Radiosensitive positive feedback gene expression amplification vector as well as construction method and application thereof

Abstract

The invention discloses a radiosensitive positive feedback gene expression amplification vector as well as a construction method and application thereof, and belongs to the technical field of tumor gene therapy, the vector is pE6-LuxI-LuxR, and has a nucleotide sequence as shown in SEQ ID NO.1; the radiosensitive positive feedback gene expression amplification vector pE6-LuxI-LuxR disclosed by the invention can drive a p53 gene to realize tumor targeting continuous high expression without endogenous interference, and target gene expression is limited to a tumor part, so that systemic toxic reaction is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor gene therapy, and in particular to a radiosensitive positive feedback gene expression amplification vector, a construction method thereof and an application thereof. Background Art

[0002] The discovery that p53 (tumor suppressor gene) dynamics determine cell fate has brought new opportunities for tumor treatment. p53 is one of the most important tumor suppressor factors in cells, known as the "guardian of the genome", and can initiate the cell self-destruction program; elephants have a very low rate of canceration due to the high number of p53 copies, and more than 50% of human malignant tumors have p53 gene mutations. Therefore, p53-targeted therapy has always been an important research direction in the field of oncology, but progress has been slow over the years.

[0003] Recently, it has been found that differences in p53 expression dynamics determine cell fate. After environmental stress, the expression dynamics of p53 in cells presents two modes: one is a pulsed oscillation, which mainly occurs in cells with low levels of DNA damage; the other is a sustained high-level expression, which is mainly caused by DNA damage that is too severe or difficult to repair. Further studies have found that the characteristics of p53 dynamic changes affect cell life and death: pulsed expression can cause increased expression of downstream molecules such as p21, leading to cell cycle arrest and gene repair, allowing cells to survive; while sustained high-level expression induces enhanced expression of PUMA and other molecules, leading cells to apoptosis or senescence.

[0004] Mathematical modeling suggests that the mechanism of p53 dynamics formation is mainly related to feedback regulation loop regulation. The pulsed oscillation changes are mainly caused by the two negative feedback loops of p53-Mdm2 and p53-Wip1-ATM, while p53 shows a sustained high level of expression when the p53-PTEN-Akt-Mdm2 positive feedback loop plays a dominant role. The above findings have deepened the understanding of the biological role of p53 and expanded the research ideas of tumor treatment.

[0005] Regulating p53 dynamics to treat tumors mainly has the following shortcomings: poor tumor targeting, weak p53 expression activity, and endogenous interference of loop components. Specifically:

[0006] First, it lacks tumor targeting: Research on p53 dynamic feedback regulation at the protein level mainly focuses on the regulation of the p53-Mdm2 negative feedback loop, using Mdm2 inhibitors to block its feedback inhibition of p53, turning negative feedback into "positive lack of tumor targeting regulation", resulting in continuous expression of p53, and ultimately leading to the death of tumor cells. Preliminary animal and clinical trials have shown that Mdm2 inhibitors such as Nutlins show good efficacy in blood tumors and various solid tumors. It has been reported that Nutlins combined with radiotherapy have shown a significant radiosensitization effect. Although radiation can also cause an increase in wild-type p53 expression under normal conditions, it often causes radiotherapy resistance due to low expression levels, suggesting that p53 dynamic regulation is expected to become a reliable solution for radiotherapy sensitivity modification. However, the problem is that the blood and organ toxicity is relatively large. The reason is that the Mdm2 negative feedback loop inhibitor lacks tumor targeting, resulting in high levels of p53 expression in normal tissues, producing unnecessary toxic reactions, which hinders clinical application;

[0007] Second, the expression activity of p53 is not strong enough: synthetic gene circuits are artificial biological systems constructed using different biological molecules and gene elements. They perform specific biological functions by sensing, integrating and processing molecular signals in living cells, such as modular functional units such as bistable switches, oscillators and cell communications. The inventors of this application previously explored the introduction of synthetic gene circuits into the transcriptional regulation of the p53 gene. Based on the hypoxic microenvironment of the tumor, they constructed a hypoxia / radiation dual-sensitivity gene circuit to induce the specific expression of the p53 gene in tumor tissues, thereby greatly improving tumor targeting. However, the main drawback of this method is that the expression activity of p53 is not strong enough, and it is not possible to obtain a sustained high level of expression of p53 in cells, and it cannot cause complete regression of the tumor;

[0008] The third is the interference of endogenous expression: Since the elements of the synthetic gene circuit mainly come from mammalian cells and have a certain homology with the host cells, they are easily interfered by endogenous expression, affecting the accuracy and stability of p53 expression regulation.

[0009] In summary, although regulating p53 dynamics for tumor treatment has good prospects, existing studies have their own shortcomings. First, tumor targeting is poor, causing systemic toxicity; second, the expression intensity is weak, affecting tumor efficacy; third, there is interference with the endogenous expression of loop components.

[0010] Therefore, it is necessary to explore new methods to precisely regulate p53 dynamics. Summary of the invention

[0011] One of the purposes of the present invention is to provide a radiation-sensitive positive feedback gene expression amplification vector to solve the above-mentioned problems.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is as follows: a radiation-sensitive positive feedback gene expression amplification vector, the vector is pE6-LuxI-LuxR, which has a nucleotide sequence as shown in SEQ ID NO.1.

[0013] Among them, pE6 has the nucleotide sequence shown in SEQ ID NO.2, and LuxI-LuxR has the nucleotide sequence shown in SEQ ID NO.3.

[0014] The second object of the present invention is to provide a method for constructing the above-mentioned radiosensitive positive feedback gene expression amplification vector, comprising the following steps:

[0015] (1) Synthetic gene fragments;

[0016] (2) Plasmid extraction and linearization of backbone vector;

[0017] (3) The target gene is constructed into a linearized expression vector;

[0018] (4) Transform the reaction products into Stbl3 competent cells;

[0019] (5) Identification of plasmid vector.

[0020] As a preferred technical solution, the specific method of step (1) is: designing and synthesizing pE6-LuxI-LuxR, and then performing PCR amplification to obtain the target gene insertion fragment.

[0021] As a further preferred technical solution, the reaction system and conditions of the PCR amplification are:

[0022] The reaction system consists of: 2x M5 HiPer SYBR Premix Es Taq 25 μL, forward primer (10uM) 1 μL, reverse primer (10uM) 1 μL, template (20-100 ng / μL) 4 μL, ddH2O to 50 μL;

[0023] Reaction conditions: pre-denaturation at 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 20 s, extension / annealing at 60°C for 30-34 s, 72°C for 3 min, and a final extension step at 72°C for 10 min.

[0024] As a preferred technical solution, in step (2), the linearization treatment method is: digest the expression vector with a restriction endonuclease, and the digestion reaction system is: 2 μg of plasmid, 5 μL of 10 x reaction buffer, 1 μL of each restriction endonuclease, make up to 50 μL with water, and incubate in a 37°C metal bath for more than 2 h. The purpose of linearization treatment is to facilitate the insertion of the target gene fragment.

[0025] The third object of the present invention is to provide the use of the above-mentioned radiosensitive positive feedback gene expression amplification vector in driving the p53 gene.

[0026] In order to further solve the tumor targeting of p53 expression, the inventors have used the radiosensitive regulatory element cArGs to induce the target gene (referring to p53) and obtained spatiotemporal specific expression, which provides a reliable method for solving the above problems. cArGs is a radiosensitive regulatory sequence CC(A+T)6GG present in the early growth response gene-1 (Egr-1) promoter. It is inactive under normal conditions, but induces a rapid and short-term increase in downstream gene expression after irradiation. cArGs is coupled to the target gene, and the target gene expression is induced during local irradiation. The target gene expression is limited to the tumor site through the spatiotemporal effect of the radiation, avoiding systemic toxic reactions. Therefore, the present application uses the radiosensitive element cArGs for the spatiotemporal specific expression regulation of p53 dynamics to obtain tumor-targeted expression.

[0027] Based on the above analysis, this application uses complementary advantages and integrated utilization of the spatiotemporal specificity of cArGs and the amplification function of the LuxR / LuxI loop to construct a radiosensitive positive feedback loop and drive the p53 gene, achieving tumor-targeted sustained high expression without endogenous interference, while overcoming the above three deficiencies. Its basic principles are:

[0028] (1) Use radiation to activate the amplification loop in cells and regulate downstream gene expression: The activation of the LuxR / LuxI feedback loop requires a certain initial level of LuxR stimulation. However, due to the lack of basal expression of LuxR in mammalian cells, this gene loop is in a "dormant" state. Therefore, it is necessary to install an induction activation device for this loop, namely the radiation response element cArGs, which can induce a transient increase in LuxR expression under the action of radiation, further activating the positive feedback loop, and finally "directedly detonating" the amplification loop at the tumor site through the "remote control" of radiation;

[0029] (2) Driving the sustained high-level expression of p53 in targeted tumors and the radiosensitization effect: Using a bicistronic expression vector, the wild-type p53 cDNA is placed downstream of the above loop and introduced into tumor cells; through the radiation-activated LuxR / LuxI amplification loop, sustained high expression of p53 in tumor cells is achieved, the cell apoptosis program is initiated, and the targeted radiosensitization effect is obtained;

[0030] In this application, the cArGs element is connected in series with the luxR / luxI loop, and a bicistronic expression vector is used to drive the expression of the reporter gene GFP to verify its radiosensitivity and amplification effect; it is then coupled with wild-type p53 cDNA and introduced into p53-deficient, mutated or wild-type lung cancer cells, and the p53 kinetic pattern, spatiotemporal distribution characteristics and targeted sensitization effect after irradiation are observed through in vivo and in vitro experiments.

[0031] Compared with the prior art, the advantages of the present invention are: the radiosensitive positive feedback gene expression amplification vector pE6-LuxI-LuxR of the present invention can drive the p53 gene to achieve tumor-targeted sustained high expression without endogenous interference, and the target gene expression is limited to the tumor site to avoid systemic toxic reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the electrophoresis diagram of pE6 synthesized in Example 1;

[0033] Figure 2 This is the electrophoresis diagram of pE6-luxI-luxR synthesized in Example 1;

[0034] Figure 3 This is the electrophoresis diagram of pLuxI-luxR synthesized in Example 1;

[0035] Figure 4 The pE6-EGFP fluorescence expression in Example 2;

[0036] Figure 5 The pLuxI-luxR-EGFP fluorescence expression of Example 2;

[0037] Figure 6 The pE6-luxI-luxR-EGFP fluorescence expression in Example 2;

[0038] Figure 7 and Figure 8 The p53 protein expression levels of different groups after irradiation for 72 hours in Example 3;

[0039] Figures 9 to 12 The p53 protein expression levels of different groups at different time points in Example 3;

[0040] Fig.13 and Fig.14This is a diagram showing the cell clone formation experiment results of Example 4;

[0041] Fig.15 and Fig.16 This is the comet experiment result diagram of Example 5;

[0042] Fig.17 This is a nude mouse transplant tumor growth curve of the in vivo tumor inhibition effect evaluation test of Example 6;

[0043] Fig.18 This is a comparison chart of tumor volumes at day 11 of treatment in the in vivo tumor inhibition effect evaluation test of Example 6. DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with the accompanying drawings.

[0045] Example 1

[0046] Plasmid vector construction, the specific method is as follows:

[0047] (1) Gene fragment synthesis

[0048] Design and synthesize pLuxI, hTP53, pE6-minP, and pE6-pLuxI fragments, and then perform PCR amplification to obtain the target gene insert fragment;

[0049] PCR amplification of target gene: Use high-fidelity PrimeSTAR enzyme to amplify the target gene. The reaction system and conditions are as follows:

[0050] The reaction system consists of: 2x M5 HiPer SYBR Premix Es Taq 25 μL, forward primer (10uM) 1 μL, reverse primer (10uM) 1 μL, template (20-100 ng / μL) 4 μL, ddH2O to 50 μL;

[0051] Reaction conditions: pre-denaturation at 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 20 s, extension / annealing at 60°C for 30-34 s, 72°C for 3 min, and a final extension step at 72°C for 10 min.

[0052] The PCR product was subjected to agarose gel electrophoresis to detect the amplification effect, and the target gene band was cut out from the gel after agarose gel electrophoresis and recovered with a gel recovery kit. The gel recovery operation refers to the kit manual;

[0053] (2) Plasmid extraction and linearization of backbone vector

[0054] Kit: Axyprep plasmid miniprep kit 250-prep (AXYGEN Co.)

[0055] Steps:

[0056] 1) Take 3-5 ml of bacterial solution and centrifuge at 10000g for 1 min at room temperature;

[0057] 2) Discard the supernatant, add 250 μl solution I / RNase A to resuspend the colonies, and mix well;

[0058] 3) Add 250 μl solution II, invert and mix gently, and incubate for 2 minutes. (The entire lysis reaction should not exceed 5 minutes);

[0059] 4) Add 250 μl of solution III and immediately invert to mix until a white flocculent precipitate is formed;

[0060] 5) Centrifuge at room temperature at 13000g for 10 min;

[0061] 6) Carefully transfer the supernatant into the HiBind miniprep column (with cannula) and centrifuge at room temperature at 10,000 g for 1 min.

[0062] 7) Discard the waste liquid, add 500 μl buffer HB, and centrifuge at room temperature at 10,000 g for 1 min;

[0063] 8) Optional step: Repeat step 7);

[0064] 9) Discard the waste liquid and centrifuge the empty tube at room temperature at 13000g for 2 minutes;

[0065] 10) Transfer the column to a new EP tube, add 30-50 μl elution buffer or 3ddH2O to the membrane, and incubate at room temperature for 1-2 minutes;

[0066] 11) Centrifuge at 13000g for 1 min at room temperature, collect the liquid from the centrifugation as the extracted plasmid and store at -20℃ for later use;

[0067] Linearization:

[0068] The expression vector was digested with restriction endonucleases. The digestion reaction system was: 2 μg of plasmid, 5 μL of 10 x reaction buffer, 1 μL of each restriction endonuclease, and water was added to 50 μL. The mixture was incubated in a 37°C metal bath for more than 2 h. The digestion product was subjected to agarose gel electrophoresis to detect the digestion effect, and the target vector band was cut from the gel after agarose gel electrophoresis and recovered with a kit. The gel recovery operation was referred to the kit instructions.

[0069] (3) Target gene is constructed into linearized expression vector

[0070] Use the seamless cloning kit to add the target gene fragment and linearized vector to a centrifuge tube in a certain ratio for recombination reaction:

[0071]

[0072] X refers to the amount of insert fragment used, and Y refers to the amount of linearized vector used. Insert fragment: vector is added at a molar ratio of 3:1. The specific amount needs to be calculated based on the actual concentration. After mixing, incubate at 37℃ for 1 h, and transform directly or store at -20℃ and thaw when needed.

[0073] (4) Transform the reaction products into Stbl3 competent cells

[0074] Add 10 μL of the reaction product to 100 μL of Stbl3 E. coli and incubate on ice for 30 minutes; heat shock the cells at 42°C for 30 seconds; immediately transfer to ice and incubate for 2 minutes; add 250 μL of SOC medium and incubate in a shaker at 37°C, 225 RPM. for 1 hour; spread 350 μL of the transformant on an LB plate containing 100 μg / mL Amp and incubate at 37°C overnight.

[0075] (5) Identification of positive bacteria (for further amplification and plasmid extraction)

[0076] Pick the transformants grown on the plate and resuspend them in 50 μL LB culture medium, and take 2 μL as a template for colony PCR identification. The reaction system and PCR cycle conditions are as follows:

[0077] The reaction system consisted of: 2x M5 HiPer SYBR Premix Es Taq 10 μL, forward primer 1 μL, reverse primer 1 μL, template 2 μL, ddH2O 6 μL;

[0078] Reaction conditions: pre-denaturation at 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 20 s, extension / annealing at 60°C for 30-34 s, 72°C for 30 min, and a final extension step at 72°C for 5 min

[0079] The reaction product was tested by gel electrophoresis to identify the positive cloned bacteria. After identification, the bacteria were expanded and then extracted using a plasmid extraction kit. After the concentration was tested, the bacteria were stored at -20°C and sequenced for further identification.

[0080] (6) Plasmid vector identification

[0081] The constructed vector was verified by DNA sequencing and gel electrophoresis. Figure 1 , Figure 2 and Figure 3 As shown, Figure 1 , Figure 2 and Figure 3 In the figure, “M” represents marker, “P” represents vector backbone, and “1” represents the corresponding gene fragment;

[0082] It should be noted that the primers used in amplifying the above different target fragments are shown in Table 1 below:

[0083] Table 1 Primer sequences used for amplification of different target fragments

[0084] .

[0085] Example 2

[0086] This example is to identify the function of the radiation-activated luxR / luxI positive feedback loop. The experimental process is as follows:

[0087] (1) Inoculate H1299 cells in the logarithmic growth phase into a 6-well plate and prepare for transfection when the cell confluence reaches 70-90%; aspirate the culture medium in the 6-well plate, add 1 ml of 1640 basal medium (GIBICO), and place in a cell culture incubator to wait for transfection;

[0088] (2) Dilute Lipofectamine™ 3000 (Invitrogen) with Opti-MEM™ medium (GIBICO) at a dilution ratio of 7.5 μl: 125 μl; dilute DNA with Opti-MEM™ medium to prepare a DNA premix, and then add P3000™ reagent (Invitrogen) at a dilution ratio of 2.5 ng: 125 μl: 5 μl;

[0089] (3) Add diluted DNA (1:1) to each tube of diluted Lipofectamine™ 3000; incubate at room temperature for 15 minutes; add 250 μl of DNA-liposome complex to each well of cells; place the transfected 6-well plate in a cell culture incubator and incubate to prepare for the next experiment;

[0090] (4) Take out the transfected 6-well plate, aspirate the old culture medium, and add new culture medium;

[0091] (5) According to the experimental purpose, 0 Gy, 1 Gy, 2 Gy, 4 Gy, and 8 Gy of X-ray irradiation were given respectively, and the fluorescence expression intensity of each well at 0 h, 3 h, 8 h, 12 h, 24 h, 36 h, 72 h, and 96 h after irradiation was observed using a fluorescence microscope;

[0092] The results are as follows Figure 4-6 As shown in the figure, it can be seen that the constructed positive feedback gene loop can target the downstream gene fluorescent protein for continuous high expression under X-ray irradiation, while it is almost not expressed without irradiation, proving that the loop is strictly regulated by radiation and has an obvious "switch" effect. In the dose range of 1Gy to 4Gy, the fluorescence expression of pE6-EGFP, pLuxI-luxR-EGFP, and pE6-LuxI-luxR-EGFP groups gradually increased, and the intensity of pE6-LuxI-luxR-EGFP was stronger than that of pE6-EGFP and pLuxI-luxR-EGFP groups in different time periods; at 8Gy, the overall fluorescence expression decreased, which may be related to the high number of cell apoptosis at this dose.

[0093] Example 3

[0094] This example is to verify the radiation-induced amplification loop driving the high expression of p53 protein. The experimental process is as follows:

[0095] (1) pE6-p53, pE6-LuxI-luxR-p53 and pluxI-luxR-p53 were transfected into H1299 cells respectively, and 4Gy X-ray irradiation was given. After incubation for 3h, 8h, 24h, 72h, 96h and 144h, the culture was terminated, the old culture medium was aspirated, and the cells were washed twice with PBS. 200ul of RIPA lysis buffer was added to each well, and the cells were mixed thoroughly by pipetting. The cells were placed on ice for 30 minutes for lysis. The lysis mixture was aspirated, centrifuged at 4℃, 15000 rpm for 10 min, the supernatant was aspirated, 50ul of SDS-PAGE protein loading buffer was added, and the cells were then placed in a water bath and heated for 10min (95℃). The protein sample preparation was completed and placed at -80℃ for later use;

[0096] (2) Electrophoresis: Fix the precast gel in a vertical electrophoresis tank, pour the prepared electrophoresis solution into the tank, and then load the samples one by one according to the experimental groups. Connect the power supply and keep the constant voltage at 130mv for 70min;

[0097] (3) Transfer: Soak the PVDF membrane evenly in ethanol for later use. Take out the precast gel that has completed electrophoresis, carefully cut open the glass and take out the gel. Stack the transfer clip from the negative electrode to the positive electrode in the order of filter paper, sponge, gel, PVDF membrane, sponge and filter paper, and clamp it to prevent bubbles from forming. Add the prepared transfer solution and ice pack to the bath, place the transfer clip in the bath, connect the power supply, and keep the constant current at 400mA for 35min;

[0098] (4) Blocking: Take out the transferred strips and place them in pre-prepared 5% skimmed milk powder, and block them on a shaker (shaking speed 60 rps) for 1 h;

[0099] (5) Incubation with antibodies: After blocking, wash once with TBST, incubate the PVDF membrane with pre-diluted antibodies p53, β-actin, and GAPDH (ProSci), and incubate overnight in a 4°C refrigerator; aspirate the antibodies the next day and wash three times with TBST (G-Biosciences), 15 minutes each time. Then incubate with secondary antibodies at room temperature for 60 minutes. After the secondary antibody incubation is complete, wash the membrane using the same method as above;

[0100] (6) Development: Soak the PVDF membrane in pre-mixed developer A and B, and then develop;

[0101] The vector backbone Lvool (purchased from Science Compass), pE6-p53, pLuxI-luxR-p53, and pE6-LuxI-luxR-p53 were transfected into H1299 cells, and the expression of p53 protein was detected by immunoblotting 72 hours after irradiation. The results are shown in Figure 7 and Figure 8 shown; from Figure 7 and Figure 8 It can be seen that the irradiated group is stronger than the non-irradiated group, and the blank group (Control) and Lvool group almost do not express p53 protein, proving that the cell background and vector skeleton do not express p53 protein; the p53 protein content of pE6-p53, pLuxI-luxR-p53 and pE6-LuxI-luxR-p53 groups in the irradiated group gradually increased, among which the p53 protein content of pE6-LuxI-luxR-p53 was significantly higher than that of other groups, and the difference was statistically significant (P < 0.05), indicating that pE6-LuxI-luxR-p53 has the effect of targeted driving high expression of target genes after irradiation.

[0102] At the same time, the inventors further tested at different time points, at 3h, 8h, 24h, 72h, 96h, and 144h. Figures 9 to 12As shown, the expression of pE6-LuxI-luxR-p53 was stronger than that of pE6-p53 and pLuxI-luxR-p53. The p53 protein content of pE6-LuxI-luxR-p53 showed a dynamic increasing trend in the time periods of 3h, 8h, 24h, 72h, and 96h. In the time period from 96h to 144h, the expression of P53 protein content weakened.

[0103] The protein expression levels of pE6-LuxI-luxR were compared with two different plasmids pE6 and LuxI-luxR. Figures 9 to 12 It can be seen from the comparison that pE6-LuxI-luxR has the strongest effect in driving p53 protein expression.

[0104] Example 4

[0105] This example is to verify the radiosensitization effect of pE6-luxR-luxI-p53, and the experimental process is as follows:

[0106] (1) H1299 cells were seeded in 6-well plates and transfected according to the following groups: blank (Control), vector skeleton (Lvool), pE6-p53, pE6-LuxI-luxR-p53 and pLuxI-luxR-p53. After 24 hours of transfection, the cells were irradiated with 0 Gy, 1 Gy, 2 Gy, 4 Gy and 8 Gy. The irradiated cells were digested with trypsin and then centrifuged and counted. Approximately 1000 cells were seeded in each well, and 1640 complete medium was added and placed in a cell culture incubator for culture. The cell growth was observed every 2 days and the medium was replaced once. The culture was terminated after 14 days.

[0107] (2) Terminate cell culture after 2 weeks, remove old culture medium, wash twice with PBS, and then fix with paraformaldehyde for 30 minutes. After fixation, remove paraformaldehyde and add crystal violet for staining for about half an hour. Recover crystal violet, wash twice with PBS, and place the 6-well plate in a ventilated dryer to dry naturally.

[0108] (3) After taking photos, use IMAGE J to count and process the data.

[0109] Experimental results and discussion: The clone formation rate of pE6-pLuxI-LuxR-p53 was significantly lower than that of other groups. The software GraphPad Prism 8.3.0 was used for nonlinear regression fitting to obtain the single-click multi-target cell survival model and its related parameters, among which Control+X-ray group: D0=2.9895, D37=5.4546; Lvool+X-ray group: D0=3.1705, D37=5.7443; pE6-p53+X-ray group: D0=2.7863, D37=4.6232; pLuxI-luxR-p53+X-ray group: D0=2.8089, D37=4.5787; pE6-LuxI-luxR-p53+X-ray group: D0=2.2614, D37=3.994, SER=1.38, as shown in Figure 5. Figure 13-14 As shown; Fig.13 In the table, C stands for Control, P stands for Lvool, E stands for pE6-p53, L stands for pLuxI-luxR-p53, and E+L stands for pE6-LuxI-luxR-p53.

[0110] The above experimental results prove that the pE6-LuxI-luxR-p53+X-ray group can improve the radiosensitivity in tumor cells.

[0111] Example 5

[0112] This example verifies the radiosensitization effect of pE6-luxR-luxI-p53, and the experimental process is as follows:

[0113] (1) Cells in 6-well plates were divided into experimental groups and transfected with lvool, pE6-p53, pE6-luxR-luxI-p53, and pLuxI-luxR-p53 using LIPO3000 (Invitrogen). After transfection, the cells were incubated in a cell culture incubator for 24 h, irradiated with 0 Gy and 4 GY, and then incubated for 48 h.

[0114] (2) First layer of gel: First, preheat the slide for 3 minutes, with the frosted surface of the slide facing up, at 45°C. Pipette 30 µl of 1% normal melting point agarose gel onto the frosted surface of the slide, cover with a coverslip, and place in a 4°C refrigerator for 10 minutes to solidify;

[0115] Second layer of gel: Digest the cells in the 6-well plate with trypsin, centrifuge, resuspend with PBS, mix 10µl of cells and 75µl of 0.7% low melting point agarose, pipette 70µl of the mixture and drop it on the first layer of gel, spread it gently with a pipette tip to cover the entire first layer of gel, cover with a coverslip, and place at 4℃ for 10 minutes to solidify;

[0116] The third layer of gel: remove the cover glass and drop 75µl of 0.7% low melting point agarose preheated at 37℃. Cover with a new cover glass and place at 4℃ for 30 minutes to solidify;

[0117] Cell lysis: Remove the cover glass, place the slide into a plate, and pour in the pre-prepared cold lysis solution. The lysis solution should cover the slide and gel, and place in a 4℃ refrigerator for 60 minutes.

[0118] (3) DNA unwinding: Take out the slide from the lysis solution and wash it with PBS three times. Place the slide in a horizontal electrophoresis tank and pour in the electrophoresis solution until it is submerged; leave it at room temperature for 60 minutes;

[0119] (4) Electrophoresis: The electrophoresis voltage is generally low voltage 25V, electrophoresis for 30 minutes, and avoid light during electrophoresis;

[0120] (5) Neutralization and staining: After electrophoresis, place the slide in a dish, add PBS, and immerse the slide. Neutralize 1-3 times, 5-10 minutes each time, and discard the neutral buffer. Add about 20µl of Propidium Iodide Solution on the slide and stain for 20 minutes in the dark; then wash with ultrapure water 3 times, cover with a coverslip, and then observe under a fluorescence microscope;

[0121] Experimental results and discussion: Radiotherapy can induce DNA double bond breakage, leading to cell cycle arrest or apoptosis. The inventors used comet electrophoresis to detect DNA damage in different groups. Fig.15 and Fig.16 As shown, there was almost no tailing phenomenon in the Control and Lvool groups, and the DNA stayed in the cell nucleus without any sign of DNA damage. There was slight tailing in the pE6-p53 and pLuxI-luxR-p53 groups, but it was not obvious, indicating that the DNA damage was relatively mild. Almost all X-ray groups had tailing, among which the Control and Lvool tailing phenomena were slight, and pE6-p53, pLuxI-luxR-p53 and pE6-LuxI-luxR-p53 all had obvious damage, among which the tailing of pE6-LuxI-luxR-p53 was the most obvious and the damage was the most serious, proving that pE6-LuxI-luxR-p53 can enhance the damage to DNA after irradiation and can play a role in radiosensitization.

[0122] Example 6

[0123] This example is an in vivo tumor inhibition effect verification test, and the test process is as follows:

[0124] H1299 cells were made into single cell suspension (5 × 10 7 / ml), the cell suspension and matrix gel were mixed in equal volumes at a ratio of 1:1, and 0.1ml was injected subcutaneously into the back of BALB / c mice until the tumor volume grew to 70mm 3 The treatment started on day 1 (d1), and 0.2 ml of plasmid liposome mixture was injected into the tumor on d1 and d3; the animals were randomly divided into 10 groups (6 animals / group): ①Control group; ②Lvool group; ③pE6-p53 group; ④pLuxI-luxR-p53 group; ⑤pE6-LuxI-luxR-p53 group; ⑥Control+IR group; ⑦Lvool+IR group; ⑧pE6-p53+IR; ⑨pLuxI-luxR-p53+IR group; ⑩pE6-LuxI-luxR-p53+IR group. The IR group was the irradiation group, and 4 Gy irradiation was given on d2 and d4, respectively. At the beginning of treatment and every 2 days thereafter, the tumor size was measured with a vernier caliper, and the tumor volume (1 / 2ab2, a: long diameter, b: short diameter) was calculated, and the tumor growth curve was drawn; the animals were killed on day 11 of treatment;

[0125] Experimental results and discussion: The volume of the transplanted tumor reached about 70 mm in about 2 weeks. 3 .like Fig.17 and Fig.18 As shown in the growth curves of the tumor during treatment, the volumes of transplanted tumors in the Control group, Lvool group, pE6-p53 group, pLuxI-luxR-p53 group and pE6-LuxI-luxR-p53 group increased with time. After two irradiations, the volumes of the Control+IR group, Lvool+IR group, pE6-p53+IR group, pLuxI-luxR-p53+IR group and pE6-LuxI-luxR-p53+IR group first decreased and then gradually increased. The volumes of the tumors in the pE6-p53+IR, pLuxI-luxR-p53+IR and pE6-LuxI-luxR-p53+IR treatment groups decreased until the 9th day of treatment and then increased slowly. Overall, the pE6-LuxI-luxR-p53+IR treatment group had the strongest long-term tumor inhibition effect, which was significantly different from other groups (p < 0.01).

[0126] It can be seen from the above experiments that the present applicant first used pE6-LuxI-LuxR-EGFP to verify whether the amplification vector pE6-LuxI-LuxR has the function of amplification, and observed the expression of EGFP, indicating that the amplification vector pE6-LuxI-LuxR can indeed make the downstream gene EGFP highly expressed; then the inventors replaced EGFP with p53 (i.e., pE6-LuxI-luxR-p53), and the final experimental results also proved that pE6-LuxI-luxR can make p53 highly expressed.

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A radiosensitive positive feedback gene expression amplification vector, characterized in that: The vector is pE6-LuxI-LuxR, which has the nucleotide sequence shown in SEQ ID NO.

1.

2. The method for constructing the radiation-sensitive positive feedback gene expression amplification vector according to claim 1, characterized in that: The steps include: (1) Synthetic gene fragments; (2) Plasmid extraction and linearization of backbone vector; (3) The target gene is constructed into a linearized expression vector; (4) Transform the reaction products into Stbl3 competent cells; (5) Identification of plasmid vector.

3. The construction method according to claim 2, characterized in that: The specific method of step (1) is: designing and synthesizing pE6-LuxI-LuxR, and then performing PCR amplification to obtain the target gene insertion fragment.

4. The construction method according to claim 3, characterized in that: The reaction system and reaction conditions of the PCR amplification are: Reaction system Composition: 25 μL of 2x M5 HiPer SYBR Premix Es Taq, 1 μL of 10uM forward primer, 1 μL of 10uM reverse primer, 4 μL of 20-100 ng / μL template, and ddH2O to 50 μL; Reaction conditions: pre-denaturation at 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 20 s, extension / annealing at 60°C for 30-34 s, 72°C for 3 min, and a final extension step at 72°C for 10 min.

5. The construction method according to claim 2, characterized in that: In step (2), the linearization treatment method is: digesting the expression vector with a restriction endonuclease, and the enzyme digestion reaction system is: 2 μg of plasmid, 5 μL of 10 x reaction buffer, 1 μL of each restriction endonuclease, and make up to 50 μL with water, and incubate in a 37°C metal bath for more than 2 h.

6. Use of the radiosensitive positive feedback gene expression amplification vector according to claim 1 in driving the p53 gene.

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