowly adding a small amount of EC L solution for 3 times to completely cover the membrane, standing for 2min, and taking a picture, wherein the result is shown in figure 5,

fusion protein I L AP1 was successfully purified.

Seventhly, removing the purified protein I L AP1 tag:

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

2. After dialysis, appropriate amount of SUMO Protease was added to the fusion protein, mixed well 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)

And 8, SDS-PAGE electrophoresis detection, wherein the result is shown in figure 6, and the active protein I L AP1 is successfully separated from the label through enzyme digestion reaction.

9. And (3) subpackaging the effluent sample or dialyzing the eluted sample to Buffer E, and subpackaging for subsequent activity verification, wherein SDS-PAGE electrophoresis shows that (shown in figure 7) the protein molecular weight is about 12.35kDa, I L AP1 is efficiently expressed in escherichia coli, the expression amount is 100 mg/L, the expression efficiency is high, and the purity of the protein reaches more than 90%.

Example 2

Protein I L AP1 inhibits lung adenocarcinoma Spca-1 cell viability assay:

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 5-10 × 10 cell concentration)⁴One/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％CO₂incubate 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% CO₂Incubating 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:

detection of growth of Spca-1 cells by MTT method after Spca-1 cells were treated with newly discovered protein I L AP1 at various concentrations for 24h, as shown in FIG. 8The survival rates of the Spca-1 cells are respectively 98.80%, 92.79%, 86.76%, 64.85% and 37.38% when the mass concentration of the protein I L AP1 is respectively 1 μ g/m L, 2 μ g/m L, 4 μ g/m L, 8 μ g/m L and 16 μ g/m L, the half inhibition concentration IC of the protein I L AP1 on the Spca-1 cells of the lung adenocarcinoma₅₀16 mu g/m L which is less than the concentration standard of 30 mu g/m L in modern tumor treatment pharmacology, and proves that the protein I L AP1 has strong lung adenocarcinoma resisting effect and has the potential of drug development.

Example 3

Protein I L AP1 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 about 35KD²⁺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) × 10⁵Per ml; adding 1ml of tumor cell suspension in 5% CO into 6-well culture plate₂Culturing 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% CO₂Culturing 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 1-5 × 10⁵A 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.

As shown in figure 9, when the protein I L AP1 acts on the Spca-1 cell, the apoptosis rate of a negative control is 3.9%, the protein I L AP1 promotes the apoptosis rate of the Spca-1 cell to be 36.0%, and the result shows that the protein I L AP1 has the effect of remarkably promoting the apoptosis of the lung adenocarcinoma cell Spca-1.

SEQUENCE LISTING

<110> Shanghai city academy of agricultural sciences

2, 1

<120> anti-tumor protein I L AP1, 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 antitumor protein I L AP1, which is characterized in that the amino acid sequence of the antitumor protein I L AP1 is shown as SEQ ID NO. 1.

2. A nucleotide sequence encoding the anti-tumor protein I L AP1 of claim 1, shown as 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 from the nucleic acid sequence shown in SEQ ID No.2, resulting in the recombinant expression plasmid pCreat-SII L AP 1.

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

6. A preparation method of an antitumor protein I L AP1 comprises the following steps:

culturing the recombinant cell of claim 5, inducing the expression of the fusion protein I L AP1, separating and purifying to obtain the antitumor protein I L AP 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 I L AP1 of claim 1 in the preparation of a medicament for the treatment or prevention of lung cancer.

9. The use of the anti-tumor protein I L AP1 of claim 1 in the preparation of a medicament for treating or preventing non-small cell lung cancer.

10. The use of the anti-tumor protein I L AP1 of claim 1 in the preparation of a medicament for treating or preventing lung adenocarcinoma.

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