Use of akr7a2 protein in the preparation of a medicament for preventing and / or treating diabetic nephropathy

Abstract

The application belongs to the field of biological medicine, and particularly relates to application of AKR7A2 protein in preparation of a medicine for preventing and / or treating diabetic nephropathy. The adeno-associated virus (AAV-AKR7A2) expressing the AKR7A2 protein can relieve a mouse model of diabetic nephropathy, significantly reduce a urine albumin creatinine ratio (UACR) of the mouse model of diabetic nephropathy, and also reduce levels of creatinine and urea nitrogen in serum, and has an effect of treating diabetic nephropathy, and the product related to the application has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and more specifically to the use of AKR7A2 protein in the preparation of drugs for the prevention and / or treatment of diabetic nephropathy. Background Technology

[0002] AKR7A2 (member A2 of the aldehyde-ketone reductase family 7) is a metabolic enzyme belonging to the aldehyde-ketone reductase superfamily (AKR). It primarily participates in the reductive metabolism of various endogenous and exogenous aldehydes, playing important physiological and pathological roles. For example, AKR7A2 converts toxic aldehydes into corresponding alcohols through NADPH-dependent reduction reactions, reducing their reactivity and promoting product excretion. AKR7A2 also mitigates oxidative stress-induced cell damage by scavenging reactive aldehydes. Furthermore, AKR7A2 participates in the metabolism of carcinogens such as polycyclic aromatic hydrocarbons (PAHs), potentially reducing their potential to induce gene mutations. However, its role in diabetic kidney disease (DKD) has not yet been reported.

[0003] Diabetic kidney disease (DKD) seriously endangers human health. It is not only a common complication of type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM), but also one of the leading causes of end-stage renal disease (ESRD), rising to the number one cause of ESRD worldwide. As a microvascular complication of diabetes, the pathogenesis of diabetic nephropathy is highly complex. In recent years, research on the pathogenesis of diabetic nephropathy has mainly focused on oxidative stress, inflammation, and autophagy. Podocytes are a key component of the glomerular filtration barrier, and podocyte damage is a significant cause of glomerular diseases such as diabetic nephropathy. Increasing research indicates that other key mechanisms besides hyperglycemia play a leading or synergistic role in the progression of diabetic nephropathy.

[0004] Currently, AAV gene therapy has made some progress in diabetes and its complications. For example, in 2022, the world's first AAV gene therapy for type 1 diabetes entered clinical trials. This gene therapy aims to express the gene encoding A20 in donor pancreatic islet cells through the GARV-AAV2-A20 viral delivery system, and then transplant these cells into the patient to achieve the therapeutic goal. AAV gene therapy has also demonstrated therapeutic efficacy in the preclinical stage of diabetic cardiomyopathy.

[0005] The inventors found in their research that after tail vein injection of AAV-AKR7A2 into db / db mice, the levels of UACR (urinary alcohol to creatinine ratio), blood urea nitrogen, and blood creatinine were significantly reduced, and kidney function was significantly improved, demonstrating a therapeutic effect on diabetic nephropathy. Summary of the Invention

[0006] To address the aforementioned technical problems, the primary objective of this invention is to provide the use of AKR7A2 protein in the preparation of drugs for the prevention and / or treatment of diabetic nephropathy, wherein the amino acid sequence of the AKR7A2 protein is shown in SEQ ID No. 1, and the nucleotide sequence of the AKR7A2 protein is shown in SEQ ID No. 2.

[0007] A second objective of this invention is to provide a recombinant plasmid expressing the AKR7A2 protein, wherein the recombinant plasmid is obtained by inserting the gene encoding the AKR7A2 protein into an expression vector, and the amino acid sequence of the AKR7A2 protein is shown in SEQ ID No. 1.

[0008] Preferably, the expression vector includes eukaryotic expression plasmid vectors, adenoviruses, adeno-associated viruses, lentiviruses, retroviruses, baculoviruses, herpesviruses, pseudorabies viruses, ZFN gene editing technology, TALEN gene editing technology, CRISPR / Cas gene editing technology, and other medically available gene editing technologies or viral vectors.

[0009] Preferably, the eukaryotic plasmid expression vector is selected from one or more of pcDNA3.1+ / -, pcDNA4 / HisMaxB, pSecTag2A, pVAX1, pBudCE4.1, pTracerCMV2, pcDNA3.1(-) / myc-HisA, pcDNA6-Myc / HisB, pCEP4, pIRES, pIRESneo, pIREShyg3, pCMV-myc, pCMV-HA, pIRES-2puro3, pIRES-neo3, pCAGGS, pSilencer1.0, pSilencer2.1-U6hygro, pSilencer3.1-H1hygro, pSilencer3.1-H1neo, and pSilencer4.1-CMVneo.

[0010] Preferably, the viral expression vector is selected from pAAV-CAG, pLKO.1, pLVXIRES-ZsGreen1, pCDH-EF1-Luc2-T2A-tdTomato, pAAV2 / 9n, pCDH-MSCV-MCS-EF1-Puro, pCDH-MSCVMCS-EF1-copGFP, pLVX-ZsGreen1-C1, pAdEasy-1, pShuttle-CMV, pShuttle, pAdTrack, pAdTrack-CMV, pShuttle-IRES-hrGFP-1, pShuttle-IRES-hrGFP-2, pShuttle-CMV-lacZ, pShuttle-CMV-EGFP-C, pXC1, pBHGE3, pAAV-MCS, pAAV-RC, pHelper, or pAAV-LacZ.

[0011] Preferably, the expression vector includes pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pPIC9K, pHIL-S1, pPICZα, pYAM75P, and PNZ8149-usp45.

[0012] Preferably, the expression vector includes pAAV-CAG, and the prepared recombinant plasmid is named pAAV-CAG-AKR7A2. The nucleotide sequence of the recombinant plasmid pAAV-CAG-AKR7A2 is shown in SEQ ID No. 3.

[0013] The third objective of this invention is to provide an AAV expressing the AKR7A2 protein, wherein the AAV-AKR7A2 is obtained by co-transfecting the prepared recombinant plasmid and helper plasmid into 293T cells for packaging.

[0014] Preferably, the co-transfection ratio is pAAV2 / 9n:pHelper:pAAV-CAG-AKR7A2-=1:1:2.

[0015] A fourth object of the present invention is to provide the use of the aforementioned AAV-AKR7A2 in the preparation of medicaments for the prevention and / or treatment of diabetic nephropathy.

[0016] Preferably, the tail vein injection dose of the AAV-AKR7A2 is 1×10⁻⁶. 11 -1×10 12 vg.

[0017] The beneficial effects of this invention are as follows: This invention provides a novel use of AKR7A2 protein for the treatment of diabetic nephropathy; it can significantly reduce UACR, serum creatinine, and blood urea nitrogen levels in db / db mice, and improve kidney function; and a recombinant plasmid of the AKR7A2 gene is constructed and transfected into 293T cells to be packaged into AAV, in which the AKR7A2 gene is effectively expressed; this invention applies AKR7A2 protein drugs to the treatment of diabetic nephropathy via gene therapy and tail vein injection, providing a new treatment method in the field and achieving significant results. Attached Figure Description

[0018] Figure 1 The results of recombinant plasmid PCR validation are shown in Figure 1.

[0019] Figure 2 Effect of different AAV packaging plasmid ratios on 293T cell transfection efficiency

[0020] Figure 3 Effect of AKR7A2 protein expression on FBG in db / db mice

[0021] Figure 4 Effect of AKR7A2 protein expression on fasting body weight in db / db mice

[0022] Figure 5 Effect of AKR7A2 protein expression on UACR in db / db mice

[0023] Figure 6 Effects of AKR7A2 protein expression on serum creatinine and blood urea nitrogen in db / db mice Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments provide a more detailed description of the invention. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0025] Example 1 Construction of recombinant plasmid

[0026] 1. Experimental reagents

[0027] Stbl3 chemocompetent cells were purchased from Beijing Qingke Biotechnology Co., Ltd.; recombinant plasmids pOTB7-AKR7A2 and pAAV-CAG-EGFP, framework plasmid pAAV2 / 9n, and helper plasmid pHelper were purchased from Miaoling Plasmid Platform; the bacterial plasmid DNA extraction kit was purchased from Zhenxuan Co., Ltd.; PrimeSTARHS (Premix, 2X) and T4 DNA ligase were purchased from Takara Bio Inc.; and the DNA endonucleases XbaI and EcoRI were purchased from Beijing NEB Co., Ltd.

[0028] The LB (Luria-Bertani) liquid culture medium is formulated as follows: 1% tryptone, 0.5% yeast extract, and 0.5% NaCl; the LB solid culture medium is formulated as follows: 1% tryptone, 0.5% yeast extract, 0.5% NaCl, and 2% agar.

[0029] 2. Primer information synthesis

[0030] The PCR primers used were purchased from Beijing Qingke Biotechnology Co., Ltd.; the primers were dissolved in sterile deionized water. The final concentration was 10 μmol / L.

[0031] 3. Construction of pAAV-CAG-AKR7A2 recombinant plasmid

[0032] 3.1 Amplification of the target gene: PCR was performed using synthesized specific primers and the pOTB7-AKR7A2 plasmid, and the results were verified by agarose gel electrophoresis. After successful verification, the PCR product was purified using a purification kit from a select company.

[0033] 3.2 Restriction Digestion and Ligation: The amplified and purified AKR7A2 gene and pAAV-CAG-EGFP plasmid were digested with XbaI and EcoRI. The digestion products were purified and ligated using T4 DNA ligase.

[0034] 3.3 Transform the recombinant plasmid pAAV-CAG-AKR7A2 into Stbl3 cells: Remove Stbl3 chemocompetent cells from the -80℃ freezer and place them on an ice box for 10-20 min to thaw. Take out 50 μL of competent cells and add 5 μL of plasmid, gently mix with your fingers a few times, and place the mixture on ice for 25 min; after heat shock at 42℃ for 45 s, quickly return them to the freezer and let them stand on ice for 2 min. Add 0.5 mL of room temperature LB liquid medium (without antibiotics), and incubate at 37℃ with shaking at 200 rpm for 1 h. Take 200 μL of the above bacterial solution and spread it on an agar plate containing 50 μg / mL ampicillin. After the bacterial solution is completely absorbed by the medium, invert the culture dish and incubate at 37℃ for 12 h.

[0035] 3.4 Verification of successful transformation colonies: Single colonies were picked and cultured in liquid medium containing 50 μg / mL ampicillin. After 12-16 h, the pAAV-CAG-AKR7A2 recombinant plasmid was extracted using a small-scale plasmid extraction kit from a select company.

[0036] 3.5 PCR Validation: Using the pAAV-CAG-AKR7A2 recombinant plasmid as a template, PCR amplification was performed using AKR7A2-specific primers. The specific system was as follows:

[0037] The PCR reaction conditions were as follows: 98.0℃ for 5 min pre-denaturation, followed by 35 cycles of denaturation, annealing, and extension. The denaturation conditions were 98.0℃ for 10 s, the annealing conditions were 64.0℃ for 5 s, extension I was 72.0℃ for 1 min, and extension II was 72.0℃ for 10 min. The PCR products were subjected to agarose gel electrophoresis (1%) at 50V for 45 min, followed by staining with 3X Gel Green solution. The PCR verification results are as follows. Figure 1 As shown, a successfully constructed pAAV-CAG-AKR7A2 recombinant plasmid PCR should have the target band of AKR7A2 (approximately 1000bp). The arrow in the figure points to the target band, indicating that the pAAV-CAG-AKR7A2 plasmid was successfully constructed.

[0038] Example 2: AAV virus packaging and harvesting

[0039] 1. Experimental reagents

[0040] HEK-293T cells were purchased from Xavier Biotech Ltd.; Lipo8000 TM Transfection reagents were purchased from Beyotime Inc.; universal virus concentration kits were purchased from Beyotime Inc.; and AAV virus titer assay kits were purchased from Takara.

[0041] 2. Cell transfection

[0042] 2.2 Before performing the following transfection steps, replace 2 mL of fresh culture medium (complete culture medium containing serum and antibiotics) in each well of a six-well plate containing 293T cells. For Lipo8000 TM The presence of antibiotics in the transfection reagent will not affect the transfection efficiency, nor will it cause cytotoxicity after cell transfection.

[0043] 2.3 Referring to the table below, take a clean, sterile centrifuge tube and add 125 μL of antibiotic- and serum-free DMEM culture medium (high-glucose DMEM or low-glucose DMEM can be used) or Opti- for each well of the six-well plate to be transfected. Add 2.5 μg of plasmid DNA to the medium solution and gently mix by pipetting; then add 4 μL of Lipo8000. TM Mix the transfection reagent gently by pipetting; do not vortex or centrifuge. Once prepared, it is stable at room temperature for 6 hours.

[0044] To improve transfection efficiency, three transfection ratios were explored, as follows: ①pAAV2 / 9n:pHelper:pAAV-CAG-=1:1:2 ②pAAV2 / 9n:pHelper:pAAV-CAG-=3:4:2 ③pAAV2 / 9n:pHelper:pAAV-CAG-=4:2:1

[0045] 2.4 Regardless of whether the cells are adherent or suspension, use 125 μL of Lipo8000 per well in a six-well plate. TM Add the transfection reagent-DNA mixture evenly to the entire well, then mix gently.

[0046] 2.5 After culturing for approximately 24-48 hours, observe the fluorescence of the EGFP group. Cell transfection results are shown below. Figure 2 .like Figure 2 As shown, 24 hours after transfection, group ① showed the strongest EGFP fluorescence and the highest transfection efficiency. Therefore, subsequent experiments used the transfection ratio of group ①, i.e., pAAV2 / 9n:pHelper:pAAV-CAG=1:1:2.

[0047] 2.6 Virus Harvesting and Concentration

[0048] 2.6.1 After packaging the virus using cultured cells, collect the virus-containing supernatant. Centrifuge at 3,500g for 10 min at 4°C to fully precipitate cell debris, and carefully aspirate the supernatant for subsequent concentration.

[0049] 2.6.2 Take out the virus precipitation reagent pre-cooled at 4℃ and mix it with virus supernatant at a ratio of 1 part virus precipitation reagent to 3 parts virus supernatant. After thorough mixing, incubate overnight at 4℃ on a mixer or side-swing shaker at low speed.

[0050] 2.6.3 Centrifuge at 4℃, 3,500g for 0.5-1h. A white precipitate is usually visible at the bottom of the centrifuge tube at this time (sometimes the precipitate is not visible). Carefully remove the supernatant, being careful not to touch the precipitate, and do not shake the centrifuge tube violently.

[0051] 2.6.4 Centrifuge at 3500g for 1 minute at 4℃, carefully aspirate any remaining liquid, and do not touch the precipitate.

[0052] 2.6.5 Add 1-10% (12%) of the original virus supernatant volume of virus resuspension (optional, depending on subsequent use), let stand for 10 minutes, then carefully pipette 20-30 times to resuspend the virus pellet. Avoid generating air bubbles during pipetting, as vigorous pipetting may inactivate the virus.

[0053] 2.6.6 Centrifuge at 12,000g for 3-5 minutes at 4℃, and collect the supernatant to obtain the concentrated virus. It can be aliquoted as needed for subsequent experiments and stored at -80℃ for later use.

[0054] 3. Virus titer detection

[0055] 3.1 Extraction of AAV genome

[0056] 3.1.1 Add DNase I to the prepared AAV virus extract as shown below to form a mixture, and react at 37°C for more than 15 minutes to remove free genomic and plasmid DNA.

[0057] 3.1.2 Heat treatment at 95℃ for 10 min to deactivate DNase I.

[0058] 3.1.3 Add an equal amount of Lysis Buffer to the 20 μL sample from step 2 above.

[0059] 3.1.4 Heat treatment at 70℃ for 10 minutes.

[0060] 3.1.5 Dilute the obtained AAV genomic extract to more than 50-fold using EASY Dilution (for Real Time PCR), and use this diluted solution as a template to perform Real Time PCR reaction to quantify the genomic DNA.

[0061] 3.2 Real-Time PCR Reaction

[0062] Take 5 μL of the DNA solution prepared in 3.1 as a template for Real-Time PCR. Simultaneously, prepare a standard curve using standards.

[0063] 3.2.1 Preparation of Standards for Standard Curves

[0064] Positive Control was serially diluted using EASY Dilution and used as a standard for the standard curve. (5 μL was used for each Real-Time PCR reaction.)

[0065] a.2×107 copies / μL (Positive Control stock solution)

[0066] b.2×10 6 copies / μL (Positive Control stock solution 5μL+EASY Dilution 45μL)

[0067] c.2×10 5 copies / μL (2×10) 6 (5 μL of solution per 100 μL + 45 μL of easy-dilution)

[0068] d.2×10 4 copies / μL (2×10) 5 (5 μL of solution per 100 μL + 45 μL of easy-dilution)

[0069] e.2×10 3 copies / μL (2×10) 4 (5 μL of solution per 100 μL + 45 μL of easy-dilution)

[0070] f.2×10 2 copies / μL (2×10) 3 (5 μL of solution per 100 μL + 45 μL of easy-dilution)

[0071] 3.3 Formulating 50X Primer mix

[0072] Prepare the 50X Primer mix according to the following recipe. Prepare the required amount of 50X Primer mix as needed; do not store.

[0073] 3.4 Preparation of reaction solution

[0074] Prepare the reaction solution on ice according to the following components. After preparing the required number of tubes + α of the following components (excluding the template), add 20 μL of the mixture to each 0.2 mL microtube and gently cap it. Add 5 μL of dH2O to one microtube as a negative control and then tighten the cap. [When using Applied Biosystems' Real-Time PCR instrument] reagents Usage TB Green Premix Ex Taq II(2X conc.) 12.5μl 50X Primer mix 0.5μl <![CDATA[ROX Reference Dye or ROX Reference Dye II * > 0.5μl <![CDATA[dH2O]]> 6.5μl template (5.0 μl) Total 25.0μl *: StepOnePlus uses ROX Reference Dye. Applied Biosystems 7500 Fast Real-Time PCR System uses ROX Reference Dye II.

[0075] 3.5 Add template (DNA solution)

[0076] Add 5 μL of the sample DNA solution or standard to the aliquot solution in section 3.4 above, and tighten the cap. After gently centrifuging the 0.2 mL reaction tube with a small centrifuge, place it on a Real-Time PCR instrument.

[0077] 3.6 Begin the Real-Time PCR reaction under the following conditions:

[0078] Example 3: Therapeutic effect of AAV-AKR7A2 on diabetic nephropathy mice

[0079] 1. Laboratory animals and reagents

[0080] SPF-grade 8-week-old male db / db mice and db / m mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd.; blood glucose meters and blood glucose test strips were purchased from Sinocare Biosensor Co., Ltd.; urine microalbumin assay kits, creatinine assay kits and blood urea nitrogen assay kits were purchased from Nanjing Institute of Biological Engineering.

[0081] 2. Experimental grouping and administration method

[0082] 2.1 Before the experiment, the animals were acclimatized for one week, given food and free access to water. The weight and blood glucose of the mice were monitored weekly (fasting but not water for 8 hours). If the fasting blood glucose (FBG) was ≥16.7 mmol / L and remained stable for at least 2 weeks, the diabetic model was considered stable and the model was successfully established.

[0083] 2.2 After the diabetes model was successfully established, 17 db / db mice and 5 db / m mice were randomly divided into 4 groups, as follows:

[0084] a.db / m group: 5 mice were injected with saline via the tail vein at 12 weeks (mouse age) and 16 weeks (mouse age).

[0085] b.db / db group: 5 mice were injected with saline via the tail vein at 12 and 16 weeks of age.

[0086] c.db / db+AAV-EGFP group: 6 mice were injected intravenously with AAV-EGFP once each at 12 and 16 weeks of age, with an injection dose of 1×10⁻⁶. 11 vg / time.

[0087] d.db / db+AAV-AKR7A2 group: 6 mice were injected intravenously with AAV-AKR7A2 once each at 12 and 16 weeks of age, at a dose of 1×10⁻⁶. 11 vg / time.

[0088] 2.3 All mice were fed to 20 weeks of age. During the drug administration period, the growth status of the mice (including fur color, movement ability, etc.) was monitored daily, and blood glucose and weight were tested weekly.

[0089] 3. Detection indicators

[0090] 3.1 Weight and FBG monitoring

[0091] Starting from week 8, after fasting for 8 hours each week but not drinking water, fasting blood glucose is measured at the same time period (9:00 am - 5:00 pm). A suitable amount of blood is taken by cutting the tail, and the blood glucose content is measured with a blood glucose meter. The blood glucose value is read and recorded. Before measuring blood glucose each week, it is necessary to weigh the blood using an electronic balance.

[0092] 3.2 Measurement of urinary microalbumin, urinary creatinine, and UACR

[0093] In week 20, 24-hour urine samples were collected from mice in each group using metabolic cages. The samples were centrifuged at 1,520g for 15 minutes at 4°C, and the supernatant was used as the urine sample. Microalbumin and creatinine levels in the urine of each group were measured according to the instructions of the microalbumin and creatinine assay kits. (One mouse died in each of the db / db and db / db+AAV-EGFP groups.)

[0094]

[0095] 3.3 Measurement of serum creatinine and blood urea nitrogen

[0096] At the end of week 20, blood was collected from the inner canthus of the eye and placed in centrifuge tubes. The tubes were incubated at room temperature for 30-60 minutes, then centrifuged at 1520g for 15 minutes at 4°C. The supernatant obtained was the serum sample. Creatinine and urea nitrogen levels in the serum of each group of mice were measured according to the instructions of the creatinine and urea nitrogen assay kits. (Two mice died in the db / db group, and one mouse died in the db / db+AAV-EGFP group.)

[0097] 4. Statistical Analysis

[0098] Experimental data were statistically analyzed using SPSS 23.0 software. Data are expressed as (x±s). One-way ANOVA and LSD-t method were used for pairwise comparisons between groups. P < 0.05 was considered statistically significant. All statistical results were plotted using GraphPad Prism 8 software.

[0099] 5. Experimental Results

[0100] The effect of AKR7A2 protein expression on FBG in db / db mice is as follows: Figure 3 As shown, at weeks 10 and 11, the FBG in db / db mice was significantly higher than that in db / m mice, and FBG > 16.7 mmol / L, indicating the successful establishment of the diabetic mouse model. After week 11, db / db mice were treated with AAV-AKR7A2. Figure 3 It can be seen that AAV-AKR7A2 has no significant effect on FBG in db / db mice.

[0101] The effect of AKR7A2 protein expression on fasting body weight in db / db mice is as follows: Figure 4 As shown, expression of AKR7A2 protein had no significant effect on fasting body weight in mice, but the body weight of db / db mice was significantly higher than that of db / m mice.

[0102] The effect of AKR7A2 protein expression on UACR in db / db mice is as follows: Figure 5 As shown, db / db vs. db / m, ### p < 0.001 indicates severe kidney damage in db / db mice, with significantly weaker glomerular filtration function compared to db / m mice, thus confirming the successful establishment of the diabetic nephropathy model. After treatment with AAV-AKR7A2, the UACR in db / db mice was significantly reduced (db / db+AAV-AKR7A2 vs. db / db, db / db+AAV-AKR7A2 vs. db / db+AAV-EGFP). *** p < 0.001 indicates that the expression of AKR7A2 protein described in this invention has the effect of alleviating diabetic nephropathy.

[0103] The effects of AKR7A2 protein expression on serum creatinine and blood urea nitrogen in db / db mice are as follows: Figure 6 As shown, db / db vs. db / m, ###p < 0.001 indicates a significant increase in the blood urea nitrogen / creatinine ratio in db / db mice, indicating impaired glomerular filtration function, consistent with the UACR results. After treatment with AAV-AKR7A2, the blood urea nitrogen / creatinine ratio in db / db mice was significantly reduced (db / db+AAV-AKR7A2 vs. db / db, **p < 0.01; db / db+AAV-AKR7A2 vs. db / db+AAV-EGFP, *p < 0.05), demonstrating that the AKR7A2 protein expressed in this invention improves glomerular filtration function and alleviates diabetic nephropathy.

[0104] In summary, this invention provides the application of AKR7A2 protein in the treatment of diabetic nephropathy. The AKR7A2 protein can significantly reduce UACR, blood urea nitrogen and serum creatinine levels in db / db mice, improve glomerular filtration function, thereby alleviating diabetic nephropathy, and has broad application prospects.

Claims

1. The use of AKR7A2 protein in the preparation of drugs for the prevention and / or treatment of diabetic nephropathy, characterized in that, The amino acid sequence of the AKR7A2 protein is shown in SEQ ID No.

1.

2. A recombinant plasmid expressing AKR7A2 protein, characterized in that, The recombinant plasmid is obtained by inserting the gene encoding the AKR7A2 protein into an expression vector, and the amino acid sequence of the AKR7A2 protein is shown in SEQ ID No.

1.

3. The recombinant plasmid as described in claim 2, characterized in that, The expression vectors include eukaryotic expression plasmid vectors, adenoviruses, adeno-associated viruses (AAVs), lentiviruses, retroviruses, baculoviruses, herpesviruses, pseudorabies viruses, ZFN gene editing technology, TALEN gene editing technology, CRISPR / Cas gene editing technology, and other medically available gene editing technologies or viral vectors, probiotic vectors, and mRNA vectors.

4. The recombinant plasmid as described in claim 3, characterized in that, The eukaryotic plasmid expression vector is selected from one or more of pcDNA3.1+ / -, pcDNA4 / HisMaxB, pSecTag2A, pVAX1, pBudCE4.1, pTracerCMV2, pcDNA3.1(-) / myc-HisA, pcDNA6-Myc / HisB, pCEP4, pIRES, pIRESneo, pIREShyg3, pCMV-myc, pCMV-HA, pIRES-2puro3, pIRES-neo3, pCAGGS, pSilencer1.0, pSilencer2.1-U6hygro, pSilencer3.1-H1hygro, pSilencer3.1-H1neo, and pSilencer4.1-CMVneo.

5. The recombinant plasmid as described in claim 3, characterized in that, The viral expression vectors are selected from pAAV-CAG, pLKO.1, pLVXIRES-ZsGreen1, pCDH-EF1-Luc2-T2A-tdTomato, pAAV2 / 9n, pCDH-MSCV-MCS-EF1-Puro, pCDH-MSCVMCS-EF1-copGFP, pLVX-ZsGreen1-C1, pAdEasy-1, pShuttle-CMV, pShuttle, pAdTrack, pAdTrack-CMV, pShuttle-IRES-hrGFP-1, pShuttle-IRES-hrGFP-2, pShuttle-CMV-lacZ, pShuttle-CMV-EGFP-C, pXC1, pBHGE3, pAAV-MCS, pAAV-RC, pHelper, or pAAV-LacZ.

6. The recombinant plasmid as described in claim 3, characterized in that, The expression vectors include pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pPIC9K, pHIL-S1, pPICZα, pYAM75P, and PNZ8149-usp45.

7. An AAV expressing AKR7A2 protein, characterized in that, The AAV-AKR7A2 was obtained by co-transfecting the recombinant plasmid and helper plasmid prepared according to claim 3 into 293T cells for packaging, with a co-transfection ratio of pAAV2 / 9n. pHelper:pAAV-CAG-AKR7A2=1:1:

2.

8. The use of AAV-AKR7A2 as described in claim 7 in the preparation of medicaments for the prevention and / or treatment of diabetic nephropathy.

9. The AAV-AKR7A2 as described in claim 7, characterized in that, The tail vein injection dose for AAV-AKR7A2 mice was 1×10⁻⁶. 11 -1×10 12 vg.

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