CN112940097A

CN112940097A - Survivin multi-point mutant recombinant protein and application thereof

Info

Publication number: CN112940097A
Application number: CN202110172636.4A
Authority: CN
Inventors: 马兴元; 郭伟; 郑文云; 苗蕙
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2021-02-08
Filing date: 2021-02-08
Publication date: 2021-06-11

Abstract

The invention provides Survivin multi-point mutant recombinant protein and application thereof, wherein the Survivin multi-point mutant recombinant protein is prepared by genetic engineering, N-terminal fusion transduction peptide is used for mutating two or three amino acids of 34 th threonine, 48 th threonine and 84 th cysteine of wild Survivin into alanine. The anti-tumor and sensitization effects of the multipoint mutation are superior to the effects of wild type and 34-site single mutation, and the multipoint mutant recombinant protein and the chemotherapeutic drug are combined to act on CSCs, so that the multipoint mutant recombinant protein is found to be capable of effectively promoting the killing effect of the chemotherapeutic drug and effectively reducing the usage amount of the chemotherapeutic drug, is an effective sensitizer for tumor chemotherapy, and provides a new solution for solving the drug resistance problem in the cancer treatment process.

Description

Survivin multi-point mutant recombinant protein and application thereof

Technical Field

The invention relates to the field of biological pharmacology, in particular to a high-efficiency anti-tumor Survivin mutant recombinant protein and application thereof.

Background

Survivin is one of the apoptosis inhibiting protein family members, and has the functions of inhibiting cell apoptosis, regulating cell mitosis and promoting tumor angiogenesis. Survivin is hardly detectable in normal adult tissues, but is expressed in high amounts in tumor cells. More and more researches show that survivin is expressed in higher amount in CSCs which are considered as the starting cells of tumorigenesis, and endows the CSCs with stronger anti-apoptosis and drug resistance, so that the cancer is difficult to cure. Given the high expression and multiplicity of actions of Survivin in cancer cells and CSCs, Survivin-targeted cancer therapies are more advantageous than targeting molecules in a single oncogenic pathway. Many strategies for inhibiting Survivin function have been developed, including small molecule inhibitors, antisense oligonucleotides (ASOs), small interfering rnas (sirnas), and Survivin dominant negative mutations. However, small molecule inhibitors, ASOs and sirnas may not be suitable for targeting Survivin because they do not eliminate the Survivin protein already present. In view of the advantages of being easy to prepare, being capable of directly binding to intracellular wild-type Survivin protein and inhibiting Survivin biological function at the terminal end of protein expression, Survivin dominant-negative mutants attract extensive attention.

The Survivin dominant negative mutant is a non-functional protein formed by mutating functionally essential amino acids of wild-type Survivin to non-essential amino acids. Because the wild-type survivin has similar target spots with the wild-type survivin, the function of the wild-type survivin can be competitively inhibited. The Survivin BIR domain has a plurality of protein posttranslational modification sites including phosphorylation sites, ubiquitination sites and acetylation sites, wherein the phosphorylation modification sites are mainly concentrated in the BIR domain, such as Thr34 and Thr48, and mutation of the phosphorylation sites can break the combination of Survivin and Caspase, thereby releasing the apoptosis-promoting function of Caspase. Ubiquitination modification sites are also distributed more in the BIR domain (1-88AA), such as lysine (Lys) site at position 23/62/78/79, and the modification is mainly used for regulating the interaction of Survivin and the centromere and promoting the completion of mitosis. The acetylation modification sites are mainly distributed in the alpha-helical region of Survivin, such as 120/121/122/129/130Lys positionIn this regard, the modification is primarily to maintain a balance between the monomeric and dimeric forms of Survivin, thereby mediating nuclear import and export of Survivin. Furthermore, Zn on Survivin²⁺The binding site is extremely important for maintaining the stability of its dimer, while performance of the anti-apoptotic function of Survivin relies mainly on the dimeric form. Taken together, we believe that the phosphorylation modification site of the Survivin BIR domain and Zn are mutated²⁺The binding site is able to more effectively disrupt the anti-apoptotic function of Survivin. Our previous studies found that cell permeable, dominant negative TmSm34 could induce apoptosis in a variety of cancer cells including breast cancer Bcap-37, pancreatic cancer SW1990 and liver cancer SMMC-7721 and could increase the sensitivity of a variety of breast cancers including T47-D, MCF-7 and Bcap-37 to the chemotherapeutic drug ADM, and further studies showed that TmSm34 also inhibited the growth and induced apoptosis of breast cancer CSCs. The mechanism of inducing apoptosis by the TmSm34 is that the phosphorylation site of p34cdc2-cyclinB1 is eliminated, so that the combination of Survivin and Caspase is prevented, and finally the apoptosis is induced. The Thr48 of Survivin is also a phosphorylation site, and T48A can induce tumor necrosis factor TNF-related apoptosis-inducing ligand (TRAIL) -mediated apoptosis. Cys at position 84 in the Survivin BIR domain is Zn²⁺Binding sites, whereas C84A can disrupt Zn²⁺Integrity of the coordination sphere and inhibition of Survivin interaction with Smac/DIABLO, in turn, induces apoptosis. Different Survivin mutants can induce different degrees of apoptosis in cells, however, there is no unified debate as to which Survivin mutant has the best anticancer activity.

Based on T34A, the invention mutates T48 and C84 in the Survivin BIR structure domain to Ala and mutates T48 and C84 in the Survivin BIR structure domain to Ala separately or simultaneously, optimizes a high-efficiency Survivin mutant protein through cell activity detection and apoptosis evaluation, promotes the application of Survivin dominant-negative mutant in cancer clinical treatment, and provides an effective method for solving the drug resistance problem in cancer treatment.

Disclosure of Invention

The invention aims to provide a Survivin multi-point mutant recombinant protein with high efficiency and anti-tumor property.

The second purpose of the invention is to provide the application of the Survivin multi-point mutant recombinant protein with high-efficiency anti-tumor effect.

In order to achieve the above objects, the present invention provides a Survivin multi-point mutant recombinant protein, which is prepared by genetic engineering, N-terminally fusing a transduction peptide, and mutating two or three amino acids among threonine 34, threonine 48 and cysteine 84 of wild type Survivin to alanine.

As a preferred embodiment, the wild type Survivin threonine 34 and threonine 48 were mutated to alanine to obtain TmSm 34/48.

As a preferred embodiment, the wild-type Survivin threonine 34 and cysteine 84 were mutated to alanine to obtain TmSm 34/84.

As a preferred embodiment, the wild-type Survivin threonine 34, threonine 48 and cysteine 84 are mutated to alanine to obtain TmSm 34/48/84.

As a preferred embodiment, the sequence of the transduction peptide is Tyr-Ala-Arg-Lys-Ala-Arg-Arg-Gln-Ala-Arg-Arg (SEQ ID NO: 1).

Biomacromolecule drugs play an important role in the treatment of many diseases, but due to the natural barrier action of cell membranes, only drugs with high lipid solubility or sufficiently small molecular weight can be internalized into cells, which limits the application of some drugs with therapeutic value but without cell penetration to some extent. Recently, it has been discovered that a trans-activator (TAT) derived from HIV-1, a human immunodeficiency virus, is effective in inducing peptides or proteins to penetrate cell membranes. In the invention, a transduction peptide mutant from HIV-1TAT is preferably inserted into the N terminal, the 11 amino acid transduction sequence of the TAT is the shortest sequence of the TAT protein transduction structural domain found so far, the transduction capability of the TAT is not worse than that of the full-length TAT sequence, and the TAT protein mutant is characterized by high transduction speed and high efficiency. At present, with the development of protein engineering, the TAT mutant minimum segment is formed by 9-11 amino acids which are artificially modified and optimized, has the characteristics of mediating protein, polypeptide and gene transmembrane which are 10-100 times higher than that of natural HIV-TAT, has no cytotoxicity, and is an excellent transfer vehicle. It is reported in the literature that the model structure of TAT is a strong amphipathic helix, and if arginine in amino acids 47-57 of TAT transmembrane peptide is replaced by alanine (TATm: YARKARRQARR) (SEQ ID NO:1), the TAT transmembrane peptide has stronger transmembrane efficiency due to the highest alpha-helix stability value of alanine in all amino acids.

In order to realize the second purpose, the invention provides an application of Survivin multi-point mutant recombinant protein in preparing tumor chemotherapeutic drug sensitizer.

In a preferred embodiment, the tumor is lung cancer and the chemotherapeutic agent is Adriamycin (ADM).

In a preferred embodiment of the present invention, when the Cancer Stem Cells (CSCs) are a549 CSCs, the effective dose of protein sensitization is 1.05-2.09 μ M.

The invention provides application of Survivin multi-point mutant recombinant protein in preparation of antitumor drugs, wherein the tumor is a tumor over-expressing Survivin.

As a preferred embodiment, the tumor includes, but is not limited to, lung cancer, liver cancer, breast cancer, pancreatic cancer, and bladder cancer.

The most common method for applying Survivin dominant negative mutant is adenovirus transfection at present, but the method not only is easy to generate immunogenicity in vivo, but also is expensive. We therefore chose to express Survivin mutant proteins in vitro using e. Compared with adenovirus transfection, the protein drug has better biocompatibility and targeting property and lower cost. By comparing the influence of different Survivin mutant proteins on the activity and apoptosis of tumor cells, more efficient Survivin mutant proteins are further optimized, and the clinical application of Survivin dominant-negative mutants in cancer treatment is finally promoted. The Survivin double-point mutant TmSm34/84 with the strongest anticancer activity is preferably obtained through cell activity detection and apoptosis evaluation, and can enhance the sensitivity of Cancer Stem Cells (CSCs) to chemotherapeutic drugs. The TmSm34/84 protein is not only a high-efficiency anti-tumor protein drug, but also can be used as a novel chemosensitizer in tumor chemotherapy.

The invention has the advantages that the invention obtains the Survivin multi-point mutant recombinant protein with high-efficiency anti-tumor, two or three amino acids in 34 th threonine, 48 th threonine and 84 th cysteine of the wild Survivin are mutated into alanine, the anti-tumor and sensitization effects of the multi-point mutation are better than the effects of the wild Survivin and 34 single mutation, and the multi-point mutant recombinant protein and the chemotherapeutic drugs are combined to act on CSCs, so that the multi-point mutant recombinant protein can effectively promote the killing effect of the chemotherapeutic drugs and effectively reduce the usage amount of the chemotherapeutic drugs, is an effective sensitizer for tumor chemotherapy, and provides new solution thinking and methods for solving the drug resistance problem in the cancer treatment process.

Drawings

FIG. 1 shows the construction of recombinant plasmid and the expression and purification of recombinant protein. Wherein, A: schematic diagram of recombinant plasmid construction. B: PCR electrophoresis of recombinant plasmid. C: DNA sequencing map. D: SDS-PAGE electrophoretic analysis is carried out after six proteins are purified.

Fig. 2 is a cytotoxicity assay of six Survivin mutant proteins against a549 cells. Wherein, A: cell viability assay of six Survivin mutant proteins acting on a549 cells at the same concentration gradient (0, 2.09, 4.19, 6.28 and 8.37 μ M) for 24 h. B: IC of six Survivin mutant proteins on A549 cells₅₀The value is obtained. Data are expressed as Mean ± SD (n ═ 3). P<0.05 and P<0.01。

FIG. 3 shows the ability of flow-testing six Survivin mutant proteins to induce apoptosis of A549 cells. After six Survivin mutant proteins act on A549 cells for 24h with the same concentration gradient (0, 4.19 and 8.37 mu M), apoptosis is detected by using a flow cytometer. Data are expressed as Mean ± SD (n ═ 3). P <0.05, P <0.01 and P <0.001 compared to the TmSm34/84 treatment group.

FIG. 4 shows the effect of Western blot analysis on the Survivin and clear Caspase-3 content in A549 cells by three kinds of multi-point mutant proteins. After three Survivin mutant proteins act on A549 cells for 24h with the same concentration gradient (0, 4.19 and 8.37 mu M), the change of protein content is detected by Western blot. Data are expressed as Mean ± SD (n ═ 3). P <0.05 and P < 0.01.

FIG. 5 shows the enrichment and "dryness" identification of A549 CSCs. Wherein, A: morphological observation of CSCs obtained by serum-free suspension culture. B: immunofluorescence and flow detection of expression of CD133 on the cell membrane surface of the enriched CSCs.

FIG. 6 shows the in vitro proliferation capacity identification of A549 CSCs. A: MTT assay CSCs 7 day proliferation curve. B: the clonogenic assay measures the proliferative capacity of CSCs in vitro. Data are expressed as Mean ± SD (n ═ 3). P < 0.05.

FIG. 7 shows the drug resistance detection of A549 CSCs. A: cell viability assay after 24h of various concentrations of ADM (0, 0.2, 0.4, 0.6, 0.8 and 1.6. mu.M) on A549 CSCs. B: and detecting the expression quantity of apoptosis related protein and drug resistance related protein in A549 CSCs by Western blot. Data are expressed as Mean ± SD (n ═ 3). P <0.05 and P < 0.01.

FIG. 8 is a graph showing the cell viability assay of TmSm34/84 after being applied to A549 CSCs at various concentrations (0, 1.05, 2.09, 4.19, 6.28 and 8.37. mu.M) for 24 h. Data are expressed as Mean ± SD (n ═ 3). P <0.05, P <0.01 and P < 0.001.

FIG. 9 shows the cell viability assay of ADM alone and TmSm34/84 (1.05. mu.M) alone and in combination on A549 CSCs for 24 h. Data are expressed as Mean ± SD (n ═ 3). P < 0.05.

FIG. 10 shows the effect of ADM alone and TmSm34/84 (1.05. mu.M) alone and in combination on the cell morphology of A549 CSCs after 24 h.

FIG. 11 shows the effect of ADM alone and TmSm34/84 (1.05. mu.M) alone and in combination on apoptosis of A549 CSCs for 24 h. Data are expressed as Mean ± SD (n ═ 3). P <0.01 and P < 0.001.

FIG. 12 shows the effect of ADM alone and TmSm34/84 (1.05. mu.M) alone and in combination on the expression level of apoptosis-related proteins and drug resistance-related proteins in A549 CSCs after 24 h. Data are expressed as Mean ± SD (n ═ 3). P <0.05 and P < 0.01.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for the following examples, in which specific conditions are not specified, are generally carried out under conventional conditions, for example, as described in molecular cloning instructions written by J. Sambruke (Joseph Sambrook), et al, or under conditions recommended by the manufacturer.

Example 1 construction of recombinant plasmid and purification of recombinant protein expression

Utilizing pET-24a (+) -TmSm34 plasmid which is available in a laboratory as a template, amplifying TmSm34/48 and TmSm34/84 gene fragments by an overlap extension PCR technology respectively, and then carrying out enzyme digestion and ligation to construct two recombinant expression plasmids of pET-24a (+) -TmSm34/48 and pET-24a (+) -TmSm 34/84; similarly, pET-24a (+) -TmSm34/48 is used as a template to construct pET-24a (+) -TmSm34/48/84 recombinant expression plasmid. The recombinant plasmid construction is schematically shown in FIG. 1A.

Reagents and kits: premix Taq DNA polymerase, Pyrobest DNA polymerase, restriction enzymes (New I, Xho I), Protein molecular weight standard Premixed Protein Marker (Low), Protein Loading Buffer 4 XProtein SDS PAGE Loading Buffer, DNA Marker purchased from Takara Bio Inc. (Dalian). isopropyl-B-D-thiogalactopyranoside (IPTG), kanamycin were purchased from Sigma-Aldrich (USA) and Ni-NTA column was purchased from GE (USA). Other biochemical reagents belong to domestic conventional analytical pure reagents.

Strains and plasmids: coli DH5 alpha and BL21(DE3) (Invirogen, USA) were used as plasmid clones and expression strains, respectively, and recombinant plasmids pET-24a (+) -TmSm34, pET-24a (+) -TmSm48 and pET-24a (+) -TmSm84 were constructed in this laboratory, and Invirogen was purchased from pET-24a (+).

Kanamycin (50. mu.g/mL): weigh 1g, dissolve in 20mL ddH₂O, filtering and sterilizing by using a sterile filter membrane of 0.22 mu m, subpackaging and storing at the temperature of minus 20 ℃. When culturing Escherichia coli, each 1mL LB medium is addedmu.L of kanamycin solution (1: 1000) was added.

isopropyl-beta-D-thiogalactopyranoside (IPTG) mother liquor (1 mol/L): 2.38g of IPTG was weighed and dissolved in 10mL of ultrapure water to prepare a 238mg/mL aqueous solution, which was sterilized by filtration through a 0.22 μm sterile filter membrane and then dispensed into 1 mL/piece of the tube and stored at-20 ℃ for use. At the time of induction, 0.75. mu.L of IPTG mother liquor was added to 1mL of LB medium. The final concentration was 0.75 mM.

LB liquid medium: weighing 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, fixing the volume to 1L by deionized water, sterilizing at 121 ℃ under high pressure for 20min, and storing at normal temperature.

5 × protein electrophoresis buffer (Tris-glycine): weighing 15.1g of Tris base, 94g of glycine, 5g of SDS and ddH₂And O is metered to 1L.

15% protein electrophoretic separation gel: measuring ddH₂O2.3 mL, 30% acrylamide 5.0mL, Tris-HCI (pH 6.8)2.5mL, 10% SDS 100. mu.L, 10% APS 100. mu.L, TEMED 100. mu.L, mix well.

5% protein electrophoresis concentrated gel: measuring ddH₂O3.15 mL, 30% acrylamide 0.75mL, Tris-HCl (pH 6.8)0.57mL, 10% SDS 45. mu.L, 10% APS 45. mu.L, TEMED 4.5. mu.L, mix well.

Coomassie brilliant blue staining solution: weighing 2g of Coomassie brilliant blue (R-250), weighing 200mL of ethanol, 100mL of glacial acetic acid and 750mL of deionized water, and storing at room temperature.

PBS buffer: 8g NaCl, 0.2g KCl, 0.24g KH₂PO₄，1.44g Na₂HPO₄And deionized water is added to the volume of 1L.

0.2M ph7.0 phosphate buffered mother liquor (PB): 43.69g of Na were weighed out separately₂HPO₄ ^.12H₂O (Mw 358.14) and 12.17g NaH₂PO₄ ^.2H₂O (Mw 156.01) was made to 1L with ultrapure water.

Inclusion body wash: 20mmol/L glycine-sodium hydroxide buffer, 0.5% Triton X-100, 0.5mol/L urea, 1mol/L sodium chloride, 2.5mmol/L EDTA, pH 10.0.

Inclusion body dissolution solution: 20mmol/L PB, 8mol/L urea, pH 7.0. Weighing 500mL of 20mmol/L PB, and weighing240.24g of urea dissolved. By means of a pH meter with NaOH solution or H₃PO₄The solution was adjusted to pH 7.0.

And (3) an equilibrium buffer: 100mL of 0.2M PB, 125mL of 4M NaCl, 480.48g of urea, 1.3616g of imidazole and ultrapure water are added to the solution to be constant volume of 1L.

Elution buffer: 100mL of 0.2M PB, 125mL of 4M NaCl, 480.48g of urea, 27.23g of imidazole and ultrapure water were added to the reaction solution to obtain a volume of 1L.

Dialysate A: 100mL of 0.2M PB, 125mL of 4M NaCl, 50mL of glycerol, 500mL of 8M urea and constant volume of ultrapure water to 1L.

Dialysate B: 100mL of 0.2M PB, 125mL of 4M NaCl, 50mL of glycerol, 250mL of 8M urea and constant volume of ultrapure water to 1L.

Dialysate C: 100mL of 0.2M PB, 125mL of 4M NaCl, 50mL of glycerol, 125mL of 8M urea and constant volume of ultrapure water to 1L.

Dialysate D: 100mL of 0.2M PB, 125mL of 4M NaCl, 50mL of glycerol, 62.5mL of 8M urea and constant volume of ultrapure water to 1L.

Bradford assay for solutions related to protein concentration:

BSA protein Standard solution (mg/mL): 10mg BSA was dissolved in 10mL sterile water.

Bradford working solution: weighing 100mg of Coomassie brilliant blue G-250, adding 50mL of 95% ethanol, 100mL of 85% phosphoric acid and deionized water to a constant volume of 1L, and storing in dark place.

Construction of recombinant plasmid

Designing a primer:

TABLE 1.1 primer sequences

PCR reaction system and reaction conditions:

TABLE 1.2 PCR reaction System

TABLE 1.3 PCR reaction conditions

Respectively amplifying gene products with overlapped chains in the upstream and downstream parts by taking pET-24a (+) -TmSm34 constructed in a laboratory as a template, and then amplifying by taking the gene products of the two parts as the template to obtain the TmSm34/48 gene product. The TmSm34/84 gene product can be obtained by the same method.

The amplification of the TmSm34/48 and TmSm34/84 genes were analyzed by agarose gel electrophoresis, and the DNAs were purified from the PCR reaction solution by an agarose gel DNA recovery kit. And carrying out double enzyme digestion on the recovered target fragment and the extracted expression vector, wherein a double enzyme digestion system is shown in a table 1.4. The enzyme cutting temperature is 37 ℃, and the enzyme cutting time is 3-5 h. After the digestion, the digestion reaction solution of the PCR product is recovered according to the above steps. And connecting the target gene and the expression vector after double enzyme digestion recovery according to a connection system shown in the table 1.5. The joining temperature was 16 ℃ and the joining time was 10 h.

TABLE 1.4 double enzyme digestion System

TABLE 1.5 systems for ligation of genes of interest and vectors

And after the ligation reaction is finished, centrifuging the PCR reaction tube, placing the centrifugal tube on ice, and performing a subsequent transformation experiment. The next day, a single colony was picked and cultured in 5mL of LB liquid medium containing kanamycin for 16 hours, and then 1mL of the bacterial solution was put in a sterile EP tube and subjected to sequencing by Shanghai Ruizian Biotech Co. Subsequently, the above-described operation was repeated to successfully construct the plasmid pET-24a (+) -TmSm34/48/84 using the constructed pET-24a (+) -TmSm34/48 as a template (FIGS. 1B and 1C).

Expression and purification of recombinant proteins

Expression of the protein: (1) the glycerol cells were removed from-80 ℃ and streaked overnight for culture. (2) The single clone was inoculated in 5mL of non-resistant LB medium and cultured overnight at 37 ℃ and 200 rpm. (3) Inoculating the bacterial liquid into LB culture medium containing kanamycin resistance in an inoculation amount of 2.5%, culturing at 37 ℃ for 2h at 200rpm, adding 150 mu L IPTG (final concentration of 0.75mM) after OD600nm reaches 0.6-0.8, and performing induced expression at 30 ℃ and 200rpm for 10h to obtain the bacterial liquid.

Breaking thallus and washing inclusion body: (1) collecting bacterial liquid, centrifuging at 12000rpm for 1min, and collecting thallus. (2) The cells were washed with PBS, and 100mL of the cells were washed with 10mL of PBS. (3) PBS resuspend the cells and sonicate under the conditions: 200W, over 5s, stopping for 5s, 99 times, and circulating three rounds. (4) The disrupted cells were centrifuged at 12000rpm at 4 ℃ for 20min, and the supernatant was discarded to obtain crude inclusion bodies. (5) The crude inclusion bodies were washed. Washing conditions are as follows: 400rpm, magnetically stir for 15 min. (6) Centrifuging the washed precipitate under the following conditions: 4 ℃, 12000rpm, 15 min. And (5) discarding the supernatant, collecting the precipitate, and repeating the steps 5 and 6 twice to obtain the refined inclusion body. (7) The inclusion bodies were purified by dissolving in 8M urea. Stirring for 1h in ice bath to obtain an inclusion body dissolving solution. (8) The solution was centrifuged at 12000rpm for 20min at 4 ℃ and the supernatant was collected by discarding the precipitate. (9) The supernatant was filtered through a 0.45 μm filter and the resulting liquid was used for subsequent protein purification.

And (3) purifying the target protein by using an AKTA protein purifier: (1) starting the AKTA purifier, placing A, B two pipelines into a blue-mouth bottle filled with ultrapure water, adjusting the parameters of a display screen to be 'set B100%', and setting the flow rate to be 10 mL/min. After the click is started, after the volume of the liquid in the pipeline B is flushed to 20mL, the parameter of the display screen is changed to 'set B0%', after the click is started, after the liquid in the pipeline A is flushed to 20mL, the pipeline is cleaned, and the nickel column can be loaded. (2) The method comprises the following steps of putting a nickel column (5mL) on an AKTA (alkyl ketene dimer) purifier, putting a pipeline A into a blue-mouth bottle filled with a balance buffer solution, putting a pipeline B into a blue-mouth bottle filled with an elution buffer solution, adjusting the parameter of a display screen to be 'set B0%', setting the flow rate to be 3mL/min, starting to clean impurities on the nickel column after clicking is started, and cleaning until a baseline (conductance and ultraviolet) is balanced. (3) And (3) unloading the nickel column from the AKTA purifier, sucking the protein by using a syringe, pushing away bubbles, slowly pushing a proper amount of protein into the nickel column by using the syringe, simultaneously measuring the protein flowing through the nickel column by using the Bradford solution until the flowing protein and the Bradford solution are back-strained to blue, which shows that the maximum protein carrying capacity of the nickel column is reached at present, and stopping sample loading. (4) And (3) loading the loaded nickel column on an AKTA (alkyl ketene dimer) purifier, adjusting display screen parameters to be 'set B0%', setting the flow rate to be 3mL/min, starting impurity washing, adjusting the display screen parameters to be 'Target B100% and 50 mL' in a gradient elution mode after a time of impurity washing peak and rebalancing of a base line, setting the flow rate to be 3mL/min, and connecting the EP tube to elute the Target protein after the peak appears so as to perform subsequent protein electrophoretic identification. (5) After purification is finished, the pipeline A and the pipeline B are placed in a blue-mouth bottle filled with ultrapure water, parameters of a display screen are set to be set B50%, the flow rate is set to be 10mL/min, the whole pipeline is washed, after washing of the ultrapure water is finished, the pipeline A and the pipeline B are placed in the blue-mouth bottle filled with 20% ethanol, the whole pipeline is washed, the whole pipeline is filled with the ethanol, and the nickel column is also stored with the 20% ethanol. (6) The nickel column was removed from the AKTA purifier, stored at 4 ℃ and the AKTA purifier was powered off.

Renaturation of inclusion bodies: (1) and (6) pretreating the dialysis bag. Placing a newly purchased dialysis bag in the dialysis bag treatment liquid A to boil for 10min, placing the newly purchased dialysis bag in the dialysis bag treatment liquid B to boil for 10min after being washed by ultrapure water, placing the newly purchased dialysis bag in a 20% ethanol solution after being washed by ultrapure water, and storing the newly purchased dialysis bag at 4 ℃. (2) Diluting the protein to be dialyzed to 0.4-0.6mg/mL, adding into a dialysis bag washed with ultrapure water, and then putting the dialysis bag into dialysate A (the ratio of the volume of the protein to the volume of the dialysate is 1:10) for dialysis for 6 h. (3) After 6h, the dialysis bag was removed and placed in dialysate B, and dialysate D was dialyzed in this order. The dialysis time is 6h each time, and the above operations are carried out in ice bath or under stirring at 4 ℃. (4) After dialysis was complete, the fluid in the dialysis bag was centrifuged at 12000rpm for 20min at 4 ℃. (5) The supernatant was protein concentrated by ultrafiltration tube (3kDa), protein concentration was determined by Bradford and stored at-80 ℃.

SDS-PAGE electrophoresis detection of protein expression: (1) and cleaning the glass plate with ultrapure water and drying. (2) And (5) assembling the glue making devices in sequence, and adding water to check leakage. (3) And (4) preparing separation gel. The formulations were prepared as follows, mixed well and injected into a gum maker. (4) And after the separation gel is injected into a gel maker, absolute ethyl alcohol is injected to carry out liquid seal on the separation gel. Waiting for 30min, separating, gelatinizing, pouring off anhydrous ethanol, and absorbing excessive ethanol with absorbent paper. (5) The concentrated gum was prepared as in table 2.17, stirred well and slowly poured into the gum maker and slowly inserted into the comb, taking care not to generate bubbles. (6) Waiting for 30min at room temperature, and adding electrophoresis buffer solution after the protein gel is solidified. (7) The electrophoresis comb is pulled out vertically and slowly, and the deformed sample adding hole is pulled by a needle head. (8) Mixing 4 xSDS-PAGE loading buffer with protein sample in proper proportion, boiling for 10min, mixing the sample with low speed centrifuge, and slowly adding into the sample adding hole. (9) After 80V electrophoresis, the voltage is changed to 120V after the sample enters the separation gel. (10) And (4) after the bromophenol blue is about to run out of the separation gel, basically completing protein electrophoresis, and stopping electrophoresis according to the situation. (11) After prying the glass plate open, the albumin glue was slowly flushed down with water. (12) After staining the protein gel with Coomassie brilliant blue for 30min, the gel is put into a microwave oven and boiled with high fire for 10min, and then the gel is placed into a gel imager for photographing. As shown in fig. 1D, TmSm protein was obtained with a purity of 95% or more by nickel column purification.

Example 2 optimization of Survivin mutant proteins

Reagents and kits: RPMI 1640 medium, fetal bovine serum, pancreatin purchased from Gibco, Inc. (USA); triantion, dimethyl sulfoxide (DMSO), MTT from solibao (beijing); 0.2 μm PVDF membrane was purchased from Biyun (Shanghai); annexin V-FITC/PI apoptosis kit was purchased from Elabsciences (Shanghai); anti-Caspase 3 monoclonal antibody (rabbit derived), anti-Survivin monoclonal antibody (rabbit derived), anti-beta-actin monoclonal antibody (mouse derived), goat anti-rabbit/goat anti-mouse IgG-HRP secondary antibody were purchased from Proteintech (USA). Other biochemical reagents belong to domestic conventional analytical pure reagents.

Cell lines: human lung adenocarcinoma cell A549 cultured at 37 deg.C and 5% CO₂The concentration of the medium was RPMI 1640 medium (containing 10% fetal bovine serum).

Preparing a reagent:

RPMI 1640 complete medium: the RPMI 1640 basic culture medium 89mL, the fetal bovine serum 10mL and the three antibodies 1mL are measured and uniformly mixed, and the mixture is stored at 4 ℃.

5mg/mL MTT solution: MTT 250mg is weighed, PBS is added to 50mL to a constant volume, sterile filtration is carried out by a sterile filter membrane with the diameter of 0.22 μm, and the mixture is preserved in the dark at the temperature of-20 ℃.

In vitro Activity assay

Culture of a549 cells: (1) and (3) recovering the cells: removing the frozen cells, rapidly thawing the cells in warm water at 37 deg.C, adding to a culture flask containing cell culture medium at 37 deg.C and 5% CO₂Culturing for 6h under the condition, and replacing fresh culture medium. (2) Passage of cells: after the cell confluence reached 80-90%, the cells were rinsed 2 times with sterile PBS, followed by incubation in a flask (25 cm)²) Adding 500 μ L pancreatin, standing at 37 deg.C and 5% CO₂Digesting for 2min under the condition. And after the cells become round, sucking away pancreatin, blowing and pumping the cells on the wall of the bottle to enable the cells to fall off completely, and sucking part of the cells to a new cell culture bottle. (3) Liquid change of cells: old medium was decanted, cells rinsed with PBS and an appropriate volume of fresh medium was added.

Survivin mutant protein inhibition a549 cell growth activity assay: (1) taking out A549 cells, rinsing the cells with PBS for 2 times, and adding appropriate amount of pancreatin for digestion for 2 min. (2) Removing pancreatin, adding appropriate amount of cell culture medium, blowing to remove cells, and regulating cell density to 2 × 10⁵Per/mL, 100. mu.L per well were added to a 96-well plate. (3) After 24h, old medium was aspirated off and proteins diluted to 2.09, 4.19, 6.28 and 8.37 μ M in cell culture medium were added, 180 μ L per well, and blank control was replaced with fresh medium, respectively. (4) After 24h of protein action, 20. mu.L of MTT solution (final concentration 0.5mg/mL) was added to each well. (5) After 4h, the 96-well plate was carefully aspirated, 150. mu.L of DMSO was added to dissolve formazan deposited in the 96-well plate, the solution was placed on a shaker, shaken for 15min, and then placedDetermination of OD under enzyme-labeled Instrument_490nmThe value is obtained.

As can be seen from FIG. 2A, the 3 Survivin multi-point mutant proteins (TmSm34/48, TmSm34/84 and TmSm34/48/84) have a greater ability to inhibit the proliferation of A549 cells than the 3 Survivin single-point mutant proteins (TmSm34, TmSm48 and TmSm 84). When the concentration of the protein drug is 8.37 mu M, the inhibition rates of the 3 Survivin multi-point mutant proteins on the proliferation of A549 cells respectively reach 60.76%, 71.06% and 62.71%, which are higher than that of the 3 Survivin single-point mutant proteins (50.23%, 42.45% and 47.75%). Half maximal Inhibitory Concentration (IC) of cells was determined using SPSS software₅₀) Referring to FIG. 2B, it can be seen that 3 Survivin multi-point mutant proteins act on the IC of A549 cells₅₀Less than 3 Survivin single point mutant proteins.

Annexin V-FITC/PI double staining for detecting apoptosis: (1) regulating A549 cell density to 2 × 10⁵2mL of each well was added to a 6-well plate. (2) After 24h, old medium was aspirated and protein diluted to 4.19 and 8.37 μ M using cell culture medium was added, 2mL per well, and only fresh medium was replaced in the blank control group. (3) After 24h of protein exposure, cells were harvested and washed 2 times with PBS. (4) Adding 200 mu L of binding buffer solution in an Annexin V-FITC/PI double-staining kit, gently blowing and beating the cells to disperse the cells into single cells, respectively adding 5 mu L of Annexin V-FITC and PI, and incubating at 4 ℃ in a dark place for 20min to obtain the product for on-machine detection.

As can be seen from fig. 3, all 6 Survivin mutant proteins induced apoptosis of a549 cells in a concentration-dependent manner. Wherein, the capability of inducing A549 cell apoptosis of the 3 Survivin multi-point mutant proteins is stronger than that of the 3 Survivin single-point mutant proteins. When the protein concentration is 8.37 mu M, the A549 cell apoptosis rates caused by the 3 Survivin multi-point mutant proteins respectively reach 60.30%, 73.50% and 63.40%, which are higher than that of the 3 Survivin single-point mutant proteins (50.60%, 37.86% and 45.80%).

Western blot detection of content changes of apoptosis-related proteins Survivin and Caspase-3: (1) a549 cells at 2X 10⁵The density of individual/mL was seeded in 6-well plates, 2mL per well. (2) After 24h, the old medium was aspirated off and 2mL of protein diluted to 4.19 and 8.37. mu.M with cell culture medium was added per well. (3) After 24h of drug action, cells were harvested and washed with PBS. (4) Adding appropriate amount of cell lysate RIPA (PMSF final concentration of 1mM) according to cell precipitation amount, and performing ice lysis for 20-30min, and shaking with vortex instrument when not needed. (5) After the lysis is finished, centrifuging at 13000rpm for 15min, taking the supernatant to determine the protein concentration (the specific concentration determination step refers to the BCA method trace protein concentration determination kit instruction), and according to the determined protein concentration, measuring a certain volume to ensure that the protein loading amount is 30 mug uniformly. (6) Protein gel is prepared according to the experimental method for detecting the expression of the protein by SDS-PAGE electrophoresis in the embodiment 1, a protein sample and 4 XProtein SDS PAGE loading buffer are mixed uniformly and boiled for 10min, then the sample can be loaded, and 5 mu L of the protein is pre-stained on a marker. And (4) carrying out 80V electrophoresis for 30min, changing the voltage to 120V when the bromophenol blue runs to the separation gel, and stopping electrophoresis until the bromophenol blue is 1-2cm away from the glass plate. (7) And stripping protein gel from the glass plate, cutting the gel containing the target protein according to the size of the target strip, shearing a PVDF membrane, and activating in methanol for 5 min. (8) And (3) assembling the membrane rotating device according to a correct sequence, placing the membrane rotating device in a membrane rotating instrument, paying attention to the correspondence of the anode and the cathode, adding a membrane rotating buffer solution, and adjusting the membrane rotating current to be 200mA and the membrane rotating time to be 50 min. (9) After the membrane transfer was completed, the PVDF membrane was washed 3 times with TBST for 5min each time. (10) The PVDF membrane is placed in 3 percent skim milk powder, and is sealed at the normal temperature for 2 hours at 80 rpm. (11) The membrane was washed 3 times 5min each time with TBST. The primary antibody is prepared by using 3% skimmed milk powder, the dilution concentration refers to the instruction, the primary antibody is incubated overnight at 4 ℃, and the primary antibody can be repeatedly used. (12) The membrane was washed 3 times with TBST for 5min each, followed by incubation of the secondary antibody at ambient temperature. (13) The membrane was washed 3 times 5min each time with TBST. The PVDF film was then placed under a gel imager for photography. (14) And (3) during color development, placing the PVDF membrane in a gel imager, uniformly dropwise adding the prepared luminescent liquid on the PVDF membrane, and selecting proper exposure time to photograph.

As can be seen from FIG. 4, all of the 3 Survivin multipoint mutant proteins can cause the change of the expression level of Survivin and clear Caspase-3 in A549 cells in a concentration-dependent manner, which indicates that the Survivin mutant protein causes the change of the clear Caspase-3 content in the cells by reducing the expression level of Survivin in the A549 cells, and finally causes the apoptosis of the A549 cells. In conclusion, the Survivin multi-point mutant protein is considered to have better anticancer activity than the single-point mutant protein.

Example 3 chemosensitization of Tm34/84 to A549 CSCs

Reagents and kits: DMEM/F12 medium, B27 from Gibco (USA); epidermal growth factor EGF, basic fibroblast growth factor bFGF purchased from Peprotech (usa); annexin V-FITC/7-AAD apoptosis kit was purchased from Elabsciences (Shanghai); FITC-labeled anti-human CD44, PE-labeled anti-human CD133, FITC isotype control, and PE isotype control were purchased from David (Shanghai); anti-P-gp monoclonal antibody (rabbit-derived), anti-Bcl-2 monoclonal antibody (rabbit-derived) was purchased from Proteintech (USA); other biochemical reagents belong to domestic conventional analytical pure reagents.

Preparing a reagent:

200 μ g/mL EGF concentrate: dissolving 100 μ g EGF lyophilized powder in 500 μ L0.1% BSA solution to obtain 200 μ g/mL concentrated solution, packaging with EP tube at volume of 10 μ L per tube, and storing at-20 deg.C. In use, 10. mu.L of the concentrate was added to 100mL of CSCs medium under sterile conditions to prepare a final EGF solution at 20 ng/mL.

200 μ g/mL bFGF concentrate: dissolving 10 μ g EGF lyophilized powder in 50 μ L0.1% BSA solution to obtain 200 μ g/mL concentrated solution, packaging with EP tube at volume of 10 μ L per tube, and storing at-20 deg.C. In use, 5. mu.L of the concentrate was added to 100mL of CSCs medium under sterile conditions to prepare a final EGF solution at a concentration of 10 ng/mL.

CSCs medium: weighing 89mL of RPMI 1640 basic culture medium, 10mL of fetal bovine serum, 1mL of three antibodies, 10 mu L of EGF and 5 mu L of bFGF, uniformly mixing, and storing at 4 ℃.

Enrichment and identification of A549 CSCs

Enrichment of a549 CSCs: (1) taking the A549 parent cells in good growth state for pancreatin digestion, centrifuging at 1000rpm for 5min after the culture medium is resuspended, and discarding the supernatant. (2) PBS was added to resuspend the cells, re-centrifuged, the supernatant discarded, and the procedure repeated once. (3) Adding stem cell culture medium to resuspend the cell pellet, adjusting cell concentration to 1 × 10⁴one/mL. (4) Transfer of single cell suspension into low adsorption 6 wellsPlates, 2mL per well, were incubated in an incubator. (5) Half liquid changing operation is carried out on the cells for about 2 to 3 days, and the cells are subjected to subculture operation when the cell diameter reaches 100 mu m. When the liquid is changed in half amount, the cells can be centrifuged or placed still for 30min, then half of the old culture medium is removed by suction, and half of the fresh culture medium is added; and (3) centrifuging to remove the old culture medium during passage, adding Accutase for digestion until the cell balls are in a single cell state, adding a new stem cell culture medium, and performing cell culture according to the ratio of 1: passage 2. (6) Observing the formation of cell balls at any time, and taking the third generation cell balls for subsequent experiments.

As can be seen from FIG. 5A, as the number of passages increases, the shape of the spherical cells tends to be regular, and the number of cell debris and single cells also gradually decreases, indicating that the CSCs are enriched in the serum-free cell culture medium, and the spherical cells can be collected by centrifugation for the third generation for subsequent experiments.

Identification of a549 CSCs: 1. immunofluorescence and flow cytometry detection of expression of stem cell surface marker CD 133: (1) inoculating third generation A549 CSCs into a confocal culture dish containing cell slide (pretreated by polylysine), inoculating A549 parental cells into a common confocal culture dish, and adding RPMI 1640 complete culture medium to culture for about 6 h. (2) After the cells were completely attached to the wall, the dish was removed and rinsed 3 times with PBS. (3) The cells were fixed with 4% paraformaldehyde for 15min and rinsed again. (4) 3% BSA (diluted in TBST) was blocked at room temperature for 1h, rabbit anti-human CD133 antibody (1:100) was added, and the mixture was allowed to stand overnight at 4 ℃. (5) After the primary antibody working solution was aspirated, PE-goat anti-rabbit IgG (1:250) was added and incubated at room temperature for 1h in the dark. (6) And (4) absorbing and removing the secondary antibody, rinsing the cells for 3 times by TBST, labeling cell nuclei by Hoechst 33342, rinsing for 3 times by TBST after 15min, and observing under a confocal microscope. Detecting the expression of a stem cell surface marker CD133 by a flow cytometer: (1) and (3) digesting the third generation cell balls in suspension growth and the A549 parent cells in a good state until the cells are completely dispersed, centrifuging the cells at 1000rpm for 5min, and discarding the supernatant. (2) The pellet was washed 2 times with PBS, and 100. mu.L of PBS was added to resuspend the pellet and adjust the cell concentration to l.times.10⁶one/mL. (3) Monoclonal antibody CD133 (PE-labeled, mouse anti-human) 5. mu.L was added to the EP tube, isotype control antibody was added to the control groupIncubate at 4 ℃ in the dark for 30 min. (4) The supernatant was centrifuged and washed 3 times with PBS. (5) Each tube was tested by adding 200. mu.L PBS to the tube.

As shown in FIG. 5B, the third generation spherical cells showed strong red fluorescence, and the expression level of CD133 on the cell surface was 45.10%.

MTT and plate clone formation experiments to detect the in vitro proliferation capacity of A549 CSCs: (1) digesting A549 cell balls and parental cells with good growth condition, and adjusting cell concentration to 2 × 10⁴200. mu.L/mL of each well was added to a 96-well plate. Five culture plates are inoculated together, and each culture plate is divided into A549 cell balls + l 0% serum and A549 parent cells + 10% serum. (2) The inoculated cells were placed in an incubator and incubated continuously for 0, 1, 3, 5 and 7 days, with exchange of the medium every 48 h. (3) Subsequent experimental procedures refer to the experiments of the Survivin mutant protein inhibiting the growth activity of L-02 and A549 cells in example 2. Plate clone formation experiment for detecting the in vitro proliferation capacity of CSCs: (1) accutase digests well grown A549 cell spheres and parental cells, resuspends the complete medium and adjusts the cell density to 500 cells/well, 2mL per well in 6-well plates. (2) Shaking the inoculated cells evenly, placing the cells in an incubator gently for continuous culture, changing the liquid every 3 days, observing the cell state, observing the size of cell clone under a microscope, and taking a picture. (3) The cells were continued to be cultured until the number of cells of most individual clones in the well was greater than 50, the culture was stopped, the supernatant was discarded, and the cells were washed 1 time with PBS. (4) 1mL of 4% paraformaldehyde was added to each well, and the cells were fixed at 4 ℃ for 15min and washed 3 times with PBS. (5) Cells were stained with 1mL of 1 XGimas stain for 10min per well. (6) ddH₂After O-washing the cells, the number of clones was counted and photographed.

As can be seen from fig. 6A, the proliferation curve of the third generation spherical cells is always located below the a549 parent cells, indicating that the proliferation ability is weaker than that of the a549 parent cells. As can be seen in fig. 6B, the ability of the third generation spheroids to form clones was weaker than the a549 parental cells.

MTT detection of the drug resistance of CSCs to ADM: (1) resuspending A549 parental cells and CSCs in complete culture medium, adjusting cell density to 2 × 10⁵one/mL, 100. mu.L per well in 96 wellsAnd (3) a plate. (2) After the cells adhered to the wall, different concentrations (0.1, 0.2, 0.4, 0.8 and 1.6 μ M) of ADM drug diluted in RPMI 1640 complete medium were added to the drug-added group according to the experimental pre-design, and only fresh medium was replaced in the blank control group. (3) Subsequent experimental procedures the Survivin mutant protein of reference example 2 inhibited the growth activity of L-02 and A549 cells. (4) IC calculation of ADM for A549 and CSCs by SPSS₅₀The value is obtained. Wherein the Drug Resistance Index (RI) ═ IC of CSCs to ADM₅₀) /(IC of A549 vs. ADM₅₀)。

As can be seen from fig. 7A, the drug sensitivity curves of CSCs to ADM consistently lie above the a549 parental cells, indicating that CSCs are less sensitive to ADM than a549 cells are. Using SPSS software, one can calculate: IC when ADM acts on A549 cells₅₀4.296 μ M; when ADM acts on CSCs, IC₅₀The drug resistance index RI is 2.46 when the drug resistance index RI is increased to 10.576 mu M, which indicates that the tolerance of the CSCs to the chemotherapeutic drug ADM is stronger than that of A549 parent cells, and the theory is identical with that the CSCs have stronger drug resistance and the traditional chemotherapeutic drug can not act on the CSCs.

Western blot for detecting the expression of apoptosis and drug resistance related proteins in CSCs: (1) and centrifuging to collect third generation cell balls growing in suspension and A549 parent cells in logarithmic growth phase. (2) The cells are washed 2 times by PBS, and the steps of cell lysis and subsequent Western blot can be referred to in example 2, Western blot is used for detecting the content change of apoptosis-related proteins Survivin and Caspase-3.

As can be seen from FIG. 7B, the expression levels of the apoptosis-related proteins Survivin, Bcl-2 and the drug-resistance-related protein P-gp in the third generation of spherical cells were all up-regulated compared to the A549 cells, and were 1.79 times, 2.43 times and 1.58 times higher than those of the A549 cells, respectively. In conclusion, the research finds that the third generation spherical cells have higher expression levels of CD133, Survivin, Bcl-2 and P-gp, slower cell growth speed and stronger chemotherapy resistance compared with the A549 parent cells, and the third generation spherical cells obtained by serum-free suspension culture are A549 CSCs.

Chemosensitization effect of Tm34/84 on A549 CSCs

Detection of the effect of TmSm34/84 on the proliferation activity of CSCs: (1) a is to be549 parental cells and CSCs in complete culture medium, adjusting cell density to 2 × 10⁵one/mL, 100. mu.L per well in 96-well plates. (2) After the cells adhered, TmSm 34/84180. mu.L diluted in RPMI 1640 complete medium was added to the dosing group at different concentrations (2.09, 4.19, 6.28 and 8.37. mu.M) and the blank group was changed with fresh medium only. (3) The subsequent experimental procedures refer to example 2 "experiment of Survivin mutant protein inhibiting a549 cell growth activity".

As shown in FIG. 8, TmSm34/84 was able to inhibit the proliferative activity of CSCs with high Survivin expression. Wherein when the TmSm34/84 concentration is 1.05 mu M, the activity of the CSCs is reduced by 7.23%, so that 1.05 mu M is selected as a non-cytotoxic dose in the study, and the effect of the TmSm34/84 on the chemotherapy sensitization of the CSCs is studied. The non-cytotoxic dose is an important index for researching chemotherapy sensitization, does not inhibit cell activity, but can change a cell drug resistance mechanism, such as influencing the expression and distribution of drug resistance protein or influencing a channel for a drug to enter cells, thereby assisting the chemotherapy drug to play a role.

MTT assay effect of non-cytotoxic dose TmSm34/84 on CSCs resistance: (1) resuspending A549 parental cells and CSCs in complete culture medium, adjusting cell density to 2 × 10⁵one/mL, 100. mu.L per well in 96-well plates. (2) After the cells were adherent, a non-cytotoxic dose of TmSm34/84 was added to different concentrations (0.1, 0.2, 0.4, 0.8 and 1.6. mu.M) of ADM drug diluted with cell culture medium, with a total volume of 180. mu.L per well, and the blank control group was changed with fresh medium only. (3) The subsequent experimental procedures refer to example 2 "experiment of Survivin mutant protein inhibiting a549 cell growth activity". (4) SPSS calculation of IC of CSCs/ADM after TmSm34/84 is combined with ADM₅₀Calculating the reversal multiple of drug resistance as IC₅₀(CSCs/ADM)/IC₅₀(CSCs/ADM+TmSm34/84)。

As shown by the MTT results in fig. 9, when CSCs were treated with different concentrations of ADM in combination with non-cytotoxic doses of TmSm34/84, the activity of the CSCs was reduced compared to ADM alone. IC with ADM acting on CSCs₅₀A drop to 1.51. mu.M indicated a resistance reversal factor of 7.01 for the non-cytotoxic dose of TmSm 34/84.

For the MTT experimental results of fig. 9, the grouping is as follows: control, 1.05. mu.M TmSm34/84, 1.6. mu.M ADM treated alone and in combination. After 24h of action on CSCs, the morphological changes of the cells are shown in fig. 10: compared with a control group, 1.05 mu M of TmSm34/84 alone has no great influence on the morphology of the CSCs, 1.6 mu M of ADM treated group alone has irregular morphology, a small amount of CSCs has lytic death phenomenon, while most of the cells of the combined drug group have obvious morphological changes, the cytospheres become loose, and floating dead cells are further increased, which shows that the TmSm34/84 can enhance the sensitivity of the CSCs to ADM.

Flow assay for the effect of non-cytotoxic doses of TmSm34/84 on drug resistance of CSCs: (1) collecting CSCs by centrifugation, digesting with Accutase, and adjusting cell density to 5 × 10⁵2mL of each well was added to a 6-well plate. (2) After the cells were attached, a non-cytotoxic dose of TmSm34/84 and a concentration of ADM drug diluted in RPMI 1640 complete medium were added and the blank control group replaced with fresh medium only. (3) After 24h of drug action, cells were harvested and the pellet washed 2 times with PBS. (4) The cells were resuspended in binding buffer, adjusted to a concentration of 1X 10⁶one/mL. (5) Sucking 100 μ L of cell suspension into a new tube, adding 5 μ L of Annexin V-FITC and 5 μ L of 7-AAD, mixing, incubating at 4 deg.C in dark for 30min, and detecting.

The flow results as shown in FIG. 11 also further indicate that the non-cytotoxic dose of TmSm34/84 is capable of increasing the sensitivity of CSCs to ADM: when 1.6 mu M ADM is used alone, the apoptosis rate of the CSCs reaches 21.81 percent, and when the ADM is used in combination with a non-cytotoxic dose TmSm34/84, the apoptosis rate of the CSCs reaches 42.2 percent.

Western blot to detect the influence of non-cytotoxic dose TmSm34/84 on CSCs apoptosis and drug resistance related protein expression: (1) the CSCs were collected by centrifugation and cell density was adjusted to 5X 10⁵2mL of each well was added to a 6-well plate. (2) After the cells are attached to the wall, 3 experimental groups (a non-cytotoxic dose TmSm34/84 treatment group, an ADM treatment group and a combined treatment group) are set, corresponding drugs are respectively added, a blank control group is only required to replace a culture medium, and 3 parallel holes are set for each concentration. (3) After 24h of drug action, cells were collected by centrifugation and the pellet was washed 2 times with PBS. (4) Cell lysis and subsequent Western blot procedureThe change in the contents of apoptosis-related proteins Survivin and Caspase-3 was examined with reference to "Western blot" in example 2.

As shown in FIG. 12, the combined use of 1.05. mu.M TmSm34/84 and 1.6. mu.M ADM significantly down-regulated the expression levels of Survivin, Bcl-2 and P-gp in CSCs (P <0.05) and significantly up-regulated the expression level of the pro-apoptotic protein Cleaved Caspase-3 (P <0.05) compared to the ADM treated group alone, indicating that the mechanism by which the non-cytotoxic dose of TmSm34/84 increased the sensitivity of CSCs to ADM may be related to its down-regulation of the expression of Survivin, Bcl-2 and P-gp, up-regulated clear Caspase-3.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of east China's college of science

<120> Survivin multi-point mutant recombinant protein and application thereof

<130> /

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Tyr Ala Arg Lys Ala Arg Arg Gln Ala Arg Arg

1 5 10

<210> 2

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggaattccat atgtacgctc gtaaagctcg t 31

<210> 3

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tctggctcgt tctcggcggg gcagtggatg 30

<210> 4

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

catccactgc cccgccgaga acgagccaga 30

<210> 5

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ccgctcgaga tccatggcag ccagctgctc g 31

<210> 6

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggaattccat atgtacgctc gtaaagctcg t 31

<210> 7

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cagaaaggaa agcggcaccg gacgaatgct 30

<210> 8

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agcattcgtc cggtgccgct ttcctttctg 30

<210> 9

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ccgctcgaga tccatggcag ccagctgctc g 31

<210> 10

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggaattccat atgtacgctc gtaaagctcg t 31

<210> 11

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

agaaaggaaa gcggcaccgg acgaatgctt 30

<210> 12

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aagcattcgt ccggtgccgc tttcctttct 30

<210> 13

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ccgctcgaga tccatggcag ccagctgctc g 31

Claims

1. The Survivin multi-point mutant recombinant protein is characterized in that the protein is prepared by genetic engineering, N-terminal fusion transduction peptide is adopted, and two or three amino acids of 34 th threonine, 48 th threonine and 84 th cysteine of wild Survivin are mutated into alanine.

2. The Survivin multi-point mutant recombinant protein according to claim 1, wherein threonine 34 and threonine 48 of wild type Survivin are mutated to alanine.

3. The Survivin multi-point mutant recombinant protein according to claim 1, wherein threonine 34 and cysteine 84 of wild type Survivin are mutated to alanine.

4. The Survivin multi-point mutant recombinant protein according to claim 1, wherein threonine 34, threonine 48 and cysteine 84 of wild type Survivin are mutated to alanine.

5. The Survivin multi-point mutant recombinant protein according to claim 1, wherein the sequence of the transduction peptide is Tyr-Ala-Arg-Lys-Ala-Arg-Gln-Ala-Arg.

6. The Survivin multipoint mutant recombinant protein of claim 1 for use in preparing tumor chemotherapeutic drug sensitizers.

7. The use of Survivin multipoint mutant recombinant protein according to claim 6, wherein the tumor is lung cancer and the chemotherapeutic drug is Adriamycin (ADM).

8. The Survivin multi-point mutant recombinant protein as claimed in claim 1, wherein the tumor is a tumor over-expressing Survivin.

9. The Survivin multi-point mutant recombinant protein as claimed in claim 8, wherein the tumor includes but is not limited to lung cancer, liver cancer, breast cancer, pancreatic cancer and bladder cancer.