Microvascular endothelial cell ATF2 site mutation plasmid vector, ATF2 K296R high-expression cell model and application

Abstract

The invention belongs to the technical field of medical molecular biology and genetic engineering, and particularly discloses a microvascular endothelial cell ATF2 site mutation plasmid vector, an ATF2K296R high-expression cell model and application. According to the application, expression of ATF2K296R protein in microvascular endothelial cells is improved by applying a point mutation plasmid technology, a microvascular endothelial cell model with high ATF2K296R expression is constructed, the effect of ATF2 myristic acylation modification in occurrence and development of an endothelial barrier is researched, and it is proved that the myristic acylation modification of ATF2 has negative effects on the endothelial cell barrier. After ATF2K296 point mutation and overexpression, the myristic acylation modification level of ATF2 can be obviously reduced, the purpose of protecting the barrier function of endothelial cells is achieved, and hopes are brought to clinical targeted therapy of endothelial barrier function imbalance and treatment of cardiovascular diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of medical molecular biology and genetic engineering, and specifically relates to a microvascular endothelial cell ATF2 site mutation plasmid vector and an ATF2 K296R high-expression cell model and application. Background Art

[0002] A major factor in increased cardiovascular risk is endothelial cell barrier dysfunction, which includes a series of maladaptive changes in the functional phenotype of endothelial cells that are associated with increased cardiovascular risk. The endothelial cell layer of the cardiovascular system provides a semipermeable barrier that regulates the exchange of fluids, molecules, and cells and plays an important role in maintaining vascular health. Vascular endothelial cell dysfunction in large arteries is an early event in atherosclerosis. Endothelial cells prevent unnecessary platelet activation and activation by participating in the regulation of primary and secondary hemostasis. Endothelial barrier dysfunction can also lead to vascular aging and decreased compliance. Therefore, protecting the function of the endothelial barrier plays an important role in the prevention and treatment of cardiovascular disease.

[0003] Recent in vivo and in vitro studies have successfully identified the important role of myristoylation in regulating endothelial dysfunction. Studies have reported that myristoylation plays an important role in animal models of ischemia-reperfusion injury and atherosclerosis. However, there is currently a lack of methods to intervene in the modification sites of myristoylation-modified substrate proteins to treat cardiovascular diseases.

[0004] Activating transcription factor 2 (ATF2) regulates the transcription of various genes, including apoptosis, cell growth and DNA damage response, depending on its binding partners to the cAMP response element or AP-1. It plays a strict transcriptional regulatory role in a variety of endothelial pathophysiological conditions, such as apoptosis, inflammation, angiogenesis and atherosclerosis. So far, no studies have reported the role of myristoylation modification of ATF2 in endothelial cells.

[0005] Plasmid is a small double-stranded circular DNA molecule that is naked, simple in structure, independent of bacterial nucleoid DNA, and has the ability to self-replicate. As a carrier of the target gene in genetic engineering, the plasmid carrying the exogenous DNA fragment enters the recipient cell, integrates into the chromosomal DNA, and replicates synchronously with the chromosomal DNA. Site-directed mutagenesis refers to the technology of introducing mutations that characterize favorable directions into the exogenous DNA fragment carried by the target plasmid through polymerase chain reaction (PCR), including base addition, deletion, point mutation, etc. Site-directed mutagenesis can quickly and efficiently improve the properties and characteristics of the target protein expressed by DNA. Summary of the invention

[0006] The present invention aims to increase the expression of ATF2 with myristoylation modification site K296 mutation by means of a point mutation plasmid, thereby protecting the endothelial barrier function of endothelial cells under high-fat conditions, and constructing a microvascular endothelial cell model with overexpression of ATF2K296R. The K296R mutation refers to a mutation that replaces lysine (K) at position 296 in protein kinase R (PKR) with arginine (R).

[0007] The specific technical solution adopted by the present invention is as follows:

[0008] In the first aspect, the present invention provides a microvascular endothelial cell ATF2 site mutation plasmid vector, i.e., an ATF2K296R overexpression plasmid vector, which is obtained by inserting a target DNA fragment of human ATF2 K296 site mutation into a plasmid (pcDNA3.1-CMV-MCS-3flag-EF1-ZsGreen-T2A-Puro); the sequence of the target DNA fragment is shown in SEQ ID NO: 1, and the sequence of its amplification primer is shown in SEQ ID NO: 2-SEQ ID NO: 5. The specific preparation method of the ATF2 K296R overexpression plasmid vector is as follows:

[0009] S1. Design sequence: Based on the sequence of human ATF2 in the NCBI GenBank database (www.ncbi.nlm.nih.gov / gene), design the target DNA fragment of ATF6 K296 mutation and its amplification primers;

[0010] S2. Target fragment amplification: The target DNA fragment ATF2 K296R is denatured at high temperature in vitro, and the double strands are dissociated into single strands. Then, the primers are induced to bind to the single strand DNA according to the principle of base complementary pairing at low temperature, and the temperature is adjusted to the optimal reaction temperature of DNA polymerase (about 72°C). In addition, DNA polymerase synthesizes complementary strands along the direction from phosphate to pentose (5'-3'), and this process is repeated until a sufficient number of target DNA fragments ATF2K296R are obtained.

[0011] S3. Vector digestion: Use the same restriction endonuclease to digest the plasmid and the DNA containing the target fragment to obtain linear DNA with the same sticky ends or blunt ends;

[0012] S4. Linking of the target fragment and the vector: The above-mentioned cloning vector and the ATF2 K296R target DNA fragment were mixed in an appropriate ratio, and HB-infusionTM Master mix was added. The DNA exonuclease, DNA polymerase and ligase in the reaction system were reacted at 50°C for 20 minutes to quickly complete the linking of the ATF2K296R target fragment and the vector, thereby completing the construction of the ATF2K296R point mutation overexpression vector.

[0013] In a second aspect, the present invention provides an engineered bacterium containing the above-mentioned ATF2 K296R overexpression plasmid vector, and the preparation process thereof is as follows:

[0014] S1. Transformation: Thaw the competent cells and immediately place them on ice. Add the ATF2 K296R point mutation high expression plasmid DNA solution and place on ice. After heat shock, quickly place them on ice to cool and add LB liquid culture medium to mix and shake to recover the bacteria. Finally, culture the bacterial solution on a resistant culture dish to screen for colonies containing the ATF2 K296R point mutation high expression plasmid.

[0015] S2. PCR identification of bacterial liquid: The colony of the ATF2 K296R point mutation high expression plasmid was added to the resistant LB liquid medium for culture, and the obtained bacterial liquid was subjected to PCR amplification to obtain positive clones;

[0016] S3. Sequencing and comparison: Sequence the positive clones and compare the sequencing results with the target fragment sequence. If the two are consistent, the engineering bacteria containing the ATF2 K296R point mutation high expression plasmid are successfully constructed.

[0017] In a third aspect, the present invention provides a microvascular endothelial cell model with high expression of ATF2 K296R, and the preparation method thereof is as follows: human microvascular endothelial cells (Humandermal microvascular endothelial cells-1, HMEC-1) are transfected with the ATF2 K296R overexpression plasmid vector extracted as described above to obtain a microvascular endothelial cell model with high expression of ATF2K296R.

[0018] In a fourth aspect, the present invention provides an application of the above-mentioned ATF2 K296R overexpression plasmid vector, which may be at least one of A1)-A4):

[0019] A1) preparing a product for treating microvascular endothelial cell barrier damage under hyperlipidemia;

[0020] A2) preparing a product for constructing a microvascular endothelial cell model with high expression of ATF2 K296R;

[0021] A3) Protect the microvascular endothelial cell barrier under high lipid conditions;

[0022] A4) Construct a microvascular endothelial cell model with high expression of ATF2 K296R.

[0023] In a fifth aspect, the present invention provides that the application of the above-mentioned engineering bacteria containing the ATF2 K296R overexpression plasmid vector can be at least one of B1)-B4):

[0024] B1) preparing products for treating conditions where microvascular endothelial cell barrier disruption occurs;

[0025] B2) preparing a product for constructing a microvascular endothelial cell model with high expression of ATF2 K296R;

[0026] B3) Protect the microvascular endothelial cell barrier under high-lipid conditions;

[0027] B4) Construct a microvascular endothelial cell model with high expression of ATF2 K296R.

[0028] In a sixth aspect, the present invention provides an application of the above-mentioned microvascular endothelial cell model with high expression of ATF2 K296R, which can be at least one of C1)-C4):

[0029] C1) preparing a product for treating microvascular endothelial cell barrier damage under hyperlipidemia;

[0030] C2) preparing a product for constructing a microvascular endothelial cell model with high expression of ATF2 K296R;

[0031] C3) Protect the microvascular endothelial cell barrier under high-lipid conditions;

[0032] C4) Construct a microvascular endothelial cell model with high expression of ATF2 K296R.

[0033] The present invention has the following beneficial effects:

[0034] The ATF2 K296R overexpression plasmid of the present invention can increase the expression of ATF2 K296R in human microvascular endothelial cells, inhibit the endothelial barrier damage of HMEC-1 under high-fat conditions, and reduce the inflammatory response of endothelial cells. The mechanism is as follows: the myristoylation modification of ATF2 has a negative impact on the endothelial cell barrier, and after the ATF2K296 point mutation and overexpression, the myristoylation modification level of ATF2 can be significantly reduced, thereby achieving the purpose of protecting the endothelial cell barrier function.

[0035] The present invention uses point mutation plasmid technology to increase the expression of ATF2 K296R protein in microvascular endothelial cells, constructs a microvascular endothelial cell model with high expression of ATF2 K296R, and studies the role of ATF2 myristoylation modification in the occurrence and development of endothelial barrier, which helps to reveal the mechanism of the occurrence and development of endothelial barrier dysfunction and provides useful clues for the treatment of cardiovascular diseases caused by endothelial barrier dysfunction. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1: Example 1 Target vector pcDNA3.1-CMV-MCS-3flag-EF1-ZsGreen-T2A-Puro map.

[0037] Figure 2 : Sequence comparison results in Example 2.

[0038] Figure 3 : The relative mRNA expression results of the ATF2 gene in HMEC-1 cells transfected with the ATF2 K296R point mutation overexpression plasmid in Example 3.

[0039] Figure 4 : Results of immunoprecipitation (upper) and quantitative analysis (lower) of the myristoylation modification level of HMEC-1 cells transfected with ATF2 K296 point mutation overexpression plasmid in Example 3. In the figure, ALK14 represents myristoylated analog, ATF2wt OE represents wild-type ATF2 (control group), ATF2kr OE represents point mutant ATF2 (experimental group), "+" represents corresponding treatment, and "-" represents no corresponding treatment.

[0040] Figure 5 : Results of immunoprecipitation (left) and quantitative analysis (right) of endothelial barrier-related proteins in HMEC-1 cells transfected with ATF2 K296 point mutation overexpression plasmid in Example 3. DETAILED DESCRIPTION

[0041] Although the treatment of cardiovascular disease has been rapidly developed, it is still a disease with a high mortality rate, which brings huge health and economic burden to society. The destruction of endothelial cell barrier function can lead to the occurrence and development of various cardiovascular diseases such as atherosclerosis, myocardial infarction, and myocardial fibrosis. Therefore, exploring the molecular mechanism and signaling pathway of endothelial cell barrier function and finding effective targets of endothelial cell barrier function are of great significance for the prevention and treatment of cardiovascular diseases. Previous studies have found that ATF2 is involved in the occurrence and development of cardiovascular diseases, but the role of its K296 myristoylation modification and the molecular mechanism involved need to be studied and improved.

[0042] Published studies have found that ATF2 is involved in multiple physiological functions of vascular endothelial cells, but its K296 myristoylation modification and the role and signaling pathway of ATF2 K296 myristoylation modification in endothelial cells have not been reported. The present invention increases the content of ATF2 K296R in endothelial cells through point mutation plasmid overexpression technology, thereby further contributing to the study of the relationship and molecular mechanism between ATF2 myristoylation modification and endothelial barrier function, bringing hope for the clinical targeted treatment of endothelial barrier function imbalance and the treatment of cardiovascular diseases.

[0043] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0044] Example 1: Preparation of ATF2 K296R overexpression plasmid vector

[0045] 1. Design and synthesis of target DNA primers for TF2 K296R point mutation overexpression plasmid

[0046] By searching the sequence of human ATF2 in the NCBI GenBank database (www.ncbi.nlm.nih.gov / gene), the target DNA for overexpression of ATF2 K296R point mutation was designed, and its sequence is shown in SEQ ID NO:1.

[0047] The design principles are as follows: (1) Check the homology (homolo>90) and conservation (the sequences of the 10 sites before and after the target site are consistent) between the human ATF2 sequence and the mouse ATF2 sequence; (2) Mutate lysine K at the target site to arginine R; (3) Strictly follow the amino acid codon table to design the gene site mutation; (4) Avoid 5'UTR and 3'UTR, i.e., the non-coding regions of the transcript; (5) Avoid the region within 50-100bp of the start codon and the stop codon; (6) Avoid stretches of 4 or more identical bases; (7) Avoid regions with a G or C base content of <30% or >60%; (8) Avoid single nucleotide polymorphism sites.

[0048] According to the above principles, the sense strand and antisense strand of ATF2 K296R were designed, and the primers were synthesized by Shanghai Hanheng Biotechnology Co., Ltd. The sense strand and antisense strand of ATF2 K296R are shown in Table 1.

[0049] Table 1

[0050]

[0051] 2. Amplification of the target DNA fragment of ATF2 K296R point mutation overexpression plasmid

[0052] Prepare the following system, mix gently, and place in a PCR instrument for reaction; the target fragment PCR amplification system is shown in Table 2, and the PCR program is shown in Table 3.

[0053] Table 2

[0054]

[0055] Table 3

[0056]

[0057] Note: 1) Annealing temperature is the Tm value of the primer, and annealing temperature directly determines amplification specificity; 2) If the amplification specificity is found to be poor, the annealing temperature can be appropriately increased by +2°C each time; 2) Prolonging the extension time appropriately helps to increase amplification yield.

[0058] 3. Linking of ATF2 K296R point mutation overexpression plasmid target DNA fragment with vector

[0059] The target vector pcDNA3.1-CMV-MCS-3flag-EF1-ZsGreen-T2A-Puro used in the present invention is shown in Figure 1 .

[0060] HB infusion kit TM One-step cloning and ligation system:

[0061] Prepare the following reaction system in an ice-water bath. If the liquid accidentally sticks to the tube wall, it can be centrifuged briefly to sink to the bottom of the tube. After the ligation reaction solution reacts at 50℃ for 30 minutes, place it on ice for 5 minutes and transform immediately. The formula is shown in Table 4.

[0062] Table 4

[0063]

[0064] Note: 1) It is recommended that when 2-3 fragments are connected, the total amount of DNA fragments used is 0.02-0.5pmols (generally, the amount of added fragments is 100-150ng, and the amount of vector is 50-100ng), and the total amount of DNA added when 4-6 fragments are connected is 0.2-1.0pmols. The efficiency of DNA splicing gradually decreases with the increase of the number of spliced ​​fragments or the increase of the length of the spliced ​​fragments.

[0065] Example 2: Preparation of bacterial solution containing ATF2 K296R point mutation high expression plasmid

[0066] 1. ATF2 K296R point mutation overexpression plasmid transformation

[0067] Transform the ATF2 K296R point mutation overexpression plasmid according to the following 6 steps:

[0068] 1) After taking out the DH5α competent cells from the -80℃ refrigerator, they should be immediately placed on ice to thaw. The competent cell packaging process should be gentle to reduce mechanical damage to them;

[0069] 2) After the competent medium is thawed, aliquot it into 50 μL volumes per tube (20 μL is sufficient for plasmid transformation). After aliquoting, add the ligation product in an amount not exceeding 1 / 10 of the competent medium volume (currently add 5 μL of ligation product) and place it on ice for 20-30 minutes.

[0070] 3) Heat shock at 42℃ for 90s (this time must be very strict), and immediately put it on ice for 2-3min after heat shock;

[0071] In a clean bench, add 500 μL LB medium (note that it must be LB medium without antibiotics) and gently invert it upside down 3-5 times;

[0072] 5) Incubate at 37°C, 230 rpm with shaking for 45-60 min;

[0073] 6) Apply the bacterial solution evenly to an ampicillin-resistant solid plate, then place the plate upside down in a 37°C incubator and culture for 12-16 hours.

[0074] 2. PCR identification of ATF2 K296R point mutation overexpression plasmid transformed bacteria

[0075] 1) In a clean bench, pick the colony on the plate in step 6 above, add it to 2 ml of LB medium containing ampicillin resistance, and shake for 3 to 5 hours.

[0076] 2) Take 2ul of the sample from the clean bench and transfer it to a labeled PCR tube for PCR identification.

[0077] The PCR system for identifying the ATF2 K296R point mutation overexpression plasmid transformation solution is shown in Table 5, and the identification procedure is shown in Table 6.

[0078] Table 5

[0079]

[0080] Note: When configuring the mix, the proportions in the mix should be amplified in equal proportions. The selected verification primers 1 and 2 are the aforementioned 2F and 2R combinations, and the sequences are shown in SEQ ID NO:4-SEQ ID NO:5.

[0081] Table 6

[0082]

[0083] 3. Sequence the positive clones screened from the ATF2 K296R point mutation overexpression plasmid transformation solution

[0084] The amplified product obtained in step 2 is sequenced and the sequencing result is compared with the target fragment sequence. Figure 2 As shown, the green area is the part that matches the target sequence (the red boxed area involves the site mutation part). The two are consistent, and the engineering bacteria containing the ATF2 K296R point mutation high expression plasmid are successfully constructed.

[0085] The matching sequence in the sequencing result is as follows (i.e., SEQ ID NO: 1):

[0086]

[0087] Example 3: Preparation of microvascular endothelial cell model with high expression of ATF2 K296R and effect testing

[0088] 1. Cell plating

[0089] (1) HMEC-1 cells were seeded in a 6-well plate and cultured in a 37°C, 5% CO2 incubator using MCDB131 microvascular endothelial cell culture medium containing 10% fetal bovine serum and 1% double antibody (streptomycin and penicillin). Transfection was performed after the cell density reached 70-80%.

[0090] (2) Add 2ug of ATF2K296R point mutation overexpression plasmid and empty control plasmid (i.e., the initial plasmid that has not been expressed) to the culture medium of step (1) above using the lipo3000 kit; replace with fresh complete culture medium after 12h; collect cell samples 48h after transfection for subsequent testing.

[0091] 2. RT-qPCR identification of the effect of ATF2 K296R point mutation overexpression therapy on ATF2 overexpression

[0092] (1) Total RNA from human microvascular endothelial cells (HMEC-1) transfected with ATF2 K296R point mutation overexpression plasmid and control plasmid was extracted using Trizol (Takara, Japan), and cell cDNA was obtained by reverse transcription. The specific steps of reverse transcription are as follows:

[0093] ① Digest DNA: Add 1 μg of total RNA from HEMC-1 infected with the virus, 2 μl of 5×gDNA Eraser Buffer and 1 μl of gDNA Eraser into a PCR tube and fill up to 10 μl with nuclease-free water. Then mix the reaction system thoroughly and centrifuge it, and incubate it in a 42°C metal bath for 2 minutes to digest the DNA.

[0094] ②Prepare the reverse transcription reaction system: add 4μl PrimeScriptRTBuffer 2, 4μl RT primer Mix, 1μl RNase Free dH2O and 1μl PrimeScriptRT Enzyme MixI to each tube after DNA digestion in step ①, mix well by pipetting and centrifuge.

[0095] ③ Place the above reaction system in a PCR instrument and perform reverse transcription reaction under the conditions of 37°C, 15min, 85°C, 5s to obtain cDNA of HMEC-1 infected with the virus, which is then stored at 4°C.

[0096] (2) qPCR was used to identify the overexpression effect of ATF2 K296R overexpression plasmid on ATF2.

[0097] Prepare qRT-PCR reaction system (10ul): 0.3μl upstream primer, 0.3μl downstream primer, 5μl TB Green Premix Ex TaqII (Tli RNaseH Plus), 4ul RNase free dH2O and 0.4μl cDNA template obtained in step (1). Place the prepared reaction system on a fluorescent quantitative PCR instrument for qPCR amplification reaction. Using the β-actin gene as the internal reference gene, the 2-ΔΔCT method was used to detect the relative mRNA expression of the ATF2 gene in HMEC-1 after transfection with the ATF2 K296R point mutation overexpression plasmid.

[0098] (3) Experimental results

[0099] Test results such as Figure 3 The results showed that compared with HMEC-1 transfected with the control plasmid, the expression of ATF2 mRNA in cells transfected with the ATF2K296R point mutation overexpression plasmid was significantly increased, indicating that the overexpression effect of the ATF2 K296R point mutation overexpression plasmid was significant.

[0100] 3. Click chemistry experiment to identify the effect of mutation of ATF2 K296 myristoylation modification site

[0101] According to the C10276 click chemistry reaction kit, the myristoylation modification degree of ATF2 in HMEC-1 human microvascular endothelial cells transfected with ATF2 K296 point mutation overexpression plasmid and control cells was detected after intervention with myristoylated analog ALK14 (10ug / ml) for 12 hours. The results are as follows Figure 4 As shown, for the wild-type ATF2 group, after the addition of the myristoyl analog ALK14, its myristoylation modification level increased significantly, which proved that the wild-type ATF2 was modified by myristoyl, and compared with the myristoylation modification level of the wild-type ATF2, the myristoylation modification level of the point mutant ATF2 decreased significantly under the intervention of ALK14, which proved the successful mutation of the myristoylation modification site in the ATF2 K296 point mutation overexpression plasmid.

[0102] 4. Effects of ATF2 K296 overexpression in high-fat-induced human microvascular endothelial cells HMEC-1 on endothelial barrier function

[0103] The expression of FLAG, which represents the level of exogenous ATF2, PRKCD, a molecule that regulates the endothelial barrier downstream of ATF2, and VE-Cadherin, a marker of vascular permeability, were detected in the HMEC-1 endothelial cell line treated with ALK14. Figure 5 As shown in the figure, the FLAG levels of each group were consistent. Under the condition of equivalent exogenous ATF2 content, the expression of PRKCD and VE-Cadherin was significantly inhibited by ALK14. This result suggests that fatty acyl groups may have a negative impact on endothelial barrier function, and the ATF2 / PRKCD / VE-Cadherin pathway plays an important role in it. Next, compared with the control group overexpressing wild-type ATF2, the expression of PRKCD and VE-Cadherin in cells overexpressing ATF2 K296R was significantly increased, which proves that the ATF2K296R site mutation protects the transcriptional activity of ATF2 under high-fat conditions, increases the expression of downstream PRKCD and VE-Cadherin, and protects the endothelial barrier function of human microvascular endothelial cells under fat treatment conditions.

[0104] This specific implementation is only an explanation of the present invention, not a limitation of the present invention. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A plasmid vector for microvascular endothelial cell ATF2 site mutation, characterized in that: It is an ATF2K296R overexpression plasmid vector, which is obtained by inserting a target DNA fragment with a mutation at the human ATF2 K296 site into a plasmid; the sequence of the target DNA fragment is shown in SEQ ID NO:1, and the sequence of its amplification primer is shown in SEQ ID NO:2-SEQ ID NO:

5.

2. The plasmid vector for microvascular endothelial cell ATF2 site mutation according to claim 1, characterized in that: The plasmid is pcDNA3.1-CMV-MCS-3flag-EF1-ZsGreen-T2A-Puro.

3. The plasmid vector for microvascular endothelial cell ATF2 site mutation according to claim 1, characterized in that: The preparation method comprises the following steps: S1. Sequence design: Based on the sequence of human ATF2 in the NCBI GenBank database, the target DNA fragment and its amplification primers for the ATF6K296 site mutation were designed; S2. Target fragment amplification: The target DNA fragment ATF2 K296R is denatured at high temperature in vitro, and the double strands are dissociated into single strands. Then, the primers are induced to bind to the single strand DNA according to the principle of base complementary pairing at low temperature, and the temperature is adjusted to the optimal reaction temperature of DNA polymerase. In addition, DNA polymerase synthesizes complementary chains along the direction from phosphate to pentose, and this process is repeated until a sufficient number of target DNA fragments ATF2 K296R are obtained. S3. Vector digestion: Use the same restriction endonuclease to digest the plasmid and the DNA containing the target fragment to obtain linear DNA with the same sticky ends or blunt ends; S4. Linking of the target fragment and the vector: Mix the above cloning vector and the ATF2 K296R target DNA fragment, add HB-infusionTM Master mix, and react at 50°C for 20 minutes to quickly complete the linking of the ATF2 K296R target fragment and the vector, and complete the construction of the ATF2 K296R point mutation overexpression vector.

4. An engineered bacterium containing the ATF2 K296R overexpression plasmid vector according to claim 1.

5. The engineered bacteria according to claim 4, characterized in that The preparation method comprises the following steps: S1. Transformation: Thaw the competent cells and immediately place them on ice. Add the ATF2 K296R point mutation high expression plasmid DNA solution and place on ice. After heat shock, quickly place them on ice to cool and add LB liquid culture medium to mix and shake to recover the bacteria. Finally, culture the bacterial solution on a resistant culture dish to screen for colonies containing the ATF2K296R point mutation high expression plasmid. S2. PCR identification of bacterial liquid: The colony of the ATF2 K296R point mutation high expression plasmid was added to the resistant LB liquid medium for culture, and the obtained bacterial liquid was subjected to PCR amplification to obtain positive clones; S3. Sequencing and comparison: Sequence the positive clones and compare the sequencing results with the target fragment sequence. If the two are consistent, the engineering bacteria containing the ATF2 K296R point mutation high expression plasmid are successfully constructed.

6. A microvascular endothelial cell model with high expression of ATF2 K296R, characterized in that: The ATF2 K296R overexpression plasmid vector of claim 1 is used to transfect human microvascular endothelial cells HMEC-1 to obtain the protein.

7. Use of the ATF2 K296R overexpression plasmid vector according to any one of claims 1 to 3, the engineered bacteria containing the ATF2 K296R overexpression plasmid vector according to any one of claims 4 to 5, or the ATF2 K296R highly expressed microvascular endothelial cell model according to claim 6, characterized in that: At least one of the following 1)-4): 1) Preparation of products for treating microvascular endothelial cell barrier damage under hyperlipidemia; 2) preparing a product for constructing a microvascular endothelial cell model with high expression of ATF2 K296R; 3) Protect the microvascular endothelial cell barrier under high-fat conditions; 4) Construct a microvascular endothelial cell model with high expression of ATF2 K296R.

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