CN102242120B - A kind of people's blood vessel eNOS gene promoter modifier and its preparation method and application - Google Patents

Abstract

The invention discloses a human vascular eNOS gene promoter transformation body, a preparation method and application thereof. The nucleotide sequence of the recombinant human vascular eNOS gene promoter described in the present invention is shown in SEQ ID NO:1. The present invention fundamentally changes the inherent weak promoter characteristics of the eNOS gene by transforming the human vascular eNOS gene promoter, improves the basic transcription activity and stimulated transcription activity of the eNOS gene promoter, and establishes a method for clarifying its function and regulation mechanism A useful tool with potential applications in the field of gene therapy.

Description

A human vascular eNOS gene promoter transformant and its preparation method and application

technical field

The invention relates to the technical field of genetic engineering, in particular to a human vascular eNOS gene promoter transformation body and its preparation method and application.

Background technique

Human vascular endothelial nitric oxide synthase (eNOS) not only participates in physiological processes such as blood pressure regulation, vascular permeability regulation, prevention of leukocyte adhesion and platelet aggregation, but also involves in hypertension, atherosclerotic heart disease, vascular stenosis and The occurrence and development of many serious pathological processes such as obstruction and ischemic stroke. The eNOS gene is inherently expressed in cardiovascular endothelial cells. More and more studies have proved that eNOS is essential for maintaining blood pressure homeostasis and vascular integrity and tension in the presence of harmful factors. Abnormal expression of eNOS is associated with hypertension, atherosclerosis , The occurrence and development of vascular dysfunction-related diseases are crucial. At present, the research on human vascular eNOS gene mainly focuses on: the stimulating factors of up-regulation or down-regulation of eNOS activity, the function of cis-regulatory elements in eNOS gene promoter, eNOS gene polymorphism and the signal regulation pathway of eNOS gene expression.

It is traditionally believed that the eNOS gene mainly responds to the stimuli of shear stress on the vessel wall, and later found that many stimuli can significantly change the expression of the eNOS gene, such as hypoxia, lysophosphatidylcholine, celiprolol, flavonoids, and fenofibrate , ceramide and oxidized low-density lipoprotein, etc. Although an increasing number of factors have been found to affect human vascular eNOS gene expression, little is known about how they up- or down-regulate eNOS gene expression.

Regarding the potential link between polymorphisms of the eNOS gene and related diseases, literature has shown that the G894→T polymorphism can lead to the Glu298Asp polymorphism of the encoded amino acid, and the missense of Glu298Asp leads to a change in the conformation of eNOS here, by The α-helix becomes tightly folded, so it is speculated that this mutation may affect the function of eNOS protein, reduce NO production, lead to enhanced platelet aggregation, leukocyte adhesion to endothelium, smooth muscle cell proliferation and other pathophysiological phenomena.

Regarding the signal regulation pathway of eNOS gene expression, it was newly found that activating the extracellular signal-regulated kinase ERK1/2 signaling pathway, a member of the mitogen-activated protein kinase family, can significantly up-regulate the transcriptional activity of the eNOS gene. Other stimulating factors such as lysophosphatidylcholine can also up-regulate the transcriptional activity of eNOS gene by activating the c-jun nitrogen-terminal kinase JNK signaling pathway, while the activation of the mitogen-activated protein kinase p38 signaling pathway can significantly down-regulate its activity. The effect can be abolished by targeting siRNA to silence the p38α gene.

SeiichiroWariishi et al. (1995) studied the function of eNOS gene promoter cis-regulatory elements and found that the SP1 element plays a key role in transcriptional activity. The SP1 binding site is also regulated by the GATA site located at the upstream -203 site. When the GATA site is mutated, the activity of the eNOS gene promoter is only slightly decreased, but when the SP1 site is mutated, the promoter activity will be sharp Decrease, suggesting that the GATA cis-regulatory element can only function when the SP1 site is intact. William C. Sessa et al. used the PCR point mutation method to discover that the SP1 and TATA elements determine the basic transcriptional activity of the eNOS promoter, which was further confirmed by FotulaKarantzoulis-Fegaras et al. and KatarzynaCieslik et al.

Human vascular eNOS gene is inherently expressed in cardiovascular endothelial cells and plays an important role in maintaining vascular tone, and its low expression is closely related to diseases related to vascular dysfunction. However, it is known that the eNOS gene promoter structure itself lacks the core promoter recognition sequence, that is, the traditional TATA BOX and the initiator, and relies on the GC BOX combined with the SP1 transcription factor to assist in transcriptional regulation, resulting in low basal activity and stimulating activity. level, which significantly increases the difficulty of ascertaining its function, and also makes it difficult to advance its gene therapy. Therefore, based on the theory that SP1 is the key to maintaining the activity of the eNOS gene promoter, we explored a method to insert key regulatory elements at the appropriate site of the eNOS gene promoter to modify the structure of the eNOS gene promoter so as to increase the activity of the eNOS gene promoter from the source . So far, there is no relevant report about increasing the activity of the eNOS gene promoter by modifying its structure at home and abroad.

Contents of the invention

The object of the present invention is to provide a recombinant human vascular eNOS gene promoter with significantly improved basic activity and stimulating activity according to the existing problems such as low activity of the human vascular eNOS gene promoter.

The object of the invention is achieved through the following technical solutions:

The present invention uses the PCR insertion sequence mutation technology to add a universal recognition sequence of the regulatory element SP1 to the human eNOS gene promoter, so as to modify the promoter structure, and the purpose is to improve the transcription activity of the promoter; HUVEC-12 cells constructed by cobalt chloride The hypoxia model and the dual fluorescent reporter system completed the transfection and detection of the promoter variant, and analyzed the transcriptional activity changes of the promoter and its variant, which preliminarily verified the function of the variant (Figure 1).

Specifically, the present invention includes the following technical solutions:

A human vascular eNOS gene promoter transformation body, its nucleotide sequence is shown in SEQ ID NO: 1.

The preparation method of the human vascular eNOS gene promoter variant of the present invention comprises the following steps:

(1) According to the eNOS gene promoter sequence, design a pair of primers with opposite directions of 5' ends. The nucleotide sequence of the eNOS gene promoter is shown in SEQ ID NO: 2, and the primer sequence is shown in SEQ ID NO: 3~4 shown;

(2) The insertion sequence mutation of the eNOS gene promoter is carried out by PCR, and the reaction system of the PCR is as follows:

Template 1ng/μL 5μL

Each dNTPs 2.5mmol/L 2μL

20 μmol/L of each primer 1 μL

10× buffer 2.5μL

Polymerase 5U/μL 0.1μL

Sterilized triple distilled water 13.5μL;

The PCR reaction program is: 95°C for 5min, (94°C for 45s, 55°C for 45s, 72°C for 5min) 30 cycles, 72°C for 10min, 4°C for 24h;

Through the above steps, the human vascular eNOS gene promoter transformant is obtained.

The human vascular eNOS gene promoter transformation body described in the present invention provides a useful tool for elucidating the function and regulation mechanism of the human vascular eNOS gene promoter. The human vascular eNOS gene promoter is a weak promoter. So far, the stimulating factors or methods used for research can only slightly increase the activity of the eNOS gene promoter, so it is difficult to explain a certain kind of activity by observing the change of its activity. The influence of endogenous or exogenous factors and the revealing of the inner process and rules of this influence lead to the research on the function and regulation mechanism of the eNOS gene promoter lagging behind other members of the NOS family. On the other hand, it is known that in diseases related to vascular dysfunction, the inherent expression of eNOS gene is up-regulated through the body's compensatory mechanism, but it is not enough to counteract the invasion of pathological damage factors. The effect of exogenous stimuli is extremely limited. If the activity of the eNOS gene promoter can be fundamentally increased from the source of gene regulation, gene therapy for diseases related to vascular dysfunction will become possible. Therefore, the present invention obtains human The vascular eNOS gene promoter transformation body can be used to prepare medicines for treating diseases related to vascular dysfunction.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The traditional research method of human eNOS gene promoter is to find out which part of the sequence and regulatory elements are inherent in the promoter by deleting the sequences of different regions of the full length of the promoter or making point mutations on the cis-regulatory elements Play a key role in determining the activity of the promoters, ie the purpose is to elucidate their role or function. In contrast, the present invention uses the insertion sequence mutation technology to add key regulatory elements to the eNOS gene promoter at an appropriate position, thereby improving the basic transcription activity and stimulating transcription activity of the eNOS gene promoter from the source of determining gene activity. It provides a new tool or method for studying the transcription regulation mechanism of eNOS gene promoter and the exploration of gene therapy. Therefore, research ideas and strategies, techniques and methods, purposes and meanings are completely different;

(2) The results showed that the transcriptional activity of the modified promoter was improved, and its basic transcriptional activity reached 2.3 times that of the original promoter. In the environment stimulated by adding cobalt chloride while transfecting HUVEC-12 cells, the modified The transcriptional activity of the promotor still maintains a higher transcriptional activity than that of the original promoter; moreover, the promotor can lead to an increase in the content of NO released by HUVEC-12 cells. These results suggest that our method and technique of skillfully inserting regulatory element sequences to improve the activity of human eNOS gene promoter by inserting sequence mutagenesis technology has obvious application value.

Description of drawings

Fig. 1 is the flow chart of the structural modification of the human eNOS gene promoter modified body of the present invention;

Figure 2 is the double-enzyme digestion electrophoresis identification diagram of the human eNOS gene promoter (pGL2-eNOS-p), where M is a 1kb DNA marker; 1 is pGL2-eNOS-p; 2 is pGL2 and eNOS-p after digestion;

Fig. 3 is the schematic diagram of point mutation experiment;

Figure 4 is the gel-cut recovery electrophoresis identification diagram, wherein, M is 1kb DNA marker; 1 is pGL2-eNOS-p; 2 is the gel-cut recovery product;

Figure 5 is the electrophoretic identification diagram of pGL2-eNOS-Mut-p, wherein, M is 1kb DNA marker; 1 is pGL2-eNOS-p; 2 is pGL2-eNOS-Mut-p;

Figure 6 shows the changes in the transcriptional activity of eNOS gene promoter variants (stimulated by different concentrations of cobalt chloride), in which, 1): vs pGL2-eNOS-p (Control), P<0.01; 2): vs pGL2-eNOS- p (50μmol/L), P<0.05; 3): vs pGL2-eNOS-p (100μmol/L), P<0.05; 4): vs pGL2-eNOS-p (200μmol/L), P<0.05; 5 ): vs pGL2-eNOS-p (400μmol/L), P<0.05; 6): vs pGL2-eNOS-p (800μmol/L), P<0.05; 7): vs pGL2-eNOS-p (Control), P<0.01; Time: 48hr; n=2;

Figure 7 shows the changes in transcriptional activity of eNOS gene promoter variants (stimulated by cobalt chloride at different times), among which, 1): vs pGL2-eNOS-p (Control), P<0.01; 2): vs pGL2-eNOS- p(12hr), P<0.05; 3): vs pGL2-eNOS-p(24hr), P<0.05; 4): vs pGL2-eNOS-p(36hr), P<0.05; 5): vs pGL2-eNOS -p (48hr), P<0.05; 6): vs pGL2-eNOS-p (72hr), P<0.05; CoCl ₂ : 200μmol/L; n=2;

Figure 8 is the effect of different concentrations of cobalt chloride stimulation on the release of NO from HUVEC-12 cells that have been transfected with eNOS variants, wherein, 1): vs Control, P<0.01; 2): vs Control, P<0.05; Time : 48hr; n=2;

Figure 9 is the effect of cobalt chloride stimulation at different times on the release of NO from HUVEC-12 cells transfected with the eNOS variant; 1): vs Control, P<0.05; 2): vs Control, P<0.01; CoCl ₂ : 200 μmol/L; n=2.

Detailed ways

The present invention is further explained below in conjunction with the examples, but the examples do not limit the present invention in any form.

Example 1 Preparation of Human eNOS Gene Promoter Transformer

Identification of Human eNOS Promoter by Double Enzyme Digestion

The pGL2-eNOS-p vector was double digested with Xho I and Kpn I restriction enzymes. The reaction system was 20 μL, the vector pGL2-eNOS-p was about 500 ng, XhoI and KpnI were 1 μL each, sterilized triple-distilled water was added to make the total volume 20 μL, and the reaction was carried out in a 37°C water bath for 4 hours. Take 50× TAE mother solution and dilute it into 1× TAE working solution, weigh 0.1g agarose and mix with 10mL 1× TAE, add to dissolve in microwave oven. When the temperature drops to about 60°C, add ethidium bromide to a final concentration of 1 μg/mL, insert a comb, and let stand at room temperature for 30 minutes. Take 2 μL of the digested product for electrophoresis, the voltage is fixed at 5V/cm, and the electrophoresis is stopped when bromophenol blue moves to the middle of the gel. The gel block was carefully taken out, the bands were observed under ultraviolet light, and the experimental results were recorded with a gel imager (Figure 2).

Design of primers for site-directed insertion sequence mutagenesis

The sequence of the human eNOS gene promoter (Gene BankTM, Accession Number: AF387340) (SEQ ID NO: 2) was found from GeneBank, and an appropriate site was selected for mutation according to literature reports and kit instructions (Figure 3). Design a pair of primers with 5' ends connected in opposite directions, named as "variation introduction primer" (SEQ ID NO: 3) and "corresponding primer" (SEQ ID NO: 4). The designed primers were tested by DNA Club analysis software and synthesized by Shanghai Bioengineering Co., Ltd.

Insertion sequence mutagenesis of the promoter

The PCR point mutation experiment was carried out, and the MutanBEST Kit (D401) mutation kit of TaKaRa Company was used for PCR. Take the pGL2-eNOS-p plasmid that has been extracted, and its OD260:OD280 range is controlled between 1.80~1.85, and diluted to 1ng/μL as a PCR reaction template; the primers are diluted to 20μmol/L, and stored in - Standby at 20°C. Perform PCR reaction with primers designed according to method 5.2.

Prepare a PCR reaction system (25 μL reaction volume) in a 0.5 mL sterilized Eppendorf tube:

Components Volume

Template (1ng/μL) 5μL

dNTPs Mixture (2.5mmol/L each) 2μL

Variation introduction primer (20 μmol/L) 1 μL

Corresponding primer (20 μmol/L) 1 μL

10×Pyrobest Buffer II 2.5μL

Pyrobest polymerase (5U/μL) 0.1μL

Sterilized triple distilled water 13.5μL

After mixing well, place it on the PCR machine, and react according to the following procedures:

95°C for 5min, (94°C for 45s, 55°C for 45s, 72°C for 5min) 30 cycles, 72°C for 10min, 4°C for 24h.

Electrophoresis identification, gel cutting and recovery of target bands

(1) Recover and purify the PCR product from the agarose gel using the gel recovery kit of Shanghai Shenergy Bocai Biotechnology Co., Ltd.;

(2) Take 1 μL of the recovered and purified product, add it into sterilized three-distilled water and dilute 100 times, measure the concentration of the recovered product with a nucleic acid analyzer, and take 3 μL of the product for identification by 1% agarose gel electrophoresis (Figure 4).

reaction

(1) Prepare the connection reaction system according to the following system:

Recycled product from rubber cutting About 1pmol

10×Blunting Kination Buffer 2μL

Blunting Kination Enzyme Mix 1μL

Make up to 20μL with dH ₂ O;

(2) React at 37°C for 10 minutes;

(3) React at 75°C for 10 minutes.

reaction

(1) Add 5 μL (about 0.25 pmol) of the reaction product in step 5.5 into a microcentrifuge tube;

(2) Add 5 μL of Ligation Solution I and mix evenly;

(3) React in a water bath at 16°C for 16 hours.

7. Transformation of E. coli

Competent cells were prepared by the calcium chloride method, and the Ligation reaction product was transformed into competent cells by the heat shock method.

(1) Take a DH5α strain preserved in glycerol, inoculate it in 4mL LB according to the volume ratio of bacterial liquid: LB of 1:1000, incubate in a constant temperature shaking incubator at 37°C for 12 hours, and make a negative control tube adding Amp at the same time ;

(2) Use a pipette to take 0.2 mL of the overnight culture solution under aseptic conditions, inoculate it in fresh LB according to the volume ratio of bacteria solution:LB of 1:16, and culture the bacteria solution in a constant temperature shaking incubator at 37°C until OD600= 0.5;

(3) Divide the bacterial liquid into microcentrifuge tubes according to 0.8mL per tube, and then centrifuge at 7000rpm at 4°C for 2min to collect the bacteria;

(4) The bacteria were resuspended and mixed with 0.1mol/L pre-cooled calcium chloride solution, and placed in an ice bath for 30 minutes;

(5) Take the bacteria solution out of the ice bath, and centrifuge at 7000rpm at 4°C for 2min to collect the bacteria

(6) Discard the supernatant, add pre-cooled calcium chloride solution containing 15% glycerol according to 0.1mL per tube, gently suspend the cells, and place them on ice for a few minutes to form a competent cell suspension. Store at ℃.

(7) Add 10 μL of the Ligation reaction product to a competent cell, mix carefully and then ice-bath for 30 minutes;

(8) Add the competent cells to a 42°C water bath for 90s heat shock, then quickly take them out and insert them on ice for about 1-2 minutes; carefully add 1mL of LB medium, and incubate at 37°C at about 150-200rpm for 1hr;

(9) Take out the bacterial solution and centrifuge at 7000rpm at room temperature for 1min, discard 1mL of the supernatant, and use the remaining supernatant to resuspend the bacteria, and spread all the remaining bacterial solution on the LB plate containing ampicillin (Amp);

(10) Place in a biochemical incubator at 37°C for 15 minutes, and incubate upside down for 16-20 hours after the bacteria solution is completely absorbed;

(11) Take out the petri dish, observe the transformation, and store it upside down in a 4°C refrigerator.

Plasmid extraction

Extract plasmids using silica gel column separation:

(1) Take out the ampicillin (Amp) LB plate that has grown colonies, pick a single colony and inoculate it in LB medium containing 0.1mg/mL Amp, and culture at 37°C at about 220~240rpm for 12~14hr;

(2) Take 1 mL of overnight cultured bacteria, centrifuge at 10,000 rpm for 1 min, and remove the supernatant completely;

(3) Add 200 μL of Solution I and mix thoroughly;

(4) Add 200 μL of Solution II, immediately and slowly invert the microcentrifuge tube up and down to lyse the bacteria, and place it at room temperature until the solution becomes clear;

(5) Add 300 μL of Solution III, and immediately mix the liquid gently to concentrate the white precipitate;

(6) Centrifuge at 13000rpm for 10min, transfer all the supernatant to a silica gel column, try to avoid inhaling the white goose precipitate; then centrifuge at 10000rpm for 1min, discard the waste liquid in the collection tube;

(7) Add 600 μL of Buffer HB to the silica gel column, centrifuge at 10,000 rpm for 1 min, and discard the waste liquid in the collection tube;

(8) Add 600 μL of Wash Solution to the silica gel column, centrifuge at 10,000 rpm for 1 min, and discard the waste liquid in the collection tube;

(9) Repeat step (8) once;

(10) Centrifuge at 12000rpm for 2min, discard the waste liquid again; put the silica gel column into a sterilized microcentrifuge tube, add 30μL of sterile triple-distilled water preheated to 50°C to the center of the membrane in the silica gel column, and centrifuge at 10000rpm 1min;

(11) Store the eluted solution at -20°C for future use.

Electrophoretic identification

(1) Weigh 0.1g of agarose powder, add 12~13mL of 1×TAE buffer solution, heat in a microwave oven until completely dissolved, and when the temperature drops to about 60°C, add 1μL of EB solution (10mg/ml), fully After mixing, pour the glue, insert the comb ruler, and let it stand for more than 30 minutes;

(2) Take 1 μL of the plasmid extracted in step 5.8, dilute it 10 times and use it for electrophoresis, and the loading volume is 0.5 μL;

(3) The electrophoresis condition is 5V/cm, and the electrophoresis time is 40~50min;

(4) After electrophoresis, take out the gel block and place it in the gel imaging analysis system for observation and recording (Figure 5).

Sequencing analysis

(1) Take the samples whose plasmid size meets the requirements after electrophoresis identification and send them for sequencing identification; the samples are sent to Shenzhen Huada Gene Company for sequencing;

(2) The sample for sequencing is 1mL bacterial liquid containing the plasmid to be tested. The requirement for sequencing is to reverse-sequence one reaction from the downstream, using pGL universal primers;

(3) BLAST analysis of the sequencing results and the eNOS gene promoter sequence on the NCBI website;

(4) The new vector that was successfully sequenced and met the requirements of subsequent experiments after comparison and analysis was named pGL2-eNOS-Mut-p, and the mutated promoter on this vector became the variant of the eNOS gene promoter.

Example 2 Effects of Different Concentrations of Cobalt Chloride on the Activity of the Human eNOS Gene Promoter Modifier

HUVEC-12 cell line was cultured in Dulbecco's Modified Eagle Media (DMEM) medium containing 5% newborn bovine serum, 100mol/L penicillin, and 100mg/L streptomycin in a 37°C incubator until the cells grew Passage to 80%~90% confluence.

When experiments are required, cells are plated in a 24-well plate at a density of 8×104/well. After the cells grow to 50% to 60% confluent, discard the cell supernatant, wash the cells once with PBS buffer, and then add different Concentration of cobalt chloride in fresh DMEM medium. The cells were divided into Control group, cobalt chloride stimulation group with different concentrations and cobalt chloride stimulation group with different time. Control group was not stimulated with cobalt chloride; different concentrations of cobalt chloride were stimulated with different concentrations of cobalt chloride, so that the final concentration of cobalt chloride reached 0, 50, 100, 200, 400 and 800 μmol/L; The cobalt stimulation group was added with the same concentration of cobalt chloride, so that the final concentration reached 200 μmol/L.

Cell transfection: the cells were divided into control transfection group, different concentrations of cobalt chloride stimulated transfection group and different time cobalt chloride stimulated transfection group. The transfection conditions of each group are shown in Table 1:

Table 1

The transfection experiment was completed using SunBioTM-EZ transfection reagent from Shanghai Shengbo Medical Bioengineering Co., Ltd. Before transfection, the cell growth confluence was controlled between 60% and 70%, and the concentration of the plasmid used for transfection was about 0.2~0.25 μg/μL , OD260: The range of OD280 is controlled between 1.80 and 1.85.

(1) Dilute the transfection reagent and plasmid in different microcentrifuge tubes with serum-free and antibiotic-free DMEM medium. The amount of transfection reagent per well is 1 μL, and the amount of plasmid is 1 μg; ensure that the total volume of each tube after dilution is 30 μL;

(2) Add the diluted transfection reagent into the diluted plasmid tube, mix well with a micro-injector, and let stand at room temperature for 20 minutes to form a transfection complex;

(3) Gently add 240 μL of DMEM medium containing serum and antibiotics into the tube containing the transfection complex, then add a corresponding amount of cobalt chloride solution according to the stimulation concentration of cobalt chloride corresponding to each well, and then slowly inhale for 2 ~3 times;

(4) Discard the old medium in the well, wash the cells once with PBS at room temperature, after aspirating and discarding the PBS, slowly add the mixed medium in step (3) along the well wall;

(5) Put the culture plate into a 37°C incubator and continue to culture for the corresponding time.

Promoter activity detection:

Promega's Dual-Luciferase Reporter Assay System was used to detect the Luciferase activity of the sample, and the expression activity of the eNOS promoter was analyzed according to the results of the activity detection.

(1) Stop the culture of cells in the control transfection group 48 hours after transfection, stop the culture of cells in other groups according to the corresponding time stimulated by cobalt chloride, and lyse the cells, add 100 μL of 1× PLB (Passive Lysis Buffer) to each well of cells, Gently shake at room temperature for 15-30 minutes, collect the cell lysate in a clean microcentrifuge tube, and store it at -70°C for later use;

(2) Add 100 μl of LAR II (Luciferase Assay Reagent II) to a white opaque 96-well plate;

(3) Centrifuge the cell lysate at 10,000 rpm for 1 min, take 100 μL of the supernatant, add it to the well and pipette evenly;

(4) Measure the intensity of visible light for 10s with a bioluminescent multifunctional detector (PerkinElmer 1420 type) (the light read at this time is emitted by firefly Luciferase produced by transcription and translation on the pGL vector);

(5) Take the plate out, continue to add 100 μL of Stop & Glo Reagent to the well, and pipette evenly;

(6) Measure the intensity of visible light for 10s (the light read at this time is emitted by Renilla Luciferase transcribed and translated on the internal reference pRL-TK vector)

(7) Analyze the obtained data after standardization.

Cells were divided into Control transfection group and cobalt chloride stimulated transfection group at different times for transfection experiments. The results suggested that the modified eNOS gene promoter showed higher transcriptional activity than the original promoter no matter under normal conditions or under different concentrations of drug stimulation environment. In the Control group, the fluorescence intensity of pGL2-eNOS-Mut-p was about 2.3 times that of pGL2-eNOS-p. Different doses of cobalt chloride stimulation had different effects on the transcriptional activity of eNOS gene. When the stimulation level was 50 μmol/L, the transcriptional activity was still at a high level, and the fluorescence intensity of pGL2-eNOS-p was up-regulated compared with the Control group , the fluorescence intensity of pGL2-eNOS-Mut-p decreased slightly compared with the Control group; when the stimulation level reached 100-800 μmol/L, the eNOS gene transcription activity decreased significantly. Comparing the results of different groups stimulated by cobalt chloride, pGL2-eNOS-Mut-p had higher expression than pGL2-eNOS-p in the same group (Figure 6).

Example 3 Effects of Cobalt Chloride on the Activity of Human eNOS Gene Promoter Modifier at Different Time

Cell treatment and transfection methods are the same as in Example 2. Cells were divided into Control transfection group and cobalt chloride stimulated transfection group at different times for transfection experiments. The reporter gene activity of firefly luciferase and Renilla luciferase was measured, and the ratio of the two was calculated to correct the transfection efficiency. The fluorescence activity of the pGL2-eNOS-p/pRL-TK co-transfection well in the Control group after correction was taken as 100, and the results of the other wells were compared with it to obtain the relative fluorescence intensity.

The results indicated that the modified eNOS gene promoter exhibited higher transcriptional activity than the original promoter no matter under normal conditions or under drug stimulation at different times. In the Control group, the fluorescence intensity of pGL2-eNOS-Mut-p was about 2.3 times that of pGL2-eNOS-p. When the stimulation concentration of cobalt chloride was maintained at 200 μmol/L, both pGL2-eNOS-p and pGL2-eNOS-Mut-p decreased significantly, and the transcriptional activities of both increased with the increase of stimulation time. Among them, when the stimulation time is from 12 to 48 hours, the fluorescence intensity of pGL2-eNOS-p is positively correlated with the stimulation time (r=0.9846, P<0.01), and the fluorescence intensity of pGL2-eNOS-Mut-p is also positively correlated with the stimulation time (r= 0.9682, P<0.05); compared with the results of cobalt chloride stimulation at different times, pGL2-eNOS-Mut-p had higher expression than pGL2-eNOS-p in the same group ( Figure 7).

Example 4 Effects of Different Concentrations of Cobalt Chloride on the Release of NO from Cells Transfected with Human eNOS Promoter Modifiers

The cell treatment method and transfection method are the same as in Example 1. The cells were divided into Control transfection group and cobalt chloride stimulation transfection group with different concentrations. The control group and cobalt chloride stimulation group with different concentrations were cultured for 48 hours after transfection to collect 100 μL of supernatant and measure the NO concentration. Nitric oxide detection kit from Nanjing KGI Company was used to detect NO content in the supernatant.

(1) Preheat each reagent at 37°C for 5 minutes, then mix Buffer A and Buffer B in equal amounts to form a mixed reagent AB, and transfer the supernatant to each tube according to the volume ratio supernatant:mixed reagent AB=1:2 Add 200 μL of mixed reagent AB to the solution, mix thoroughly, and react in a water bath at 37°C for 1 hour; the reactants in the blank tube and standard tube were replaced with 100 μL of triple-distilled water and standard solution;

(2) Add 100 μL of Buffer C and 50 μL of Buffer D to the microcentrifuge tube successively, mix vigorously, let stand at room temperature for 40 minutes, then centrifuge at 4000 rpm for 10 minutes, carefully suck out 250 μL of the supernatant and put it into another clean microcentrifuge tube;

(3) Dissolve reagent F in a water bath at 90°C, prepare the color developer according to the ratio of reagent E: reagent F: Buffer G =2.5:1:1, then add 350 μL of color developer to each tube, mix well, and let it stand for 10 minutes On-board inspection;

(4) Use a UV spectrophotometer, adjust to zero with distilled water, and measure the absorbance of each tube at 550 nm.

(5) NO conversion formula:

The results of different concentrations of cobalt chloride stimulation group indicated that when the stimulation concentration was 50 and 800 μmol/L, the NO concentration was lower than that of the control group, and there was no correlation between NO concentration and cobalt chloride concentration in the range of stimulation concentration 50-800 μmol/L. When the stimulation concentration was 50-200 μmol/L, the concentration of cobalt chloride increased and the concentration of NO also increased, but the concentration of cobalt chloride continued to increase, and the concentration of NO appeared to decrease (Figure 8).

Example 5 Effect of Cobalt Chloride on NO Release from Cells Transfected with Human eNOS Promoter Modifier at Different Time

The cell culture method and transfection method are the same as in Example 2, and the method for measuring NO concentration is the same as in Example 4. The cells were divided into Control transfection group and cobalt chloride stimulation transfection group with different concentrations. In the Control group, 100 μL of supernatant was collected at 48 hours after transfection, and in the cobalt chloride stimulation group at different times, they were cultured at 12 hours after transfection. , 24, 36, 48 and 72 hr, the supernatant 100 μL was collected and the NO concentration was measured. The results of the cobalt chloride stimulation group at different times indicated that when the stimulation time was 12 and 24 hr, the NO concentration was lower than that of the control group, and the NO concentration was positively correlated with the cobalt chloride stimulation time in the range of 12-72 hr (r= 0.9637 , P<0.01) (Figure 9).

SEQUENCE LISTING

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<110> Jinan University

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<120> A Human Vascular eNOS Gene Promoter Transformer and Its Preparation Method and Application

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tctctgcctc tcccagtctc tcagcttccg tttctttctt aaactttctc tcagtctctg 600

the

aggtctcgaa atcacgaggc ttcgacccct gtggaccaga tgcccagcta gtggcctttc 660

the

tccagcccct cagatggcac agaactacaa accccagcat gcactctggc ctgaagtgcc 720

the

tggagagtgc tggtgtaccc cacctgcatt ctgggaactg tagtttccct agtcccccat 780

the

gctccccacca gggcatcaag ctcttccctg gccggctgac cctgcctcag ccctagtctc 840

the

tctgctgacc tgcggccccg ggaagcgtgc gtcactgaat gacagggtgg gggtggaggc 900

the

actggaaggc agcttcctgc tcttttgtgt cccccacttg agtcatgggg gtgtgggggt 960

the

tccaggaaat tggggctggg aggggaaggg ataccctaat gtcagactca aggacaaaaa 1020

the

gtcactacat ccttgctggg cctctatccc caagaaccca aaaggactca agggtgggga 1080

the

tccaggagtt cttgtatgta tggggggagg tgaaggagag aacctgcatg accctagagg 1140

the

tccctgtggt cactgagagt gtgggctgcc atcccctgct acagaaacgg tgctcacctt 1200

the

ctgcccaacc ctccagggaa aggcacacag gggtgaggcc gaaggccctt ccgtctggtg 1260

the

ccacatcaca gaaggacctt tatgacccccc tggtggctct accctgccac tccccaatgc 1320

the

cccagccccc atgctgcagc cccagggctc tgctggacac ctgggctccc acttatcagc 1380

the

ctcagtcctc acagcggaac ccaggcgtcc ggccccccac ccttcaggcc agcgggcgtg 1440

the

gagctgaggc tttagagcct cccagccggg cttgttcctg tcccattgtg tatgggatag 1500

the

gggcggggcg agggccagca ctggagagcg gggcggggcc ccctcccact gccccctcct 1560

the

ctcggtcccc tccctcttcc taaggaaaag gccagggctc tgctggagca ggcagcagag 1620

the

tggacgcaca gtaacatggg caacttgaag agcgtggccc aggagcctgg gccaccctgc 1680

the

ggcctggggc tggggctggg ccttgggctg tgcggcaagc ag 1722

the

the

<210> 2

<211> 1712

<212> DNA

<213> Artificial sequence

the

<400> 2

atctgatgct gcctgtcacc ttgaccctga ggatgccagt cacagctcca ttaactggga 60

the

cctaggaaaa tgagtcatcc ttggtcatgc atttcaaa tggtggctta atatggaagc 120

the

cagacttggg atctgttgtc tcctccagca tggtagaaga tgcctgaaaa gtaggggctg 180

the

gatcccatcc cctgcctcac tgggaaggcg aggtggtggg gtggggtggg gcctcaggct 240

the

tggggtcatg ggacaaagcc caggctgaat gccgcccttc catctccctc ctcctgagac 300

the

aggggcagca gggcacacta gtgtccagga gcagcttatg aggccccttc accctccatc 360

the

ctccaaaact ggcagacccc accttcttgg tgtgacccca gagctctgag cacagcccgt 420

the

tccttccgcc tgccggcccc ccaccccaggc ccaccccaac cttatcctcc actgcttttc 480

the

agaggagtct ggccaacaca aatcctcttg tttgtttgtc tgtctgtctg ctgctcctag 540

the

tctctgcctc tcccagtctc tcagcttccg tttctttctt aaactttctc tcagtctctg 600

the

aggtctcgaa atcacgaggc ttcgacccct gtggaccaga tgcccagcta gtggcctttc 660

the

tccagcccct cagatggcac agaactacaa accccagcat gcactctggc ctgaagtgcc 720

the

tggagagtgc tggtgtaccc cacctgcatt ctgggaactg tagtttccct agtcccccat 780

the

gctccccacca gggcatcaag ctcttccctg gccggctgac cctgcctcag ccctagtctc 840

the

tctgctgacc tgcggccccg ggaagcgtgc gtcactgaat gacagggtgg gggtggaggc 900

the

actggaaggc agcttcctgc tcttttgtgt cccccacttg agtcatgggg gtgtgggggt 960

the

tccaggaaat tggggctggg aggggaaggg ataccctaat gtcagactca aggacaaaaa 1020

the

gtcactacat ccttgctggg cctctatccc caagaaccca aaaggactca agggtgggga 1080

the

tccaggagtt cttgtatgta tggggggagg tgaaggagag aacctgcatg accctagagg 1140

the

tccctgtggt cactgagagt gtgggctgcc atcccctgct acagaaacgg tgctcacctt 1200

the

ctgcccaacc ctccagggaa aggcacacag gggtgaggcc gaaggccctt ccgtctggtg 1260

the

ccacatcaca gaaggacctt tatgacccccc tggtggctct accctgccac tccccaatgc 1320

the

cccagccccc atgctgcagc cccagggctc tgctggacac ctgggctccc acttatcagc 1380

the

ctcagtcctc acagcggaac ccaggcgtcc ggccccccac ccttcaggcc agcgggcgtg 1440

the

gagctgaggc tttagagcct cccagccggg cttgttcctg tcccattgtg tatgggatag 1500

the

gggcggggcg agggccagca ctggagagcc ccctcccact gccccctcct ctcggtcccc 1560

the

tccctcttcc taaggaaaag gccagggctc tgctggagca ggcagcagag tggacgcaca 1620

the

gtaacatggg caacttgaag agcgtggccc aggagcctgg gccaccctgc ggcctggggc 1680

the

tggggctggg ccttgggctg tgcggcaagc ag 1712

the

the

<210> 3

<211> 29

<212> DNA

<213> Artificial sequence

the

<400> 3

gccccgcccc gctctccagt gctggccct 29

the

the

<210> 4

<211> 18

<212> DNA

<213> Artificial sequence

the

<400> 4

cccctcccac tgccccct 18

the

Claims (1)

1. A preparation method for a human vascular eNOS gene promoter transformant, characterized in that it comprises the steps: (1) According to the eNOS gene promoter sequence, design a pair of primers with opposite directions of the 5' ends. The nucleotide sequence of the eNOS gene promoter is shown in SEQ ID NO: 2, and the primer sequence is shown in SEQ ID NO: 3~4 shown; (2) The insertion sequence mutation of the eNOS gene promoter is carried out by PCR, and the reaction system of the PCR is as follows: Template 1ng/μL 5μL Each dNTPs 2.5mmol/L 2μL 20 μmol/L of each primer 1 μL 10× buffer 2.5μL Polymerase 5U/μL 0.1μL Sterilized triple distilled water 13.5μL; The PCR reaction program is: 95°C for 5min, 94°C for 45s, 55°C for 45s, 72°C for 5min, 30 cycles, 72°C for 10min, 4°C for 24h;  After the above steps, the recombinant human vascular eNOS gene promoter was obtained.