Application of deacetylated NDP52 protein in preparing medicine for controlling salmonella infection

Abstract

The invention relates to the technical field of genetic engineering, in particular to application of deacetylated NDP52 protein in preparation of a medicine for controlling salmonella infection. The invention provides a deacetylated NDP52 protein, provides a gene for coding the deacetylated NDP52 protein and a nucleotide sequence of the gene, provides a recombinant vector, a transgenic cell line or a recombinant virus containing the gene, and a substance capable of realizing deacetylation of the NDP52 protein, and further provides the gene, the protein and the recombinant vector containing the gene, the transgenic cell line or the recombinant virus containing the gene and the substance capable of realizing deacetylation of the NDP52 protein. The invention relates to application of a substance for realizing NDP52 protein deacetylation in preparation of salmonella infection control drugs, and provides a scientific basis and a potential tool for treatment of bacterial infection diseases by expressing NDP52 and a deacetylated variant thereof in mouse liver to effectively promote removal of liver bacterial infection and relieve hepatic necrosis.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to application of a deacetylated NDP52 protein in the preparation of a drug for controlling salmonella infection. Background Art

[0002] Salmonella is a common Gram-negative bacterium that is widely present in the intestines of animals and humans and can be transmitted through the fecal-oral route, causing a variety of diseases. Currently, the clinical treatment of Salmonella infection mainly relies on antibiotics, but because Salmonella shows strong tolerance in the internal environment, especially under conditions of nutrient deficiency, antibiotics are not ideal for the treatment of diseases caused by Salmonella.

[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, encapsulate it in autophagosomes, and degrade it through the xenophagy pathway, thereby preventing the bacteria from multiplying in large numbers within the cell and 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, how to enable NDP52 to bind ubiquitinated Salmonella more effectively, thereby enhancing its ability to recognize and encapsulate bacteria, improving the efficiency of its autophagy process, and enabling it to more effectively eliminate Salmonella from cells has become the current main research direction.

[0006] Especially in drug preparation, the use of NDP52 may also become a potential therapeutic strategy. By enhancing the autophagy of host cells, invading bacteria can be more effectively removed, thereby reducing the severity of infection and the risk of transmission. In addition, it can also be used in combination with other antibiotics to improve the therapeutic effect and reduce the development of antibiotic resistance.

[0007] Further research on NDP52 and its application in the defense mechanism of Salmonella infection is expected to provide new ideas and directions for the development of new anti-Salmonella drugs and control strategies. Summary of the invention

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

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

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

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

[0012] In order to achieve the purpose of promoting the elimination of Salmonella and provide a new solution for the treatment of bacterial infections such as Salmonella, a deacetylated NDP52 protein is provided in the present application.

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

[0014] MEETIKDPPTSAVLLDHCHFSQVIFNSVEKFYIPGGDVTCHYTFTQHFIPRRKDWIGIFRVGWKTTREYYTFMWVTLPIDLNNKSAKQQEVQFKAYYLPKDDEYYQFCYVD EDGVVRGASIPFQFRPENEEDILVVTTQGEVEEIEQHNKELCKENQELKDSCISLQKQNSDMQAELQKKQEELETLQSINKKLELKVKEQKDYWETELLQLKEQNQKMSSEN EKMGIRVDQLQAQLSTQEKEMEKLVQGDQDKTEQLEQLKKENDHLFLSLTEQRKDQKKLEQTVEQMKQNETTAMKKQQELMDENFDLSKRLSENEIICNALQRQKERLEGEN DLLKRENSRLLSYMGLDFNSLPYQVPTSDEGGARQNPGLAYGNPYSGIQESSSPSPLSIKKCPICKADDICDHTLEQQQMQPLCFNCPICDKIFPATEKQIFEDHVFCHSL.

[0015] The deacetylated NDP52 protein is a deacetylated variant of the NDP52 protein, specifically, the lysine at position 202 of the NDP52 protein (codon is AAG) is mutated to arginine (codon is AGG), which is referred to in the present application as: NDP52 K202RA protein whose encoded amino acid sequence is 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 achieving deacetylation of the protein.

[0016] SEQ ID NO.1 is:

[0017] MEETIKDPPTSAVLLDHCHFSQVIFNSVEKFYIPGGDVTCHYTFTQHFIPRRKDWIGIFRVGWKTTREYYTFMWVTLPIDLNNKSAKQQEVQFKAYYLPKDDEYYQFCYVD EDGVVRGASIPFQFRPENEEDILVVTTQGEVEEIEQHNKELCKENQELKDSCISLQKQNSDMQAELQKKQEELETLQSINKKLELKVKEQRDYWETELLQLKEQNQKMSSEN EKMGIRVDQLQAQLSTQEKEMEKLVQGDQDKTEQLEQLKKENDHLFLSLTEQRKDQKKLEQTVEQMKQNETTAMKKQQELMDENFDLSKRLSENEIICNALQRQKERLEGEN DLLKRENSRLLSYMGLDFNSLPYQVPTSDEGGARQNPGLAYGNPYSGIQESSSPSPLSIKKCPICKADDICDHTLEQQQMQPLCFNCPICDKIFPATEKQIFEDHVFCHSL.

[0018] Furthermore, a gene encoding a deacetylated NDP52 protein is provided, wherein the sequence of the gene encoding the deacetylated NDP52 protein is a nucleotide sequence as 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 nucleotide sequence of a protein with the same biological function.

[0019] SEQ ID NO.2 is:

[0020]

[0021] Furthermore, a biomaterial is provided in the present application, which is a recombinant vector, a transgenic cell line or a recombinant virus containing a gene encoding a deacetylated NDP52 protein, or a substance capable of achieving the deacetylated expression of the gene.

[0022] Furthermore, the present application also provides an application, specifically the application of the following four items simultaneously or separately in the preparation of a drug for regulating Salmonella infection:

[0023] (1) Deacetylated NDP52 protein: deacetylated variant 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 a gene encoding a deacetylated NDP52 protein;

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

[0027] Specifically, the control of Salmonella infection in an organism is achieved by expressing or overexpressing at least one of the above four items.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] By using AAV viral vector to specifically express NDP52 protein and its lysine 202 deacetylation variant K202R in the liver of C57BL / 6J mice, it has a significant effect in promoting the clearance of liver bacterial infection and alleviating liver necrosis.

[0030] In the constructed bacterial infection model, the number of bacteria in the liver tissue of mice expressing NDP52 protein was significantly reduced, and the degree of liver necrosis was significantly reduced. This finding illustrates the key role of NDP52 protein in enhancing the defense mechanism against bacterial infection. In addition, mice expressing the deacetylated variant K202R showed more significant bacterial clearance and liver damage reduction effects, indicating that the deacetylation modification of lysine at position 202 further enhanced the antibacterial activity of NDP52. This research result not only provides a new direction for understanding the mechanism of NDP52 and its modification in bacterial infection defense, but also provides a strong scientific basis and potential therapeutic tool for the treatment of bacterial infections such as Salmonella. It has important clinical translation value by promoting the clearance of bacteria in the liver and reducing liver necrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the AAV9-TBG-NDP52-GFP plasmid map of the present invention;

[0032] Figure 2 This is an experimental flow chart of an embodiment of the present invention;

[0033] Figure 3 In the embodiment of the present invention, the expression of AAV is verified by fluorescence imaging of liver slices;

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

[0035] Figure 5 The results of mouse liver colony counts in the examples of the present invention;

[0036] Figure 6 This is a result diagram of observing the infection of Salmonella in the liver by fluorescence imaging in an embodiment of the present invention;

[0037] Figure 7 This is a statistical result diagram of the salmonella infection in the liver observed by fluorescence imaging in an embodiment of the present invention;

[0038] Figure 8 The HE staining results and magnified images of the liver of uninfected and infected mice in the examples of the present invention;

[0039] Fig. 9 This is the statistical result of the number of liver necrosis in the embodiment of the present invention. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0042] The wild-type WT nucleotide sequence of NDP52 described in the present application is shown in SEQ ID No. 4, and SEQ ID No. 4 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 was used as the template plasmid, and the total reaction system was 50 μL. 25 μL PrimeSTAR Max enzyme, 1 μL template plasmid, 0.2 μM upstream primer, 0.2 μM downstream primer and ddH2O were added to the PCR tube, and the liquid was flicked to mix evenly. After centrifugation, it was placed in the PCR instrument. Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 55°C for 20 s, extension at 72°C at 0.2 kb / s, denaturation to extension for 30 cycles; full extension at 72°C for 5 min, and storage at 4°C.

[0047] (2) Agarose gel electrophoresis

[0048] Prepare 50×TAE electrophoresis buffer and dilute it to 1×TAE for later use. Place the bottom plate on the gel plate, insert a suitable comb, weigh 0.6g of agarose, add 60mL of 1×TAE, heat in a microwave oven until completely dissolved, add YeaGreenNucleicAcid Gel Stain dye and shake to mix, pour the solution into the gel plate and wait for 30 minutes to solidify, and prepare 1% agarose gel. Prepare for loading, add a small amount of 6×DNA loading buffer to the PCR product, and add 10μL of Marker according to the fragment size (large fragment 15k, small fragment 2K). Turn on the electrophoresis instrument, set a constant voltage of 125V, and run for 20 to 30 minutes. After the electrophoresis is completed, take the gel block out of the mold and place it under the UV analyzer, check the bright band according to the Marker, cut the target band with a clean blade, and put it in a 2mL EP tube for gel recovery.

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

[0050] The cut agarose gel was operated 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 completely dissolved in a metal bath at 50-60°C for 10 minutes. After dissolution, it was taken out from the metal bath and cooled before being loaded onto the column. The adsorption column EZ Spin Column was placed on a 2 mL collection tube, and the dissolved gel was loaded onto the column. It was left to stand for 1 minute to improve the recovery effect. Centrifuged at 12000 rpm for 1 minute, and the liquid in the collection tube was loaded onto the column again (the same column), and centrifuged again at 12000 rpm for 1 minute, and the waste liquid was discarded. Take 500 μL of wash buffer with anhydrous ethanol added, and discard the liquid after centrifugation. This step was repeated twice. Centrifuged at 12000 rpm for 3-4 minutes to ensure that the residual wash buffer was removed. After the adsorption column was idling, the lid was opened and placed into a sterilized 1.5 mL EP tube, and the metal bath was placed at 50-60°C for 10 minutes until the alcohol evaporated. Add 30 μL preheated ddH2O and centrifuge at 12000 rpm for 3-4 minutes to elute DNA. After digestion of the recovered fragments and vector, add them to the PCR tube with Basic Mix and connect them in a PCR machine at 50°C for 20 minutes.

[0051] (4) Plasmid transformation and bacterial culture

[0052] Add an appropriate amount of competent cells to the ligation product and place on ice for 30 minutes. Heat shock in a 42℃ water bath for 45-90 seconds. After heat shock, place on ice for another 5 minutes. Add 500μL of LB medium without antibiotics and culture on a shaking table for 45 minutes. After shaking, centrifuge at 4000rpm for 5 minutes, and draw an appropriate amount of bacterial liquid and evenly spread it on the solid LB medium containing specific resistance. Invert the plate and place it in a 37℃ constant temperature incubator for overnight culture. The next day, pick a single clone from the plate, add it to 500μL of medium containing resistance and culture it for about 5 hours, then draw 50μL for sequencing. Add the correctly sequenced bacterial liquid to 200mL of liquid LB medium with corresponding resistance, and culture it on a 37℃ shaker at 220rpm for about 15 hours.

[0053] (5) Endotoxin-free plasmid extraction

[0054] Add the overnight cultured bacterial solution to a centrifuge tube, centrifuge at 4000rpm for 15min at room temperature to collect the bacteria, and remove the supernatant. The collected bacterial precipitate is operated according to the endotoxin-free plasmid extraction kit (purchased from TIANGEN). Add 8mL of solution P1 (RNaseA has been added) to the centrifuge tube with the bacterial precipitate, and vortex the oscillator to thoroughly suspend the bacterial precipitate. Add 8mL of solution P2 to the centrifuge tube, gently turn it upside down 6 to 8 times to fully lyse the bacteria, and place it at room temperature for 5min. Add 8mL of solution P4 to the centrifuge tube, gently turn it upside down 6 to 8 times, mix it thoroughly until the solution appears white dispersed flocculent precipitate, and place it at room temperature for about 10min. Centrifuge at 8000rpm for 5 to 10min to make the white precipitate separate to the bottom of the tube, pour all the solution into the filter CS1, push the push handle to filter, and collect the filtrate in a clean 50mL centrifuge tube. Add 0.3 times the volume of isopropanol to the filtrate, mix it upside down, and transfer it to the adsorption column CP6. Centrifuge it at 8000rpm for 2min, discard the waste liquid, and apply it to the column several times until all the liquid passes through the column. Add 10mL of rinsing liquid PW to the adsorption column CP6, centrifuge it at 8000rpm for 2min, discard the waste liquid, and repeat once. Add 3mL of anhydrous ethanol to the adsorption column CP6, centrifuge it at 8000rpm for 2min, and discard the waste liquid. Put the adsorption column CP6 back into the collection tube and centrifuge it at 8000rpm for 5min. Place the adsorption column CP6 in a clean 50mL collection tube, add 1mL of sterile ddH2O to the middle part of the adsorption membrane in mid-air, leave it at room temperature for 5min, and centrifuge it at 8000rpm for 2min to collect the plasmid. Transfer the eluate in the 50mL centrifuge tube to a clean 1.5mL centrifuge tube, quantify the plasmid and store it at -20℃.

[0055] Finally, wild-type AAV9-TBG-NDP52 was obtained WT -GFP and the deacetylated variant AAV9-TBG-NDP52 K202R -GFP two plasmids, NDP52 WT The nucleotide sequence of NDP52 is shown in SEQ ID No.4. K202R The nucleotide sequence is shown as 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, wild-type AAV9-TBG-NDP52 WT -GFP, deacetylated variant AAV9-TBG-NDP52 K202R-GFP three plasmids were sent to Hangzhou Guannan Biotechnology Co., Ltd., and the relevant adeno-associated viruses were customized to obtain the control, wild-type, and deacetylated variant adeno-associated viruses, with virus titers of 5.73×10 13 , 3.57×10 13 , 1.17×10 13 μg / mL.

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

[0059] C57BL / 6J mice were used as experimental materials, and the obtained AAV virus was injected into the tail vein at 200 μL / mouse, 1×10 12 μg / mL / mouse, divided into Control group (injected with control virus), NDP52 WT Group (injected with NDP52 wild-type virus), NDP52 K202R Group (injected with NDP52 deacetylated variant virus), 10 mice in each group. After one month of in vivo expression, expression verification was performed.

[0060] The livers of mice in the three groups injected with adeno-associated virus and the Blank group without virus injection were removed, frozen sections were made, and images were taken after DAPI staining. The expression of adeno-associated virus in the mouse liver was observed by observing the green fluorescence excited at 488nm. Figure 3 The results showed that the Blank group was not injected with virus, the Vector group was injected with empty vector, and NDP52 WT For the group injected with NDP52 wild type, NDP52 K202R The NDP52 protein 202 lysine site deacetylation mutant group was injected, the scale bar is 50 μm, Figure 3 It can be seen that compared with the Blank group mice, the AAV virus was successfully expressed in the livers of the 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 livers, and the tissues were broken in PBS + 0.5% Triton, centrifuged at 250xg and 4°C for 5 min, and the supernatant was taken. The supernatant was centrifuged at 15000rpm and 4°C for 30 min, and the loading buffer was added. The supernatant was then placed in a 100°C metal bath for 1 h. Western blot was then performed to verify the expression of NDP52 protein in mouse liver. Figure 4 The results showed that the control group was injected with empty virus, and NDP52 WTFor the group injected with NDP52 wild type, NDP52 K202R Compared with the control group, the NDP52 in the liver was increased in the group injected with the NDP52 protein 202 lysine deacetylation mutant. WT Group, NDP52 K202R Smooth expression.

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

[0063] After verifying that the wild-type and deacetylated variants of NDP52 proteins were successfully expressed in the mouse liver, intraperitoneal infection was performed with Salmonella typhimurium, with 1×10 6 CFU / mouse, 8 mice were infected in each group, and mice were killed and dissected 2 days later, and the liver was removed for histopathological examination and analysis. The experimental process is as follows Figure 2 As shown. Figure 2 The mouse liver obtained in the process was crushed and centrifuged at 250xg and 4°C for 5 min. The supernatant was diluted 10 3 or 10 4 times, and then take 50 μL for plate counting. After incubation at 37°C overnight, observe the growth of the colonies. The results are as follows: Figure 5 shown.

[0064] Figure 5 The left side shows the representative colony growth results, and the right side shows the statistical results (n=8). The results show that NDP52 is expressed in mouse liver. WT After 4 h, the number of bacteria in the liver decreased significantly compared with the control group (P < 0.05), and the expression of the deacetylated variant NDP52 K202R After that, compared with NDP52 WT In the control group, the number of liver bacteria 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, NDP52 WT Group, NDP52 K202R The livers of the three groups of mice infected with bacteria were frozen and sectioned, and then stained with DAPI for fluorescence imaging. The green GFP represents the expression of AAV virus, the red RFP represents Salmonella, and the blue represents the cell nucleus. The fluorescence imaging results were used to observe and analyze the Salmonella infection in the liver. Figure 6 The scale bar is 50 μm. Figure 7 The vertical axis represents the number of red Salmonella in each microscopic field, and a total of 40 fields of view from 4 mice were counted, ****P < 0.0001. Figure 6 and Figure 7 It can be seen that mouse liver expresses NDP52 WT After 4 h, the number of bacteria decreased significantly (P < 0.0001), and the expression of the deacetylated variant NDP52 K202R After that, the number of bacteria further 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 the mouse liver and has a therapeutic effect on liver bacterial infection caused by Salmonella.

[0066] Example 5 Expression of NDP52 K202R Protective effect against bacterial infection of mouse liver

[0067] Take injection control virus Control, NDP52 WT 、NDP52 K202R The livers of the three groups of mice that were not infected with bacteria and those that were infected with bacteria were embedded in paraffin and sectioned, and then stained with hematoxylin-eosin (HE) to observe the necrosis of the mouse livers. Figure 8 As shown, wherein S.Tm is S.Typhimurium (Salmonella typhimurium), Figure 8 HE staining results and magnified images of livers of uninfected and infected mice, with scale bars of 200 μm and 100 μm; Fig. 9 The results are the statistical results of the number of liver necrosis (n=10). The results showed that the liver staining of mice in the non-Salmonella infection group was normal; in the Salmonella infection group, the control group had more liver necrosis areas and expressed NDP52 WT The liver necrosis area in the group was relatively reduced, and the expression of the 202-deacetylated variant NDP52 K202R The necrotic area of ​​the liver in the control group was further reduced, indicating that the deacetylation of NDP52 protein and its 202 site can protect the liver infected by Salmonella and play a certain therapeutic role in Salmonella infection.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A deacetylated NDP52 protein, characterized in that: is (a) or (b) as follows: (a) NDP52 K202R : The lysine at position 202 of the NDP52 protein was mutated to arginine, and the amino acid sequence was SEQ ID No. 1; (b) 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 achieving deacetylation of the protein.

2. A gene encoding the deacetylated NDP52 protein as claimed in claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the gene is SEQ ID No. 2, or a nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO. 2 and encoding a protein with the same biological function.

4. A biomaterial, characterized in that: The biological material is a recombinant vector, a transgenic cell line or a recombinant virus containing the gene according to claim 2 or 3, or a substance that can deacetylate the NDP52 protein.

5. Use of at least one of the following in the preparation of a drug for controlling Salmonella infection: (1) The deacetylated NDP52 protein according to claim 1; (2) The gene according to claim 2 or 3; (3) an expression cassette, recombinant vector, transgenic cell line or recombinant virus containing the gene of claim 2 or 3; (4) Substances that achieve deacetylation of NDP52 protein.

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