Application of deacetylated NDP52 protein in the preparation of drugs for controlling Salmonella infection

Abstract

This invention relates to the field of genetic engineering technology, and in particular to the application of a deacetylated NDP52 protein in the preparation of drugs for controlling Salmonella infection. This application provides a deacetylated NDP52 protein, a gene encoding the deacetylated NDP52 protein and its nucleotide sequence, and includes a recombinant vector containing the gene, a transgenic cell line or recombinant virus, and a substance capable of deacetylation of the NDP52 protein. The application of the gene, the protein, and the substance for deacetylation of the NDP52 protein in the preparation of drugs for controlling Salmonella infection demonstrates that expressing NDP52 and its deacetylated variants in mouse liver effectively promotes the clearance of bacterial infection in the liver and reduces liver necrosis, providing a scientific basis and potential tool for the treatment of bacterial infectious diseases.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of a deacetylated NDP52 protein in the preparation of drugs for controlling Salmonella infection. Background Technology

[0002] Salmonella is a common Gram-negative bacterium that is widely found in the intestines of animals and humans and can be transmitted via the fecal-oral route, causing a variety of diseases. Currently, clinical treatment of Salmonella infection mainly relies on antibiotics. However, because Salmonella exhibits strong resistance in the body environment, especially under conditions of nutritional deficiency, the treatment effect of antibiotics on diseases caused by Salmonella is not ideal.

[0003] Cells have developed unique self-protection mechanisms during long-term evolution, among which the autophagy receptor protein NDP52 plays an important role in resisting Salmonella infection.

[0004] NDP52 can specifically recognize invading Salmonella and encapsulate it in autophagosomes, degrading it through heteroautophagy. This prevents the bacteria from multiplying in large numbers within the cell, reducing further infection and damage. This mechanism is an important component of cellular innate immunity and helps provide additional defense beyond antibiotic treatment.

[0005] In summary, the main research direction is to enable NDP52 to bind more effectively to ubiquitinated Salmonella, thereby enhancing its ability to recognize and encapsulate bacteria, improving the efficiency of its autophagy process, and enabling it to more effectively clear Salmonella from cells.

[0006] In particular, the use of NDP52 may be a potential therapeutic strategy in drug development. By enhancing the autophagy capacity of host cells, it can more effectively eliminate invading bacteria, thereby reducing the severity of infection and the risk of transmission. Furthermore, it can be used in combination with other antibiotics to improve therapeutic efficacy and reduce the development of antibiotic resistance.

[0007] Further research on NDP52 and its application in the defense mechanisms of Salmonella infection may provide new ideas and directions for the development of novel anti-Salmonella drugs and control strategies. Summary of the Invention

[0008] The purpose of this invention is to provide a deacetylated NDP52 protein;

[0009] Another object of the present invention is to provide the use of deacetylated NDP52 protein in the preparation of drugs for controlling Salmonella infection.

[0010] The technical solution adopted to achieve the purpose of this invention is:

[0011] NDP52 plays an important role in the autophagy process of host cells, especially in clearing invading Salmonella. NDP52 can specifically recognize ubiquitinated Salmonella and encapsulate it in autophagosomes, degrading it through the autophagy pathway, thereby preventing the bacteria from multiplying in large numbers within the cell.

[0012] To promote the clearance of Salmonella and provide a new treatment option for bacterial infections such as Salmonella, this application provides a deacetylated NDP52 protein.

[0013] The NDP52 has an NCBI ID of 10241, and the amino acid sequence of the NDP52 protein is shown in SEQ ID No. 3. SEQ ID No. 3 is:

[0014] MEETIKDPPTSAVLLDHCHFSQVIFNSVEKFYIPGGDVTCHYTFTQHFIPRRKDWIGIFRVGWKTTREYYTFMWVTLPIDLNNKSAKQQEVQFKAYYLPKDDEYYQFCYVD EDGVVRGASIPFQFRPENEEDILVVTTQGEVEEIEQHNKELCKENQELKDSCISLQKQNSDMQAELQKKQEELETLQSINKKLELKVKEQKDYWETELLQLKEQNQKMSSEN EKMGIRVDQLQAQLSTQEKEMEKLVQGDQDKTEQLEQLKKENDHLFLSLTEQRKDQKKLEQTVEQMKQNETTAMKKQQELMDENFDLSKRLSENEIICNALQRQKERLEGEN DLLKRENSRLLSYMGLDFNSLPYQVPTSDEGGARQNPGLAYGNPYSGIQESSSPSPLSIKKCPICKADDICDHTLEQQQMQPLCFNCPICDKIFPATEKQIFEDHVFCHSL.

[0015] The deacetylated NDP52 protein is a deacetylated variant of the NDP52 protein, specifically, the lysine residue (codon AAG) at position 202 of the NDP52 protein is mutated to arginine (codon AGG), and is referred to in this application as: NDP52. K202RA protein encoding the amino acid sequence SEQ ID No. 1, or a protein derived from SEQ ID No. 1 having one or more amino acid residues substituted and / or deleted and / or added compared to the amino acid sequence shown in SEQ ID No. 1, and capable of deacetylation of the protein.

[0016] SEQ ID NO.1 is:

[0017] MEETIKDPPTSAVLLDHCHFSQVIFNSVEKFYIPGGDVTCHYTFTQHFIPRRKDWIGIFRVGWKTTREYYTFMWVTLPIDLNNKSAKQQEVQFKAYYLPKDDEYYQFCYVD EDGVVRGASIPFQFRPENEEDILVVTTQGEVEEIEQHNKELCKENQELKDSCISLQKQNSDMQAELQKKQEELETLQSINKKLELKVKEQRDYWETELLQLKEQNQKMSSEN EKMGIRVDQLQAQLSTQEKEMEKLVQGDQDKTEQLEQLKKENDHLFLSLTEQRKDQKKLEQTVEQMKQNETTAMKKQQELMDENFDLSKRLSENEIICNALQRQKERLEGEN DLLKRENSRLLSYMGLDFNSLPYQVPTSDEGGARQNPGLAYGNPYSGIQESSSPSPLSIKKCPICKADDICDHTLEQQQMQPLCFNCPICDKIFPATEKQIFEDHVFCHSL.

[0018] Furthermore, a gene encoding deacetylated NDP52 protein is provided, wherein the sequence of the gene encoding deacetylated NDP52 protein is the nucleotide sequence shown in SEQ ID No. 2, or the nucleotide sequence of the gene has more than 90% homology with the nucleotide sequence shown in SEQ ID No. 2 and encodes a protein with the same biological function.

[0019] SEQ ID NO.2 is:

[0020]

[0021] Furthermore, this application provides a biological material, which is a recombinant vector, transgenic cell line or recombinant virus containing a gene encoding deacetylated NDP52 protein, or a substance capable of achieving deacetylated expression of the gene.

[0022] Furthermore, this application also provides an application, specifically the simultaneous or separate use of the following four items in the preparation of drugs for regulating Salmonella infection:

[0023] (1) Deacetylated NDP52 protein: Deacetylated variants of NDP52 protein;

[0024] (2) Gene: Gene encoding deacetylated NDP52 protein;

[0025] (3) An expression cassette, recombinant vector, transgenic cell line or recombinant virus containing the gene encoding deacetylated NDP52 protein;

[0026] (4) Substances that enable the deacetylation of NDP52 protein.

[0027] Specifically, Salmonella infection in organisms is controlled by expressing or overexpressing at least one of the above four items.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] By utilizing an AAV viral vector to specifically express the NDP52 protein and its lysine deacetylated variant K202R at position 202 in the liver of C57BL / 6J mice, significant effects were observed in promoting the clearance of bacterial infection in the liver and alleviating liver necrosis.

[0030] In the constructed bacterial infection model, mice expressing NDP52 protein showed a significant reduction in bacterial count and liver necrosis in their liver tissue, demonstrating the crucial role of NDP52 protein in enhancing bacterial infection defense mechanisms. Furthermore, mice expressing the deacetylated variant K202R exhibited even more significant bacterial clearance and liver damage reduction, indicating that deacetylation of lysine at position 202 further enhances the antibacterial activity of NDP52. This research not only provides a new direction for understanding the mechanisms of NDP52 and its modifications in bacterial infection defense but also offers strong scientific evidence and potential therapeutic tools for the treatment of bacterial infections such as Salmonella, possessing significant clinical translational value by promoting bacterial clearance and reducing liver necrosis. Attached Figure Description

[0031] Figure 1 This is a map of the AAV9-TBG-NDP52-GFP plasmid used in this invention;

[0032] Figure 2 This is a flowchart of the experimental process in an embodiment of the present invention;

[0033] Figure 3 In this embodiment of the invention, fluorescence imaging of liver slices is used to verify AAV expression;

[0034] Figure 4 Western blot was used to verify the expression of liver NDP52 protein in this embodiment of the invention;

[0035] Figure 5 The results of mouse liver colony counting in this embodiment of the invention;

[0036] Figure 6 This is a diagram showing the results of fluorescence imaging observation of Salmonella infection in the liver in an embodiment of the present invention;

[0037] Figure 7 This is a statistical result of fluorescence imaging observation of Salmonella infection in the liver in an embodiment of the present invention;

[0038] Figure 8 The images show HE staining results and magnified views of uninfected and infected mouse livers in this embodiment of the invention.

[0039] Figure 9 This is a statistical result of the number of liver necrosis cases in an embodiment of the present invention. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0042] The NDP52 wild-type WT nucleotide sequence described in this application is shown in SEQ ID No. 4, which is:

[0043]

[0044] Example 1: Construction of AAV9-TBG-NDP52-GFP wild-type WT and deacetylated variant K202R plasmids. The specific experimental steps are as follows:

[0045] (1) Polymerase chain reaction (PCR)

[0046] AAV9-TBG-GFP, pcDNA5-NDP52-Flag, pcDNA5-NDP52 K202R -Flag is the template plasmid, and the total reaction volume is 50 μL. Add 25 μL RimeSTAR Max enzyme, 1 μL template plasmid, 0.2 μM upstream primer, 0.2 μM downstream primer, and ddH2O to the PCR tube. Gently tap to mix thoroughly, centrifuge, and then place in the PCR instrument. Perform pre-denaturation at 95℃ for 5 min; denature at 95℃ for 15 s, anneal at 55℃ for 20 s, and extend at 72℃ at 0.2 kb / s for 30 cycles; finally, extend at 72℃ for 5 min and store at 4℃.

[0047] (2) Agarose gel electrophoresis

[0048] Prepare 50×TAE electrophoresis buffer and dilute it to 1×TAE. Place the substrate on the gel preparation plate, insert a suitable comb, weigh 0.6g of agarose, add 60mL of 1×TAE, heat in a microwave oven until completely dissolved, add YeaGreen Nucleic Acid Gel Stain dye, shake to mix, pour the solution into the gel preparation plate, and allow it to solidify for 30 minutes to obtain a 1% agarose gel. Prepare for sample loading: add a small amount of 6×DNA loading buffer to the PCR product, and add 10μL of marker according to the fragment size (15k for large fragments, 2k for small fragments). Turn on the electrophoresis apparatus, set a constant voltage of 125V, and run for 20–30 minutes. After electrophoresis, remove the gel from the mold and place it under a UV analyzer. Observe the bright bands according to the markers. Use a clean blade to cut off the target band and place it in a 2mL EP tube for gel recovery.

[0049] (3) Gel recovery, enzyme digestion and ligation

[0050] The agarose gel was prepared according to the DNA gel recovery kit (purchased from Sangon Biotech Co., Ltd.). First, 300 μL of binding buffer II was added, and the gel was incubated in a metal bath at 50–60 °C for 10 min to completely dissolve it. After dissolution, the gel was removed from the metal bath and cooled before loading onto the column. The EZ Spin Column was placed on a 2 mL collection tube, and the dissolved gel was loaded onto the column. The column was allowed to stand for 1 min to improve recovery efficiency. The column was centrifuged at 12000 rpm for 1 min. After centrifugation, the liquid in the collection tube was loaded back onto the same column, and centrifuged again at 12000 rpm for 1 min, discarding the waste liquid. 500 μL of wash buffer containing anhydrous ethanol was taken, centrifuged, and the liquid was discarded. This step was repeated twice. The column was then centrifuged at 12000 rpm for 3–4 min to ensure the removal of any residual wash buffer. The empty-run adsorption column was then opened and placed into a sterile 1.5 mL EP tube, and incubated in a metal bath at 50–60 °C for 10 min until the ethanol evaporated. Add 30 μL of preheated ddH2O and centrifuge at 12000 rpm for 3–4 min to elute the DNA. After digesting the recovered fragments and vector with enzymes, add them to a PCR tube with Basic Mix and ligate at 50 °C for 20 min.

[0051] (4) Plasmid transformation and bacterial culture

[0052] Add an appropriate amount of competent cells to the ligation product and incubate on ice for 30 min. Heat shock in a 42°C water bath for 45–90 s. After heat shock, incubate on ice for 5 min. Add 500 μL of antibiotic-free LB medium and incubate on a shaker for 45 min. After shaking, centrifuge at 4000 rpm for 5 min, and evenly spread an appropriate amount of bacterial culture onto solid LB medium containing specific antibiotics. Invert the plate and incubate overnight at 37°C. The next day, pick single colonies from the plate and add them to 500 μL of medium containing the antibiotic and incubate for approximately 5 h. Then, send 50 μL for sequencing. Add the correctly sequenced bacterial culture to 200 mL of liquid LB medium with the corresponding antibiotic and incubate at 37°C and 220 rpm for approximately 15 h.

[0053] (5) Endotoxin-free plasmid extraction

[0054] Add the overnight cultured bacterial solution to a centrifuge tube and centrifuge at 4000 rpm for 15 min at room temperature to collect the bacteria. Aspirate the supernatant. Proceed with the collected bacterial pellet according to the endotoxin-free plasmid large-scale extraction kit (purchased from TIANGEN). Add 8 mL of solution P1 (containing RNase A) to the centrifuge tube containing the bacterial pellet and vortex to thoroughly resuspend the pellet. Add 8 mL of solution P2 to the centrifuge tube and gently invert 6–8 times to fully lyse the bacteria. Incubate at room temperature for 5 min. Add 8 mL of solution P4 to the centrifuge tube and gently invert 6–8 times to mix thoroughly until a white, dispersed flocculent precipitate appears. Incubate at room temperature for approximately 10 min. Centrifuge at 8000 rpm for 5–10 min to allow the white precipitate to settle to the bottom of the tube. Pour the entire solution into filter CS1 and filter by pushing the push handle. Collect the filtrate in a clean 50 mL centrifuge tube. Add 0.3 times the volume of isopropanol to the filtrate, mix thoroughly by inverting, and transfer to adsorption column CP6. Centrifuge at 8000 rpm for 2 min, discard the waste liquid, and repeat the process multiple times until all liquid has passed through the column. Add 10 mL of PW wash buffer to adsorption column CP6, centrifuge at 8000 rpm for 2 min, discard the waste liquid, and repeat once. Add 3 mL of anhydrous ethanol to adsorption column CP6, centrifuge at 8000 rpm for 2 min, and discard the waste liquid. Return adsorption column CP6 to the collection tube and centrifuge at 8000 rpm for 5 min. Place adsorption column CP6 in a clean 50 mL collection tube, add 1 mL of sterile ddH2O dropwise to the center of the adsorption membrane, incubate at room temperature for 5 min, and centrifuge at 8000 rpm for 2 min to collect the plasmid. Transfer the eluent from the 50 mL centrifuge tube to a clean 1.5 mL centrifuge tube, quantify the plasmid, and store at -20℃.

[0055] Wild-type AAV9-TBG-NDP52 was finally obtained. WT -GFP and the deacetylated variant AAV9-TBG-NDP52 K202R -GFP two plasmids, NDP52 WT The nucleotide sequence is shown in SEQ ID No. 4, NDP52 K202R The nucleotide sequence is shown in SEQ ID No. 2.

[0056] Example 2 Wild-type NDP52 WT and deacetylated variant NDP52 K202R Preparation of adeno-associated virus

[0057] The empty control AAV9-TBG-GFP and wild-type AAV9-TBG-NDP52 were used. WT -GFP, deacetylated variant AAV9-TBG-NDP52 K202R- Three GFP plasmids were sent to Hangzhou Guannan Biotechnology Co., Ltd. to customize related adeno-associated viruses, resulting in three types of adeno-associated viruses: control, wild-type, and deacetylated variants, with viral titers of 5.73 × 10⁻⁶. 13 3.57×10 13 1.17×10 13 μg / mL.

[0058] Example 3: NDP52 in mouse liver WT and NDP52 K202R expression

[0059] Using C57BL / 6J mice as experimental subjects, the obtained AAV virus was injected via tail vein at a dose of 200 μL / mouse, 1×10⁻⁶. 12 μg / mL / mouse, divided into Control group (injected with control virus) and NDP52 group. WT Group (injected with NDP52 wild-type virus), NDP52 K202R Groups (injected with NDP52 deacetylated variant virus), 10 animals in each group. Expression was validated one month after in vivo expression.

[0060] The livers of mice in the three groups injected with adeno-associated virus (AAV) and the Blank group not injected with the virus were removed, frozen sections were prepared, stained with DAPI, and then imaged. The expression of AAV in the mouse liver was observed by observing the green fluorescence excited at 488 nm. Figure 3 The results showed that the Blank group was the group that did not receive the virus, the Vector group was the group that received the empty vector, and NDP52 WT For the NDP52 wild-type group, NDP52 K202R To inject the NDP52 protein lysine deacetylated mutant group, scale bar 50 μm, via... Figure 3 It can be seen that, compared with the Blank group of mice, the AAV virus was successfully expressed in the livers of all three groups of mice.

[0061] In the Control group, NDP52 WT Group, NDP52 K202R Three mice were randomly selected from each of the three groups of mice. The liver tissue was lysed in PBS + 0.5% Triton, centrifuged at 250xg, 4℃ for 5 min, and the supernatant was collected. The supernatant was then centrifuged again at 15000 rpm, 4℃ for 30 min, and the supernatant was collected again. Loading buffer was added, and the mixture was incubated in a 100℃ metal bath for 1 h. Western blot analysis was then performed to verify the expression of NDP52 protein in the mouse liver. Figure 4 The results showed that the Control group was the group injected with empty virus, NDP52 WTFor the NDP52 wild-type group, NDP52 K202R The group injected with the NDP52 protein deacetylated mutant at the 202 lysine site showed that, compared to the control group, NDP52 levels in the liver were significantly higher. WT Group, NDP52 K202R Smooth expression.

[0062] Example 4 expressing NDP52 K202R Promoting effect on bacterial clearance in mouse liver

[0063] After verifying successful expression of both wild-type and deacetylated NDP52 protein in mouse liver, intraperitoneal infection was performed using Salmonella Typhimurium, 1×10⁻⁶. 6 CFU / mouse, 8 mice per group were infected, and mice were sacrificed 2 days later for dissection. The livers were removed for histopathological examination and analysis. The experimental procedure is as follows: Figure 2 As shown. Will be in accordance with Figure 2 After the mouse livers obtained by the procedure were lysed, they were centrifuged at 250xg and 4℃ for 5 min, and the supernatant was diluted 10. 3 Or 10 4 Then, take 50 μL for plate counting, incubate overnight at 37°C, and observe the colony growth. The results are as follows: Figure 5 As shown.

[0064] Figure 5 The left side of the image shows representative colony growth results, while the right side shows statistical results (n=8). The results indicate that NDP52 is expressed in mouse liver. WT Subsequently, compared with the control group, the number of bacteria in the liver was significantly reduced (P < 0.05), and the expression of the deacetylated variant NDP52 was observed. K202R Afterwards, compared to NDP52 WT In the group, the number of bacteria in the liver decreased significantly (P < 0.0001). This indicates that the deacetylation of NDP52 protein and its 202 site can significantly promote the clearance of bacteria in the liver and has a certain therapeutic effect on Salmonella infection.

[0065] Take the Control group and NDP52 WT Group, NDP52 K202R Frozen sections of mouse livers from groups three after bacterial infection were prepared, stained with DAPI, and then subjected to fluorescence imaging. Green GFP represents AAV virus expression, red RFP represents Salmonella, and blue represents cell nuclei. The fluorescence imaging results were used to observe and analyze the Salmonella infection status in the liver. The fluorescence imaging results are shown below. Figure 6 As shown, the scale bar is 50 μm. Statistical results are as follows: Figure 7 The vertical axis represents the number of Salmonella rubrum bacteria in each microscopic field of view. A total of 40 fields of view from 4 mice were analyzed. ****P < 0.0001. Figure 6 and Figure 7 It can be seen that mouse liver expresses NDP52. WT Subsequently, the bacterial count decreased significantly (P < 0.0001), and the expression of the deacetylated variant NDP52 was observed. K202R Subsequently, the number of bacteria decreased significantly (P < 0.0001), indicating that deacetylation of the 202 lysine site of the autophagy receptor protein NDP52 can promote the clearance of Salmonella in mouse liver and has a therapeutic effect on liver bacterial infection caused by Salmonella.

[0066] Example 5 expressing NDP52 K202R Protective effect against bacterial liver infection in mice

[0067] Control and NDP52 viruses were injected separately. WT NDP52 K202R Liver tissues from three groups of mice, including those uninfected and those infected with bacteria, were embedded in paraffin and sectioned for hematoxylin-eosin (HE) staining to observe liver necrosis. Results are as follows: Figure 8 As shown, S.Tm stands for S. Typhimurium (Salmonella Typhimurium). Figure 8 HE staining results and magnified images of livers from uninfected and infected mice, with scale bars at 200 μm and 100 μm; Figure 9 The results show the number of liver necrosis cases (n=10). The results indicate that the livers of mice in the uninfected Salmonella group showed normal staining; in the Salmonella-infected group, the control group had more areas of liver necrosis and expressed NDP52. WT The area of ​​liver necrosis was relatively reduced in the group, and the expression of the deacetylated variant NDP52 at position 202 was increased. K202R The further reduction in the area of ​​liver necrosis indicates that the deacetylation of NDP52 protein and its 202 site can protect the liver infected with Salmonella and play a certain therapeutic role in Salmonella infection.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A deacetylated NDP52 protein, characterized in that: As shown in (a) below: (a) NDP52 K202R : The lysine at position 202 of the NDP52 protein is mutated to an arginine, and the amino acid sequence is SEQ ID No.

1.

2. A gene encoding the deacetylated NDP52 protein of claim 1.

3. The gene of claim 2, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID No.

2.

4. A biomaterial, characterized by: The biological material is a recombinant vector, a transgenic cell line or a recombinant virus containing the gene of claim 2 or 3.

5. Use of at least any one of the following in the preparation of a medicine for controlling Salmonella infection, (1) the deacetylated NDP52 protein of claim 1; (2) the gene of claim 2 or 3; (3) an expression cassette, a recombinant vector, a transgenic cell line or a recombinant virus containing the gene of claim 2 or 3.

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