CN111499695B - Functional protein MU20410, its coding sequence and application - Google Patents

Abstract

The invention relates to a functional protein MU20410, its coding sequence and application, the amino acid sequence of the protein is shown in SEQ ID NO. 1. The functional protein has good capacity of inhibiting proliferation and promoting apoptosis on lung cancer cells, and has higher anti-lung cancer tumor activity; the functional protein can be used for preparing anti-lung cancer drugs, and provides a new idea for the treatment of lung cancer.

Description

Functional protein MU20410, its coding sequence and application

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a functional protein MU20410, a coding sequence thereof and application thereof.

Background

Lung cancer is one of the most rapidly growing malignancies with the greatest threat to human health and life. In many countries, the incidence and mortality of lung cancer have been reported to be significantly higher in recent 50 years, with lung cancer incidence and mortality in men accounting for the first of all malignancies, in women accounting for the second, and mortality accounting for the second.

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, and is more likely to occur in women and patients without smoking.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a functional protein MU20410, a coding sequence and application thereof, wherein the functional protein provided by the invention has good abilities of inhibiting proliferation and promoting apoptosis on lung cancer cells, and has high anti-lung cancer tumor activity; the functional protein can be used for preparing anti-lung cancer drugs, and provides a new idea for the treatment of lung cancer.

The lung cancer resistant functional protein MU20410 has the amino acid sequence shown in SEQ ID No. 1.

The protein shown in SEQ ID No.1 has the function of resisting lung cancer, particularly lung adenocarcinoma, can better inhibit the proliferation of lung adenocarcinoma cells and promote the apoptosis of lung adenocarcinoma cells, and is a novel protein with the capacity of resisting lung cancer.

The invention also discloses a coding gene for coding the functional protein MU 20410.

On the basis of the coding gene provided by the invention, the functional protein can be obtained by recombinant expression through a genetic engineering technology, and of course, in other embodiments, the functional protein can also be prepared by a direct synthesis method, and the method is not limited by the method.

The coding gene sequence is shown in SEQ ID NO. 2.

The coding gene shown in SEQ ID NO.2 is a sequence which is subjected to codon optimization by the inventor of the invention, the functional protein has higher expression efficiency when the sequence is used for recombinant expression, and the yield of the obtained functional protein is also higher.

The invention discloses a preparation method of the lung cancer resistant functional protein MU20410 according to claim 1, which comprises the following steps:

(1) constructing a recombinant expression vector containing the coding gene;

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

(3) culturing the recombinant cell, separating and purifying to obtain the lung cancer resistant functional protein MU 20410.

The skeleton of the recombinant vector in the step (1) is a pCreat-SII plasmid vector.

The recombinant cell in the step (2) is escherichia coli.

The preparation method provided by the invention is simple to operate, the functional protein can be prepared in large quantity, and the obtained functional protein has anti-lung cancer activity, and can inhibit the proliferation of lung cancer cells and promote the apoptosis of the lung cancer cells.

The fusion protein is formed by fusing and expressing one or more of a tag protein, a fluorescent protein and an antibody molecule at two ends of the functional protein.

The invention discloses the modification based on the SEQ ID NO.1, for example, fusion of molecules such as expression labels, fluorescent protein or antibody and the like at two ends of the functional protein, and the modification belongs to the protection scope of the invention as long as the modification is carried out based on the sequence of the SEQ ID NO.1 and the obtained modified product can also play the roles of inhibiting the proliferation of lung cancer cells and/or promoting the apoptosis of the lung cancer cells.

The invention provides a pharmaceutical composition containing the protein or the fusion protein.

The pharmaceutical composition also contains pharmaceutically acceptable auxiliary materials.

The pharmaceutical composition is prepared by selecting proper auxiliary materials according to actual needs, and the auxiliary materials are all in the protection scope of the invention.

The invention provides application of the pharmaceutical composition in preparing a medicament for inhibiting lung cancer cell proliferation or promoting lung cancer cell apoptosis.

The invention provides application of the pharmaceutical composition in preparing a medicament for inhibiting lung adenocarcinoma cell proliferation or promoting lung adenocarcinoma cell apoptosis.

Advantageous effects

The functional protein provided by the invention has good abilities of inhibiting proliferation and promoting apoptosis on lung cancer cells, and has high anti-lung cancer tumor activity; the functional protein can be used for preparing anti-lung cancer drugs, and provides a new idea for the treatment of lung cancer.

The molecular weight of the protein is about 12.29kDa, MU20410 can be efficiently expressed in escherichia coli, the expression amount can reach 250mg/L, the expression efficiency is high, and the purity of the protein reaches more than 90 percent.

The protein MU20410 of the invention has good inhibition effect, generates obvious activity for inhibiting cancer cell proliferation at a concentration of more than 1 mug/mL, and has very obvious inhibition effect at 16 mug/mL.

The protein MU20410 of the invention has the function of remarkably promoting apoptosis of lung adenocarcinoma cells Spca-1.

Drawings

FIG. 1: identifying the expression of the recombinant protein MU 20410; in the figure, M: protein Marker; 1: MU20410 pre-induction samples; 2: MU20410 post-induction sample I; 3: sample II post MU20410 induction; 4: MU20410 post-induction sample III; 5: MU20410 post-induction sample IV; 6: MU20410 induced post-sample V.

FIG. 2: optimizing the heterologous expression condition of the recombinant protein MU 20410; in the figure, M: protein Marker; 1: MU20410 uninduced sample; 2-4: post-induction sample at 37 ℃ with 1.0mM IPTG; 5-7: 37 ℃ of: 0.2mM IPTG post-induction samples; 8-10: post-induction samples at 15 ℃ with 1.0mM IPTG; 11-13: post-induction samples with 0.2mM IPTG at 15 ℃.

FIG. 3: performing solubility analysis on the recombinant protein MU 20410; in the figure, M: protein Marker; 1: MU20410 uninduced sample; 2: post MU20410 induction samples; 3: precipitation of the sample after induction with 1.0mM IPTG at 37 ℃; 4: supernatant samples after induction with 1.0mM IPTG at 37 ℃; 5: precipitation of the sample after induction with 0.2mM IPTG at 37 ℃; 6: supernatant samples after induction with 0.2mM IPTG at 37 ℃; 7: precipitation of the sample after induction with 1.0mM IPTG at 15 ℃; 8: supernatant samples after induction with 1.0mM IPTG at 15 ℃; 9: precipitation of the sample after induction with 0.2mM IPTG at 15 ℃; 10: supernatant samples after induction with 0.2mM IPTG at 15 ℃.

FIG. 4: performing affinity purification analysis on the recombinant protein MU 20410; in the figure, M: protein Marker; 1: precipitating after crushing; 2: crushing and then cleaning the supernatant; 3: flowing out liquid after the Ni-NTA incubation; 4: buffer B elution sample; 5: buffer C eluted sample; 6: buffer D eluted.

FIG. 5: western Blot examination of purified protein MU 20410; m: 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 H eluted.

FIG. 7: protein MU20410 was isolated and purified from the unlabeled SDS-PAGE analysis.

FIG. 8: effect of protein MU20410 on proliferation of lung adenocarcinoma Spca-1 cells.

FIG. 9: flow cytometry test that the protein MU20410 promotes lung adenocarcinoma Spca-1 apoptosis; in the figure: NC: and (5) negative control. FIG. 10: plasmid map of vector pCreat-SII-MU 20410.

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 Co., Japan), T4 DNA ligase (TaKaRa Co., Japan), plasmid extraction kit (TIANGEN), agarose gel DNA recovery kit (TIANGEN), SUMO Protease (general biosystems (Anhui) Co., Ltd.), Annexin V-FITC/PI apoptosis detection kit (Nanjing Kai Biotech development Co., Ltd.)

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 functional protein for resisting lung cancer provided by the embodiment is shown in SEQ ID No.1, and the nucleotide sequence coded by the protein is shown in SEQ ID No. 2. In this example, the functional protein against lung cancer is designated as MU 20410.

The present example provides a method for preparing the anti-lung cancer functional protein, which comprises the following steps:

firstly, constructing a vector containing optimized codons of the protein MU 20410:

the coding sequence for protein MU20410 (SEQ ID NO.2) was inserted between the BamHI and XhoI restriction sites on the pCreat-SII vector to yield recombinant large intestine expression plasmid pCreat-SII-MU20410 (shown in FIG. 10).

Specifically, the method comprises the following steps: the amino acid sequence (SEQ ID NO.1) of the protein MU20410 is determined by molecular docking screening, virtual amino acid mutation, bioinformatics means such as protein stability evaluation and the like. According to the MU20410 amino acid sequence (SEQ ID NO.1), performing codon optimization of an Escherichia coli expression system, entrusting the optimized codon sequence (SEQ ID NO.2) to a general biological system (Anhui) limited company for gene synthesis, then placing the obtained product in 5mL LB liquid culture medium for overnight shake at 37 ℃, and placing the obtained product in 5mL LB liquid culture medium for overnight shake at 37 ℃ with a vector pCreat-SII. The plasmid was extracted, digested with BamHI and XhoI, and ligated with T4 ligase to give recombinant large intestine expression plasmid pCreat-SII-MU20410 (FIG. 10).

Secondly, the plasmid pCreat-SII-MU20410 is transformed into BL21(DE3) competent cells to obtain a recombinant strain DE3-MU20410, 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 MU20410 was normally expressed, as shown in FIG. 1.

Thirdly, the recombinant protein MU20410 heterologous expression condition is optimized:

1. selecting a single clone on a streak plate to be inoculated into 13 test tubes containing 4mL 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 MU20410 is expressed obviously under each optimized condition, and 0.2Mm IPTG is the optimal expression condition at 37 ℃.

Fourthly, performing solubility analysis on the recombinant protein MU 20410:

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 MU20410 was a soluble protein.

Fifthly, amplifying expression and affinity purification of the recombinant protein MU 20410:

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. 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. As shown in FIG. 4, the fusion protein MU20410 can be purified by Ni-NTA affinity chromatography after extensive expression at 37 ℃ under conditions of 0.2Mm IPTG, etc.

Sixthly, Western Blot test of purified protein MU 20410:

SDS-PAGE electrophoretic samples: pre-stabilized Protein Marker, post-mutagenic sample. Electrophoresis conditions are as follows: 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 the electrophoresis was completed, the gel was removed from the gel concentrate 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 the membrane 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. Pouring out PBST, putting into a closed liquid of 5% skimmed milk powder, adjusting the table to the lowest rotation speed, and sealing 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 FIG. 5, and the fusion protein MU20410 was successfully purified.

Seventhly, removing the purified protein MU20410 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 H (1 × PBS, 250mM Imidazole, ph 7.4).

SDS-PAGE electrophoretic detection. As shown in FIG. 6, the active protein MU20410 was successfully separated from the tag by the enzyme digestion.

7. And (4) subpackaging the effluent sample or the elution sample to Buffer E, and then subpackaging for subsequent activity verification. SDS-PAGE shows (see figure 7) that the molecular weight of the protein is about 12.29kDa, MU20410 is efficiently expressed in Escherichia coli, the expression level is 250mg/L, the expression efficiency is high, and the purity of the protein reaches more than 90 percent.

Example 2

And (3) detecting the activity of the MU20410 protein 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 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 of culture medium, and mixing uniformly;

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 MTT method is used for detecting the growth condition of the cells after Spca-1 cells are treated for 24 hours by the newly found protein MU20410 with different concentrations. As shown in FIG. 8, the growth inhibitory effect of the protein MU20410 on Spca-1 cells showed a good concentration dependence. When the mass concentration of the protein MU20410 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 98.89%, 94.36%, 84.77%, 66.39% and 47.55% respectively. Half inhibitory concentration IC of protein MU20410 on lung adenocarcinoma Spca-1 cells ₅₀ Is 16 MU g/mL, is less than the concentration standard of 30 MU g/mL related to modern tumor treatment pharmacology, and proves that the protein MU20410 has strong anti-lung adenocarcinoma effect and great potential to become an anti-lung adenocarcinoma drug.

Example 3

Detection of protein MU20410 promoting lung adenocarcinoma Spca-1 apoptosis:

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 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 the indexes for detecting the early apoptosis of the 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 by matching Annexin V to PI, and cells at different apoptosis stages can be distinguished. 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. regulating deviceThe whole cell concentration is 5-10 x 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 MU20410 on the apoptosis of Spca-1 cells after the protein MU20410 treats the Spca-1 cells for 36 hours. As shown in FIG. 9, when the protein MU20410 acts on Spca-1 cells, the apoptosis rate of the negative control is 3.9%, and the apoptosis rate of the protein MU20410 promoting Spca-1 cells is 13.6%, and the result shows that the protein MU20410 has the effect of remarkably promoting the apoptosis of lung adenocarcinoma cells Spca-1.

SEQUENCE LISTING

<110> Shanghai city academy of agricultural sciences

2, 1

<120> functional protein MU20410, coding sequence 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 Ala Asn Glu Ala Leu Ala Phe Leu Leu Val Ser

1 5 10 15

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

20 25 30

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

35 40 45

Lys Ala Tyr Glu Tyr Lys Val Val Val Asp Gly Val Asp Lys Gly Thr

50 55 60

Arg Arg Ala Phe Ser Val Ala Pro Asp Gly Ser Gln Lys Val Asn Phe

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser

<210> 2

<211> 342

<212> DNA

<213> Artificial sequence

<400> 2

atgagcctga ccggcgcaaa tgaagcactg gcctttctgc tggttagtca ggttaaaaag 60

attgcatttg actacacccc gaattattat cgtccgacca gcggctatac cgatgccgtg 120

acctttccga aagttctgac cgataaagcc tatgaatata aagttgtggt tgatggtgtt 180

gataaaggca cccgtcgcgc atttagtgtt gcaccggatg gcagtcagaa agttaatttt 240

ctggcatata acgccggtct gggcattccg accgcaagca gtattcaggt ttatgcagtt 300

gatccgaata ccggtattgc ctattatatt gcaaccgtga gt 342

Claims (9)

1. An anti-lung cancer functional protein MU20410, characterized in that the amino acid sequence of the protein is shown in SEQ ID NO. 1.

2. A gene encoding the functional protein MU20410 according to claim 1.

3. The encoding gene of claim 2, wherein the sequence of the encoding gene is shown as SEQ ID No. 2.

4. A method for preparing the anti-lung cancer functional protein MU20410 according to claim 1, comprising:

(1) constructing a recombinant vector containing the coding gene of claim 2;

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

(3) culturing the recombinant cell, separating and purifying to obtain the anti-lung cancer functional protein MU 20410.

5. The method of claim 4, wherein the recombinant vector has a backbone of a pCreat-SII plasmid vector.

6. The method according to claim 4, wherein the recombinant cell in the step (2) is Escherichia coli.

7. A pharmaceutical composition comprising the protein of claim 1.

8. Use of the pharmaceutical composition of claim 7 for the preparation of a medicament for inhibiting lung cancer cell proliferation or promoting lung cancer cell apoptosis.

9. Use of the pharmaceutical composition of claim 7 in the preparation of a medicament for inhibiting proliferation of lung adenocarcinoma cells or promoting apoptosis of non-small cell lung cancer.

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