CN103113468A - Anti-tumor protein - Google Patents

Abstract

The invention discloses an anti-tumor protein. The amino acid sequence of the protein is shown in the sequence listing SEQ ID NO6, and the nucleotide sequence of the protein is shown in the sequence listing SEQ ID NO5. The invention also discloses the experiment of killing tumor cells by the protein. The molecular weight of the protein described in the present invention is small, and the killing effect on tumor cells is good.

Description

an anti-tumor protein

technical field

The present invention relates to a protein. Specifically, the present invention relates to a protein capable of inhibiting tumor cell proliferation and inducing tumor cell apoptosis.

Background technique

Platelet-derived growth factor receptor-like gene (PDGFRL Platelet-derived growth factor receptor-like) is located at 8p21.3-p22, the full length of the gene is 1128bp, it is a newly discovered tumor suppressor gene in recent years, the gene is about 600kb, homologous to human and mouse , with a similarity of 90%. It is specifically and lowly expressed in tumor tissues of patients with colon cancer, breast cancer, lung cancer and liver cancer.

Studies have shown that Susanne Seitz and others confirmed that PDGFRL plays a major role in the occurrence and development of breast cancer through immunohistochemistry and other large amounts of data. The full name of the expressed protein is PDGFRL protein (Platelet-derived growth factor receptor-like protein), with a total of 375 amino acids. PDGFRL is named PDGFR-like protein because of its significant similarity with the extracellular domain of PDGFR beta, and it has been confirmed that it participates in the PDGF signaling pathway as a tumor suppressor gene (TSGTumor Suppressor Gene), and has an antagonistic effect on PDGF and PDGF. its receptor interactions.

A few scholars believe that the reason why PDGFRL can be used as TSG is that PDGFRL has a high frequency of genes close to loss of heterozygosity in non-small cell lung cancer, liver cancer and prostate, which helps its missense repair function. Studies have reported that recombinant human PDGFRL protein can effectively inhibit the proliferation and migration of colon cancer cell line HCT-116.

Since PDGFRL protein has the effect of inhibiting tumor cells, but its molecular weight is relatively large, it is not convenient for the delivery of protein drugs, and it has strong immunogenicity. In order to solve the inconvenience caused by its large molecular weight, the present invention analyzes, screens, and optimizes the protein sequence of PDGFRL protein to obtain a polypeptide that not only has a small molecular weight, but also has higher tumor cell killing activity than the full-length PDGFRL protein.

Contents of the invention

In order to solve the inconvenience caused by the large molecular weight of PDGFRL protein, the present invention solves the technical problem described by the following scheme.

The molecular weight of the active region protein D2 designed and screened in the present invention is much smaller than that of the PDGFRL protein, and its killing effect on tumor cells is better than that of the PDGFRL protein. The amino acid sequence of the active region protein D2 is shown in the sequence table SEQ ID NO6, and the activity See SEQ ID NO5 in the sequence table for the nucleotide sequence of the domain protein D2, and the sequences of the upstream and downstream primers for amplifying the active domain protein gene GD2 are SEQ ID NO3 and SEQ ID NO4, respectively.

A method for preparing active region protein D2, comprising the following steps:

1) Analyze its protein active region according to the PDGFRL gene sequence, and design primers. The sequences of the primers are shown in the sequence table SEQ ID NO3 and SEQ ID NO4, and the target gene fragment is amplified by PCR method, and the amplified target fragment is connected to the carrier On, the resulting recombinant plasmids were transformed into cloning cells, and the recombinant plasmids were extracted and sequenced;

2) Transform the expression vector containing the target gene correctly into the corresponding expression cells, induce the expression of the target protein, obtain the full-length PDGFRL protein and its active region protein, prepare protein samples, and measure the protein concentration;

3) Identify the protein obtained in the previous step by SDS-PAGE and Western Blot, and check whether the obtained protein is successfully expressed.

The carrier described in the above method can be a prokaryotic cell expression vector, a eukaryotic cell expression vector or an insect cell expression vector, and the carrier is preferably Pet28a(+); the described expression cell can be a prokaryotic expression cell, a eukaryotic expression cell or Insect cells, the expression cells are preferably E.coli BL21(DE3).

The present invention detects the influence of PDGFRL protein and its active region protein on tumor cells through the following experiments:

The inhibitory effect of PDGFRL protein and its active region protein on tumor cell proliferation was detected by SRB method. The described SRB method is a method for detecting cell proliferation. Its principle is that SRB, namely sulforhodamine B (Sulforhodamine B, SRB), is a pink anionic dye, soluble in water, and can specifically It selectively binds to the basic amino acids that make up proteins in the cell; it produces an absorption peak at a wavelength of 540nm, and the absorbance value is linearly positively correlated with the cell mass, so it can be used for quantitative detection of the cell number. The inhibitory effect of the PDGFRL protein and its active region protein in the present invention on tumor cell proliferation increases gradually as the protein concentration increases. The results show that the PDGFRL active region protein D2 designed in the present invention has more obvious inhibitory effect on cells than the full-length PDGFRL protein, and the D2 protein has the most significant inhibitory effect. BSA and empty protein were used as negative controls, which had no obvious inhibitory effect on cells.

The effect of PDGFRL protein and its active domain protein D2 on the cell cycle of colon cancer cells was detected by flow cytometry, and the results showed that both PDGFRL protein and active domain protein D2 could arrest the CT-26 cell cycle in the G2/M phase, and this The effect of the active domain protein D2 screened by the invention design is the most remarkable.

Whether PDGFRL protein and its active domain protein D2 can induce colon cancer cell apoptosis by flow cytometry, the results showed that both PDGFRL protein and its active domain protein D2 could induce CT-26 cell apoptosis, and with the stimulation time Prolonged, the apoptosis of CT-26 was more obvious. Consistent with the above cell cycle detection results, the pro-apoptotic effect of active domain protein D2 is more significant.

Another object of the present invention is to provide the application of the active region protein D2 of the present invention in the preparation of anti-tumor drugs, that is, the anti-tumor drugs with the D2 protein of the present invention as an active ingredient, especially for the treatment of colon cancer, breast cancer, lung cancer, liver cancer.

When necessary, the above-mentioned medicine may also contain one or more pharmaceutically acceptable carriers, which include common diluents, excipients, fillers, disintegrants, etc. in the field of pharmacy.

Description of drawings

Figure 1.A: Coomassie Brilliant Blue staining diagram of PDGFRL full-length protein and Western blotting detection diagram, B: Coomassie Brilliant Blue staining diagram and Western blotting detection diagram of active region protein D2;

Figure 2. SRB method to detect the proliferation inhibition of four proteins on CT-26 cells, total: PDGFRL total protein; D2: active region protein D2; BSA: bovine serum albumin; PET: pet28a(+) empty protein P<0.05 ;

Figure 3. PI staining by flow cytometry after stimulation of CT-26 cells by PDGFRL total protein and active region protein D2; pathway: FL2-A;

Figure 4.A Apoptosis detection diagram of CT-26 cells stimulated by total PDGFRL protein and active region protein D2 for 16 hours; pathway: FITC and PI; Apoptosis detection diagram; pathway: FITC and PI; Figure 4 Apoptosis detection diagram of CT-26 cells stimulated by CPDGFRL total protein and active region protein D2 for 24 hours; pathway: FITC and PI.

Detailed ways

The present invention will be further elaborated below in conjunction with specific examples, but by no means limited to the scope described in the examples. The test methods that do not show specific conditions in the following examples are generally in accordance with conventional conditions.

The extraction of embodiment 1PDGFRL protein and active region gene

1. Experimental materials

The expression strain E.coli BL21(DE3) was purchased from Yuanpinghao Biotechnology Co., Ltd.; the target gene was synthesized by Yingjun Biological Products Co., Ltd. and constructed in pMD18-T vector (Takara). ) plasmid vectors are kept in this laboratory. Agarose affinity medium nickel column (Ni) was purchased from National Biochemical Engineering Technology Research Center (Beijing); His-Taq tag antibody was purchased from Tianjin Sanjian Biotechnology Co., Ltd. (Product No.: KM8001), other reagents were imported or domestic analysis pure.

DNA sequencing was completed by Beijing Tianyi Huiyuan Biotechnology Co., Ltd. The mouse colon cancer cell line CT-26 was purchased from ATCC, USA.

2. Design primers to transfer PDGFRL gene

Firstly, primers were designed to obtain the full gene sequence of the mouse PDGFRL protein. The primers were as follows: SEQ ID NO1: TT CCATGGCATTCTCCTAAGAAC; SEQ ID NO2: AA CTCGAGGGAAAACTCAACAGT, where CCATGG is the enzyme cleavage site of SEQ ID NO1; CTCGAG is the enzyme cleavage site of SEQ ID NO2 Sites; underlined bases are protected bases.

The gene sequence length of mouse PDGFRL obtained by PCR amplification was 1128bp. Amplification program: pre-denaturation at 94°C, denaturation at 94°C for 10s, gradient annealing at 50°C, extension at 72°C for 90s, 35 cycles, and finally extension at 72°C. The obtained gene sequence of 1128bp PDGFRL was amplified by those skilled in the art based on the above primers by using the plasmid carrying the full-length sequence of mouse PDGFRL (synthesized by Yingjun Biologicals Co., Ltd.) as a template, and will not be given here.

3. Analysis of genes in the active region of PDGFRL, design of primers and transfer of corresponding genes

Search in Genebank to obtain the public information of the PDGFRL gene sequence and amino acid sequence, and use the primerpremier5.0 software to design multiple sets of upstream and downstream primers on the conserved functional domain of the exon coding region to amplify different regions of the PDGFRL gene. Screen the best active region of PDGFRL gene. Among them, the anti-tumor activity of the protein expressed by the gene amplified by the upstream primer set UP2 and the downstream primer down2 of the designed primer set is the highest. Wherein the sequence of primer UP2 is shown in SEQ ID NO3, and the sequence of primer down2 is shown in SEQ ID NO4. The gene amplified by the upstream and downstream primers is 312bp, and is named the active region gene GD2, whose sequence is shown in SEQ ID NO5, and the amino acid expressed by it is named D2, whose sequence is shown in SEQ ID NO6.

The program of the gene in the active region amplified by PCR is as follows: pre-denaturation at 94°C, denaturation at 94°C for 10s, gradient annealing at 55°C, extension at 72°C for 90s, 34 cycles, and finally extension at 72°C.

The amplified product was gel-cut and recovered after agarose electrophoresis, and the target fragment was digested with the pet28(a) vector at 37°C for 2 hours and ligated overnight. Transformation into clonal type JM109 competent cells, the steps are as follows: take out Escherichia coli competent cells JM109 from -70°C, immediately place them on ice for about 10 minutes, and let them thaw naturally. Take PDGFRL, GD2 and the pet28(a) carrier respectively and add 8μl to 100μl competent cells, mix well, ice-bath for 30min, heat shock at 42℃ for 90s, gently take out and place in ice-bath for 3min, repeat ice-bath and Heat shock process to increase conversion efficiency. In an ultra-clean bench, add 400 ml of non-resistant LB medium to the tube, place it in a shaker at 37°C for 45 minutes to revive the bacteria (shaker ≤ 225 rpm). Centrifuge at 6000rpm for 1min, discard the supernatant, and resuspend the bacteria in the remaining 200μl medium, spread evenly on the LB plate containing Amp (100μg/ml), wait for 3min, and incubate overnight in a 37℃ incubator (12-18h ) to observe the colony growth. Plate overnight. Single clones were picked, cultured and sequenced.

The sequencing results showed that the gene extraction was completely correct, without base mutations and frame shifts.

Example 2 Expression of active region protein D2

The plasmid with correct sequencing was transferred into E. coli E.coli BL21(DE3), induced by prokaryotic IPTG to obtain PDGFRL and PDGFRL active region protein D2, prepare protein samples, measure protein concentration, take 30μg samples for 12% dodecane Sodium sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), after electrophoresis, the protein bands were transferred to PVDF membrane, and the His-tagged primary antibody diluted in 5% BSA was used to incubate overnight at 4°C, and horseradish was added overnight. The secondary antibody to oxidase (HRP) was incubated at room temperature for 1 h, developed with ECL, and exposed in a dark room.

The His tag on the Pet28a(+) carrier can specifically bind to the nickel column, and the obtained protein is bound to the nickel column, eluted with imidazole, and the eluate is stained with Coomassie brilliant blue to verify whether there is protein. The results showed that the protein induced by IPTG was mainly concentrated in the supernatant of the supernatant after ultrasonic crushing, which was a soluble protein. The expressed protein was identified by SDS-PAGE and Western Blot with high purity and strong specificity.

Coomassie blue staining and western blotting indicated that PDGFRL protein and PDGFRL active region protein D2 were successfully induced (Fig. 1).

Example 3 Functional identification of PDGFRL and active region protein D2

1. Experimental method

1.1 The effect of the obtained protein detected by SRB method on the proliferation of tumor cells

Cells of CT-26 line were evenly plated in 96 wells, 2000 cells/well, 100 μl/well system, cultured at 37°C, 5% CO ₂ for 24 hours, then grouped and added blank, PDGFRL whole protein, active region protein D2, pet28a ( +) Empty protein, bovine serum albumin (BSA) treatment, the concentration is 400, 200, 100, 50, 25, 12.5, 0 (unit: μg/ml), each set up 3 parallel wells, the final volume is 200μl/ Incubate the wells at 37°C and 5% _CO2 for 24 hours, suck out the culture supernatant with a vacuum pump, add 70 μl of 0.4% SRB/well, let stand at room temperature for 40 minutes, wash the plate three times with 1% acetic acid solution, and finally wash the plate with 1% acetic acid solution for each well. 200 μl of 10 mM Tris solution was added to dissolve the dye solution, and the absorbance (OD) value at 570 nm was detected with a microplate reader. Data were processed with Prism 3.0 software.

1.2 Cell cycle detection by flow cytometry

Cells of CT-26 line were evenly plated in 6-well plates, 1× ¹⁰⁶ cells/well, cultured at 37°C, 5% CO ₂ for 24 hours, then grouped with blank, PDGFRL whole protein, active region protein D2, added The protein concentration was 50 μg/ml, the cells were collected by trypsinization, and the experiment was carried out according to 5×10 ⁵ per tube, washed twice with PBS, fixed with 70% ice ethanol at -20°C for 36 hours, washed twice with PBS, and 100 μl of 1 mg/ml RNase A was added, Incubate for 30 min at 37°C in the dark, then add 100 μl of propidium iodide (PI) staining solution and incubate for 30 min in the dark, then detect with flow cytometry, and analyze the cell cycle ratio with Cell Quest software.

1.3 Detection of cell apoptosis by flow cytometry

Cells of CT-26 line were evenly spread in 6-well plate, 1×10 ⁶ cells/well, and cultured at 37°C, 5% CO ₂ for 24h. The groups were as follows: no protein group, PDGFRL full-length protein group, active region protein D2, pet28a(+) empty protein group and bovine serum albumin group (BSA); the concentration was 100 μg/ml; two time points were set: 16h and 24h. Collect CT-26 cells by trypsinization, wash twice with normal saline; divide into 2 batches, each with 5 groups and blank and single-stained controls, protein concentration: 100μg/ml; resuspend 20ug of FITC-labeled in 100ul Binding buffer /ml Annexin V10ul, incubate at room temperature in the dark for 30min; then add 50μg/ml PI5μl, incubate at room temperature in the dark for 5min, add 400μl Binding buffer to dilute and detect on the machine.

2 Experimental results

2.1 SRB detects the inhibition of PDGFRL protein on the proliferation of colon cancer cells

The results of detecting the inhibitory rate of the target protein on CT-26 cells by the SRB method are shown in Figure 2. As shown in Figure 2, with the increase of the concentration, the inhibitory effect of PDGFRL protein and protein D2 on the cells gradually increased, and protein D2 inhibited the growth of tumor cells. Cell proliferation inhibition was more pronounced than that of the full-length protein. BSA and empty protein were used as negative controls, which had no obvious inhibitory effect on cells.

2.2 The effect of PDGFRL protein and protein D2 on the cell cycle of colon cancer cells detected by flow cytometry

According to the effect of protein concentration on cells in the SRB experiment, we designed the experimental conditions as follows: protein concentration was 50 μg/ml, cultured for 24 hours, and the groups were no protein untreated group, full-length protein group, and domain II protein group. As shown in Figure 3, both proteins can arrest CT-26 cell cycle in G2/M phase, especially protein D2 is more significant.

2.3 The effect of PDGFRL protein and protein D2 on colon cancer cell apoptosis detected by flow cytometry

The groups were as follows: no protein group, PDGFRL full-length protein group, active region protein D2 group, pet28a(+) empty protein group and bovine serum albumin group (BSA); the concentration was 100 μg/ml; two time points were set: 16h and 24h. The results showed that both PDGFRL full-length proteome and protein D2 could induce the apoptosis of CT-26 cells, and the apoptosis of CT-26 cells was more obvious with the prolongation of stimulation time. Consistent with the results of previous cell cycle assays, the pro-apoptotic effect of protein D2 was more significant (Fig. 4A, B, C).

Claims (10)

1. An anti-tumor protein, characterized in that its amino acid sequence of the protein is as shown in the sequence table SEQ ID NO: 6. 2. The protein according to claim 1, wherein the nucleotide sequence of the protein is as shown in SEQ ID NO: 5 in the sequence table. 3. a primer for amplifying the protein nucleotide sequence according to claim 2, characterized in that, said primer comprises an upstream primer and a downstream primer, and its sequence is as shown in sequence table SEQ ID NO: 3 and SEQ ID NO: 4 . 4. A method for preparing the protein according to claim 1 or 2, comprising the steps of: 1) Analyze its protein active region according to the PDGFRL gene sequence, and design primers, the sequences of the primers are shown in the sequence table SEQ ID NO: 3 and SEQ ID NO: 4, amplify the target gene fragment by PCR method, and amplify The target fragment was connected to the vector, and the obtained recombinant plasmid was transformed into cloning cells, the recombinant plasmid was extracted and sequenced, and the full-length PDGFRL gene was amplified at the same time as a positive control; 2) Transform the expression vector containing the target gene correctly into the corresponding expression cells, induce the expression of the target protein, obtain the full-length PDGFRL protein and its active region protein, prepare protein samples, and measure the protein concentration; 3) The protein obtained in the previous step was identified by SDS-PAGE and Western Blot to detect whether the protein was successfully expressed. 5. The method of claim 4, wherein the carrier is Pet28a(+). 6. The method of claim 4, wherein the expressing cell is E.coliBL21(DE3). 7. A pharmaceutical composition, characterized in that the pharmaceutical combination comprises the protein according to claim 1 or 2. 8. The use of the protein according to claim 1 or 2 in the preparation of medicines for treating tumor diseases. 9. The use according to claim 6, characterized in that the tumor is colon cancer. 10. The use according to claim 6, wherein the tumor is breast cancer.

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