CN113717289A - SAC-TRAIL fusion protein and preparation method and application thereof - Google Patents
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Abstract
The invention relates to a SAC-TRAIL fusion protein, a preparation method and application thereof, which utilize flexible link (G)4S)3Fusing anti-tumor protein TRAIL to the C-terminal of SAC protein to form SAC-TRAIL fusion protein, wherein the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1; the preparation method of the fusion protein comprises the following steps: (1) construction of pET28aSUMO-SAC-TRAIL recombinant plasmid; (2) heterologous expression of SUMO-SAC-TRAIL fusion protein by using escherichia coli BL21(DE 3); (3) the SAC-TRAIL fusion protein is efficiently separated and purified by utilizing nickel ion affinity chromatography and weak cation exchange resin. Compared with the prior art, the SAC-TRAIL fusion protein is prepared for the first time, the purity of the purified protein reaches 94 percent, and the prepared protein drug SAC-TRAIL can effectively inhibit the proliferation and IC of ovarian cancer cells SK-OV-3 and breast cancer cells MCF-750The values were 4.42ng/mL and 0.053. mu.g/mL, respectively.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a SAC-TRAIL fusion protein and a preparation method and application thereof.
Background
Among gynecological tumors, ovarian cancer and breast cancer are common malignant tumors, and the incidence rate is higher and higher. Current treatments for cancer rely primarily on chemotherapy, radiation therapy, immunotherapy, and targeted therapies, but most therapeutic strategies do not completely eliminate cancer cells. TRAIL has a broad apoptosis-inducing effect on cancer cells in malignant tumors, hardly inhibits the proliferation of normal cells, and is identified as an antitumor protein. TRAIL can bind with death receptor 4/5(DR4/5) on the surface of cancer cell, and activate a series of apoptosis signal pathways to promote apoptosis of cancer cell. TRAIL has shown good application prospects in cancer treatment, but when combined with non-death receptors (such as DcR1), TRAIL can activate, for example, NF- κ B kinase signaling pathway to stimulate pathways promoting cell survival, proliferation and migration, which is also one of the reasons for the development of TRAIL resistance by tumor cells. Clinical data also show that TRAIL has no obvious proliferation inhibition effect on tumor cells with high NF-kB activity, so that the tumor cells show drug resistance to TRAIL treatment. Par-4, a tumor suppressor, was originally found in apoptotic prostate cancer cells and is widely expressed in a variety of cells. The sequence of Par-4 was analyzed for deletions to determine the core domain to be SAC, which comprises 59 amino acids. The core domain SAC can enter the nucleus, activate caspase, inhibit NF-kB kinase signal path and selectively induce cancer cell apoptosis, has little cytotoxicity to normal cells or tissues, and is proved to be a promising candidate for cancer treatment.
SAC can effectively inhibit the activity of NF-kB, and SAC recombinant protein has the potential of being developed into an anti-tumor drug. So far, the research of SAC has focused on the research of the action mechanism, and no report has been found on the research of preparing SAC fusion protein by using genetic engineering technology and using the SAC fusion protein to overcome the TRAIL resistance of tumor cells.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a high-purity SAC-TRAIL fusion protein, a preparation method and application thereof.
The purpose of the invention can be realized by the following technical scheme:
in a first aspect of the invention, a SAC-TRAIL fusion protein is provided, the antitumor protein TRAIL being linked via a flexible linkage (G)4S)3Fused to the C-terminal of the SAC protein to form the SAC-TRAIL fusion protein, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1.
Preferably, the amino acid sequence of the SAC protein is shown as SEQ ID NO. 2, and the amino acid sequence of the anti-tumor protein TRAIL is shown as SEQ ID NO. 3.
Preferably, the nucleotide sequence of the gene encoding the fusion protein is shown in SEQ ID NO. 4.
The second aspect of the present invention provides a method for preparing the SAC-TRAIL fusion protein, comprising the steps of:
(1) constructing pET28a/SUMO-SAC-TRAIL recombinant plasmid;
(2) heterologous expression of SUMO-SAC-TRAIL fusion protein by using escherichia coli BL21(DE 3);
(3) the SAC-TRAIL fusion protein is efficiently separated and purified by utilizing nickel ion affinity chromatography and cation exchange resin.
Preferably, in the step (1), the construction of pET28a/SUMO-SAC-TRAIL recombinant plasmid comprises the following steps:
(1-1) taking an upstream primer with a sequence shown as SEQ ID NO:5 and a downstream primer with a sequence shown as SEQ ID NO:6 as reaction primers, taking a full-length SUMO-SAC gene as a template, wherein the nucleotide sequence of the SUMO-SAC gene is shown as SEQ ID NO:7,
an upstream primer with a sequence shown as SEQ ID NO. 8 and a downstream primer with a sequence shown as SEQ ID NO. 9 are taken as reaction primers, a full-length TRAIL gene is taken as a template, wherein the nucleotide sequence of the TRAIL gene is shown as SEQ ID NO. 10,
carrying out PCR reaction to obtain coding genes of SUMO-SAC and TRAIL protein;
(1-2) recovering the PCR product in the step (1) by using an upstream primer with a sequence shown as SEQ ID NO:5 and a downstream primer with a sequence shown as SEQ ID NO:9 as reaction primers, using full-length SUMO-SAC and TRAIL genes as templates, and carrying out fusion PCR reaction to obtain a coding gene of the SUMO-SAC-TRAIL fusion protein, namely a target gene;
(1-3) recovering the PCR product in the step (2) by gel, carrying out double enzyme digestion on a target gene and a pET28a expression plasmid by Bmt I and Xho I, connecting the target gene and the pET28a expression plasmid by T4 ligase to obtain a pET28a/SUMO-SAC-TRAIL recombinant plasmid, transforming the recombinant plasmid into escherichia coli BL21(DE3), selecting a positive clone, and carrying out sample sending and sequencing.
Preferably, in step (1), the conditions of the PCR reaction are: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 20s, and circulating for 30 times, and final extension at 72 deg.C for 10 min.
Preferably, in the step (2), the heterologous expression of the SUMO-SAC-TRAIL fusion protein by using Escherichia coli BL21(DE3) comprises the following steps: coli BL21(DE3) containing the pET28a/SUMO-SAC-TRAIL recombinant plasmid, which was sequenced correctly, was cultured at 37 ℃ in LB medium containing 50mg/L kanamycin resistance, when OD was determined600When the concentration reaches 0.6, isopropyl-beta-D-thiogalactopyranoside with the final concentration of 0.1mM is added, induction is carried out for 16h at 18 ℃, and the SUMO-SAC-TRAIL fusion protein is expressed in a heterologous mode.
Preferably, in the step (3), the isolating and purifying of the SAC-TRAIL fusion protein comprises the following steps:
(3-1) purification of SUMO-SAC-TRAIL fusion protein Using His tag:
utilizing nickel column affinity chromatography, selectively adsorbing SUMO-SAC-TRAIL fusion protein with His label to nickel column, purifying to obtain SUMO-SAC-TRAIL fusion protein;
(3-2) collecting the purified SUMO-SAC-TRAIL fusion protein, carrying out enzyme digestion on the fusion protein by SUMO protease, removing the SUMO label, carrying out weak cation exchange chromatography on enzyme digestion reaction liquid to remove the SUMO label, eluting and collecting SAC-TRAIL protein by PB containing 500mM NaCl, and obtaining the SAC-TRAIL fusion protein.
The third aspect of the invention provides application of the SAC-TRAIL fusion protein, and the SAC-TRAIL fusion protein is used for inhibiting proliferation of ovarian cancer cells SK-OV-3 and breast cancer cells MCF-7.
The invention provides application of the SAC-TRAIL fusion protein in preparing a therapeutic drug for ovarian cancer cells SK-OV-3 and breast cancer cells MCF-7.
The activity of the purified SAC-TRAIL fusion protein was examined by the CCK-8 method. Treating the ovarian cancer cell SK-OV-3 and the breast cancer cell MCF-7 with different concentrations, calculating the number of living cells after 72 hours, calculating corresponding cell activity, and analyzing the inhibition effect of the fusion protein on the cancer cell proliferation.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) first use of a Flexible Link (G)4S)3The core structural domain SAC of the prostate apoptosis effector-4 is fused to the N-terminal of TRAIL to form SAC-TRAIL fusion protein. Suitable linkage can not only improve the expression efficiency of the fusion protein, but also help the polypeptide chain to fold correctly and maintain the biological activity of the structural domain. Flexible Link (G) utilized in the present invention4S)3Can keep proper space distance between SAC and TRAIL structural domain, and can maximally exert the anti-tumor activity of the fusion protein.
(2) pET28a/SUMO-SAC-TRAIL recombinant plasmid is constructed, and SAC-TRAIL fusion protein is prepared through heterologous expression, separation and purification. Through two-step purification method of nickel ion affinity chromatography and weak cation exchange resin, the purity of the SAC-TRAIL fusion protein is 94%.
(3) The SAC protein of the SAC-TRAIL fusion protein can transport the fusion protein into cells to play a role. The invention firstly detects the activity of SAC-TRAIL fusion protein in vitro, and CCK-8 experimental results show that the proliferation of ovarian cancer cells SK-OV-3 and breast cancer cells MCF-7 can be effectively inhibited. Semi-lethality IC of TRAIL and SAC-TRAIL to SK-OV-3 cells50Values of 64. mu.g/mL and 4.4, respectively2 ng/mL; half-lethality IC of TRAIL and SAC-TRAIL on MCF-7 cells50The values were 53.18. mu.g/mL and 0.503. mu.g/mL, respectively. The SAC-TRAIL fusion protein prepared by the invention obviously enhances the anti-tumor activity of the TRAIL protein, and lightens the drug resistance of tumor cells to the TRAIL protein, so that the SAC-TRAIL fusion protein has great application prospect in tumor treatment.
Drawings
FIG. 1 is a plasmid map of pET28a/SUMO-SAC-TRAIL recombinant plasmid;
FIG. 2 is an SDS-PAGE analysis of expression and purification of the SUMO-SAC-TRAIL fusion protein purified using His-tag affinity chromatography;
FIG. 3 is an analysis of expression and purification of SAC-TRAIL fusion protein with the SUMO tag removed by SDS-PAGE analysis;
FIG. 4 is a graph showing the proliferation inhibitory effect of purified SAC-TRAIL fusion protein on ovarian cancer cells SK-OV-3;
FIG. 5 is a graph showing the proliferation inhibitory effect of purified SAC-TRAIL fusion protein on breast cancer cells MCF-7.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
Examples
A SAC-TRAIL fusion protein is formed by fusing TRAIL, an anti-tumor protein, to the C-terminal of a SAC protein to form a fusion protein, wherein the amino acid sequence of the fusion protein is shown as SEQ ID NO:1 is shown. The amino acid sequence of the SAC protein is shown as SEQ ID NO:2, and the amino acid sequence of the TRAIL protein is shown as SEQ ID NO. 3. The nucleotide sequence of the gene for coding the fusion protein is shown as SEQ ID NO. 4.
A method of preparing a SAC-TRAIL fusion protein, the method comprising the steps of:
(1) construction of pET28a/SUMO-SAC-TRAIL recombinant plasmid:
a. an upstream primer with a sequence shown as SEQ ID NO. 5 and a downstream primer with a sequence shown as SEQ ID NO. 6 are used as reaction primers, and a full-length SUMO-SAC gene is used as a template, wherein the nucleotide sequence of the SUMO-SAC gene is shown as SEQ ID NO. 7; taking an upstream primer with a sequence shown as SEQ ID NO. 8 and a downstream primer with a sequence shown as SEQ ID NO. 9 as reaction primers, taking a full-length TRAIL gene as a template, wherein the nucleotide sequence of the TRAIL gene is shown as SEQ ID NO. 10, and carrying out PCR reaction to obtain coding genes of SUMO-SAC and TRAIL protein;
the conditions for the PCR reaction were: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 20s, and circulating for 30 times, and final extension at 72 deg.C for 10 min. The PCR product was identified by agarose gel and the desired fragment was recovered using gel recovery kit (Omiga).
b. Recovering the PCR product, and fusing PCR reaction by using an upstream primer with a sequence shown as SEQ ID NO. 5 and a downstream primer with a sequence shown as SEQ ID NO. 9 as reaction primers and using full-length SUMO-SAC and TRAIL genes as templates to obtain an encoding gene of the SUMO-SAC-TRAIL fusion protein, namely a target gene;
c. and recovering the PCR product, performing double enzyme digestion on the target gene and the pET28a expression plasmid by using Bmt I and Xho I, connecting by using T4 ligase, and standing overnight at 16 ℃ to obtain a pET28a/SUMO-SAC-TRAIL recombinant plasmid, wherein the plasmid map of the recombinant plasmid is shown in figure 1, and the connection product is transformed into E.coli BL21(DE3) competence. Selecting positive clones, preliminarily verifying the positive clones by bacterial liquid PCR and recombinant plasmid double enzyme digestion, and carrying out sample sequencing on the recombinant bacteria which are preliminarily verified to be correct.
(2) The SUMO-SAC-TRAIL fusion protein is expressed by using escherichia coli BL21(DE3) in a heterologous manner:
coli BL21(DE3) containing the pET28a/SUMO-SAC-TRAIL recombinant plasmid, which was sequenced correctly, was cultured at 37 ℃ in LB medium containing 50mg/L kanamycin resistance, when OD was determined600When the concentration reaches 0.6, isopropyl-beta-D-thiogalactopyranoside with the final concentration of 0.1mM is added, induction is carried out for 16h at 18 ℃, and the SUMO-SAC-TRAIL fusion protein is expressed in a heterologous mode. The cells were centrifuged at 8000 Xg for 10min at 4 ℃ to collect the cells. Cells were disrupted by sonication and the supernatant was collected.
(3) Separating and purifying SAC-TRAIL fusion protein:
a. purification of SUMO-SAC-TRAIL fusion protein using His tag (see FIG. 2 for purification results);
the nickel column (2mL/min) was equilibrated with 5CV of 20mM PBS buffer. The supernatant containing the SUMO-SAC-TRAIL fusion protein was applied at a flow rate of 2mL/min, and the His-tagged SUMO-SAC-TRAIL fusion protein was selectively affinity-adsorbed. The desmin was washed away with 20mM PBS buffer (pH 7.4) at a flow rate of 2mL/min, and finally the SUMO-SAC-TRAIL fusion protein was eluted with PBS buffers containing different concentrations of imidazole (pH 7.4) at a flow rate of 2 mL/min.
b. Removing the SUMO label to obtain SAC-TRAIL fusion protein (the purification result is shown in figure 3);
the SUMO-SAC-TRAIL fusion protein was cleaved with 0.05U of SUMO protease and treated at 37 ℃ for 2h to remove the SUMO tag. The enzyme digestion reaction was subjected to weak cation exchange chromatography to remove the SUMO tag, and SAC-TRAIL protein was eluted and collected with PB containing 500mM NaCl.
(4) Quantification of purified SAC-TRAIL protein
Total protein mass was determined using the Bradford method. And (3) carrying out SDS-PAGE electrophoresis on the collected liquid in each step of the purification operation, staining the collected liquid for 30min by using Coomassie brilliant blue R-250 after electrophoresis, then decolorizing the collected liquid by using a decolorizing liquid, and finally scanning the gel by using computer software Image J to analyze the purity of the SAC-TRAIL fusion protein.
Through the two purification operations, the SAC-TRAIL fusion protein with the purity of 94% is obtained.
Use of a SAC-TRAIL fusion protein for inhibiting proliferation of ovarian cancer cells SK-OV-3 and breast cancer cells MCF-7.
The proliferation inhibition effect of the SAC-TRAIL fusion protein on ovarian cancer cells SK-OV-3 and breast cancer cells MCF-7 is tested in vitro. The CCK-8 analysis method is adopted to detect the influence of the SAC-TRAIL fusion protein on the proliferation of the ovarian cancer cell SK-OV-3 and the breast cancer cell MCF-7. The specific operation is as follows:
(1) activating and culturing ovarian cancer cells SK-OV-3 and breast cancer cells MCF-7.
(2) A cell suspension is prepared. The cells were trypsinized for 1min, and after termination, the culture was discarded by centrifugation, washed 2 times with PBS, and resuspended in 5% FBS-containing medium. Adjusting the cell density to 105One per ml.
(3) Cells were seeded. 100 μ l of the cell suspension was added to a 96-well plate, and the periphery of the 96-well plate was not inoculated with cells, and 100 μ l of PBS buffer was used instead.
(4) SK-OV-3 and MCF-7 cells were treated with SAC-TRAIL fusion protein. SK-OV-3 and MCF-7 cells were treated with different concentrations of fusion protein for 72h, respectively. And adding 10 mul of CCK-8 reagent into each hole, and detecting the absorption value of each hole at the wavelength of 490nm by using a microplate reader after 2 hours.
(6) And (5) carrying out statistical analysis on the experimental result. The activity of the cells at different concentrations was calculated. Cell activity (%) ([ a (dosed) -a (blank) ]/[ a (not dosed) -a (blank) ] × 100
A (dosed) represents the absorbance of the wells containing cells, drug solution and CCK-8;
a (blank) represents the absorbance of cells-free, medium-containing and CCK-8 wells;
a (no drug added) represents the absorption value of the drug-free wells containing cells and CCK-8.
Statistical analysis of the data shows that SAC-TRAIL fusion protein can significantly inhibit the proliferation of ovarian cancer cells SK-OV-3 and breast cancer cells MCF-7 (as shown in FIG. 4 and FIG. 5). Semi-lethality IC of TRAIL and SAC-TRAIL to SK-OV-3 cells50Values were 64. mu.g/mL and 4.42ng/mL, respectively; the half-lethality IC50 values of TRAIL and SAC-TRAIL on MCF-7 cells were 53.18. mu.g/mL and 0.503. mu.g/mL, respectively. The SAC-TRAIL fusion protein prepared by the invention obviously enhances the anti-tumor activity of the TRAIL protein, reduces the drug resistance of tumor cells to the TRAIL protein, and lays a foundation for promoting the application of the SAC-TRAIL fusion protein in the gynecological tumor treatment field.
The embodiments described above are intended to facilitate the understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Sequence listing
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gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat 240
ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggtcgtaag 300
ggcaaaggtc aaatcgagaa acgtaaactg cgcgagaaac gtcgttctac tggcgttgtg 360
aacatcccgg cggctgaatg cctggatgaa tacgaagacg atgaagcagg tcagaaagaa 420
cgtaaacgcg aagacgccat tacccagcag aatacgatcc agaacgaagc gggcggaggt 480
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aagaatgaaa aggctctggg ccgcaaaata aactcctggg aatcatcaag gagtgggcat 660
tcattcctga gcaacttgca cttgaggaat ggtgaactgg tcatccatga aaaagggttt 720
tactacatct attcccaaac atactttcga tttcaggagg aaataaaaga aaacacaaag 780
aacgacaaac aaatggtcca atatatttac aaatacacaa gttatcctga ccctatattg 840
ttgatgaaaa gtgctagaaa tagttgttgg tctaaagatg cagaatatgg actctattcc 900
atctatcaag ggggaatatt tgagcttaag gaaaatgaca gaatttttgt ttctgtaaca 960
aatgagcact tgatagacat ggaccatgaa gccagttttt tcggggcctt tttagttggc 1020
<210> 5
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gctagcatgt cggactcaga agtcaatcaa gaagctaag 39
<210> 6
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cactaccgcc tccaccagat ccacctccgc ccgcttcgtt ctggatcgt 49
<210> 7
<211> 471
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct 60
gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa 120
aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg 180
gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat 240
ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggtcgtaag 300
ggcaaaggtc aaatcgagaa acgtaaactg cgcgagaaac gtcgttctac tggcgttgtg 360
aacatcccgg cggctgaatg cctggatgaa tacgaagacg atgaagcagg tcagaaagaa 420
cgtaaacgcg aagacgccat tacccagcag aatacgatcc agaacgaagc g 471
<210> 8
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gtggatctgg tggaggcggt agtggtggag gcggtagtgt gagagaaaga ggtcctca 58
<210> 9
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ccgctcgagt tagccaacta aaaaggcccc ga 32
<210> 10
<211> 504
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gtgagagaaa gaggtcctca gagagtagca gctcacataa ctgggaccag aggaagaagc 60
aacacattgt cttctccaaa ctccaagaat gaaaaggctc tgggccgcaa aataaactcc 120
tgggaatcat caaggagtgg gcattcattc ctgagcaact tgcacttgag gaatggtgaa 180
ctggtcatcc atgaaaaagg gttttactac atctattccc aaacatactt tcgatttcag 240
gaggaaataa aagaaaacac aaagaacgac aaacaaatgg tccaatatat ttacaaatac 300
acaagttatc ctgaccctat attgttgatg aaaagtgcta gaaatagttg ttggtctaaa 360
gatgcagaat atggactcta ttccatctat caagggggaa tatttgagct taaggaaaat 420
gacagaattt ttgtttctgt aacaaatgag cacttgatag acatggacca tgaagccagt 480
tttttcgggg cctttttagt tggc 504
Claims (10)
1. A SAC-TRAIL fusion protein, characterized in that the antitumor protein TRAIL is linked via a flexible linkage (G)4S)3Fused to the C-terminal of the SAC protein to form the SAC-TRAIL fusion protein, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1.
2. The SAC-TRAIL fusion protein according to claim 1, wherein the amino acid sequence of SAC protein is shown in SEQ ID NO. 2, and the amino acid sequence of anti-tumor protein TRAIL is shown in SEQ ID NO. 3.
3. The SAC-TRAIL fusion protein according to claim 1, wherein the nucleotide sequence of the gene encoding the fusion protein is shown in SEQ ID NO. 4.
4. A method for preparing a SAC-TRAIL fusion protein according to any one of claims 1 to 3, comprising the steps of:
(1) constructing pET28a/SUMO-SAC-TRAIL recombinant plasmid;
(2) heterologous expression of SUMO-SAC-TRAIL fusion protein by using escherichia coli BL21(DE 3);
(3) and (3) separating and purifying the SAC-TRAIL fusion protein by using nickel ion affinity chromatography and weak cation exchange resin.
5. The method of claim 4, wherein the step (1) of constructing pET28a/SUMO-SAC-TRAIL recombinant plasmid comprises the steps of:
(1-1) taking an upstream primer with a sequence shown as SEQ ID NO:5 and a downstream primer with a sequence shown as SEQ ID NO:6 as reaction primers, taking a full-length SUMO-SAC gene as a template, wherein the nucleotide sequence of the SUMO-SAC gene is shown as SEQ ID NO:7,
an upstream primer with a sequence shown as SEQ ID NO. 8 and a downstream primer with a sequence shown as SEQ ID NO. 9 are taken as reaction primers, a full-length TRAIL gene is taken as a template, wherein the nucleotide sequence of the TRAIL gene is shown as SEQ ID NO. 10,
carrying out PCR reaction to obtain coding genes of SUMO-SAC and TRAIL protein;
(1-2) recovering the PCR product in the step (1) by using an upstream primer with a sequence shown as SEQ ID NO:5 and a downstream primer with a sequence shown as SEQ ID NO:9 as reaction primers, using full-length SUMO-SAC and TRAIL genes as templates, and carrying out fusion PCR reaction to obtain a coding gene of the SUMO-SAC-TRAIL fusion protein, namely a target gene;
(1-3) recovering the PCR product in the step (2) by gel, carrying out double enzyme digestion on a target gene and a pET28a expression plasmid by Bmt I and Xho I, connecting the target gene and the pET28a expression plasmid by T4 ligase to obtain a pET28a/SUMO-SAC-TRAIL recombinant plasmid, transforming the recombinant plasmid into escherichia coli BL21(DE3), selecting a positive clone, and carrying out sample sending and sequencing.
6. The method of claim 5, wherein in the step (1), the PCR reaction is performed under the following conditions: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 20s, and circulating for 30 times, and final extension at 72 deg.C for 10 min.
7. The method of claim 4, wherein the step (2) of heterologous expression of the SUMO-SAC-TRAIL fusion protein using E.coli BL21(DE3) comprises the steps of: coli BL21(DE3) containing the pET28a/SUMO-SAC-TRAIL recombinant plasmid, which was sequenced correctly, was cultured at 37 ℃ in LB medium containing 50mg/L kanamycin resistance, when OD was determined600When the concentration reaches 0.6, isopropyl-beta-D-thiogalactopyranoside with the final concentration of 0.1mM is added, induction is carried out for 16h at 18 ℃, and the SUMO-SAC-TRAIL fusion protein is expressed in a heterologous mode.
8. The method of claim 4, wherein the step (3) of isolating and purifying the SAC-TRAIL fusion protein comprises the steps of:
(3-1) purification of SUMO-SAC-TRAIL fusion protein Using His tag:
utilizing nickel column affinity chromatography, selectively adsorbing SUMO-SAC-TRAIL fusion protein with His label to nickel column, purifying to obtain SUMO-SAC-TRAIL fusion protein;
(3-2) collecting the purified SUMO-SAC-TRAIL fusion protein, carrying out enzyme digestion on the fusion protein by SUMO protease, removing the SUMO label, carrying out weak cation exchange chromatography on enzyme digestion reaction liquid to remove the SUMO label, eluting and collecting SAC-TRAIL protein by PB containing 500mM NaCl, and obtaining the SAC-TRAIL fusion protein.
9. Use of a SAC-TRAIL fusion protein according to any one of claims 1 to 3 for inhibiting proliferation of ovarian cancer cells SK-OV-3 and breast cancer cells MCF-7.
10. Use of a SAC-TRAIL fusion protein according to any one of claims 1 to 3 in the preparation of a medicament for the treatment of ovarian cancer cells SK-OV-3 and breast cancer cells MCF-7.
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Citations (4)
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|---|---|---|---|---|
| US20040213764A1 (en) * | 2003-03-28 | 2004-10-28 | William Wold | Adenovirus replication-competent vectors expressing trail |
| CN101717449A (en) * | 2008-10-09 | 2010-06-02 | 重庆富进生物医药有限公司 | Recombinant TRAIL-Fc fusion protein as well as preparation and application thereof |
| CN102757503A (en) * | 2011-04-28 | 2012-10-31 | 中国人民解放军第二军医大学 | Preparation method and application of human prostate apoptosis response protein 4 and apoptin 2 ligand fusion protein |
| CN102958940A (en) * | 2010-06-25 | 2013-03-06 | 阿达梅德公司 | Anticancer fusion protein |
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2021
- 2021-07-23 CN CN202110836049.0A patent/CN113717289A/en active Pending
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| US20040213764A1 (en) * | 2003-03-28 | 2004-10-28 | William Wold | Adenovirus replication-competent vectors expressing trail |
| CN101717449A (en) * | 2008-10-09 | 2010-06-02 | 重庆富进生物医药有限公司 | Recombinant TRAIL-Fc fusion protein as well as preparation and application thereof |
| CN102958940A (en) * | 2010-06-25 | 2013-03-06 | 阿达梅德公司 | Anticancer fusion protein |
| CN102757503A (en) * | 2011-04-28 | 2012-10-31 | 中国人民解放军第二军医大学 | Preparation method and application of human prostate apoptosis response protein 4 and apoptin 2 ligand fusion protein |
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