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

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of dephosphorylated NDP52 protein in preparation of salmonella infection control drugs. The technical scheme of the invention is as follows: a protein NDP52S162A with an amino acid sequence of SEQ ID No.1, a gene for coding the protein and with a nucleotide sequence of SEQ ID No.2, a recombinant vector, a transgenic cell line or a recombinant virus containing the gene, and a substance capable of realizing dephosphorylation of the NDP52 protein are provided; the invention also discloses application of the gene, the protein and a substance for realizing dephosphorylation of the NDP52 protein in preparation of salmonella infection control medicines, and experiments in mice show that the NDP52 protein and the 162-site serine dephosphorylation thereof promote removal of salmonella in the liver of the mice and play a role in controlling salmonella infection.

Description

Technical Field

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

[0002] Salmonella is a major pathogen that can cause a variety of illnesses, including typhoid fever, paratyphoid fever, food poisoning, diarrhea, bacteremia, and sepsis. Traditionally, antibiotics have been the mainstay of treatment for Salmonella infections. However, the widespread use and misuse of antibiotics has led to the emergence of multidrug-resistant strains, making treatment more difficult. Therefore, finding new treatments has become a key research priority.

[0003] In recent years, several novel therapies have shown potential as effective alternatives for treating Salmonella infections:

[0004] Small molecule therapies: Inhibiting Salmonella infection by designing or screening small molecule compounds that interfere with bacterial growth, metabolism, or virulence. These small molecules can target specific bacterial proteins or metabolic pathways, thereby inhibiting growth or reducing virulence.

[0005] Phage therapy: Bacteriophages are viruses that can infect and kill bacteria. Phage therapy uses specialized phages to target and eliminate Salmonella. Because phages are highly specific, they can precisely attack target bacteria without affecting host cells or other beneficial bacteria.

[0006] Attenuated vaccine therapy: By using attenuated or inactivated Salmonella strains as vaccines, the host's immune response is stimulated, providing long-term protection against Salmonella infection. Attenuated vaccines can simulate natural infection and induce the immune system to produce a specific immune response against Salmonella.

[0007] In addition, host cells have also developed a variety of defense mechanisms to fight bacterial infections during the long process of evolution. Among them, autophagy is an important cellular self-protection mechanism. NDP52, also known as CALCOCO2 (calcium-binding and coiled-coil domain 2), is an important autophagy receptor protein in mammalian cells. It plays an important role in the selective autophagy of invading pathogens (such as Salmonella typhimurium) and damaged mitochondria. The autophagy receptor protein NDP52 can specifically recognize invading bacteria and encapsulate and degrade them through the xenophagy pathway, thereby preventing the bacteria from multiplying and spreading within the cell. This process not only helps to control infection, but also reduces bacterial damage to host cells.

[0008] By further studying these novel therapeutics and host defense mechanisms, the inventors aim to develop more effective and safer treatment strategies to combat Salmonella infections and their drug resistance. Summary of the Invention

[0009] The purpose of the present invention is to improve the host animal's resistance to Salmonella by expressing or overexpressing a dephosphorylated variant of serine at position 162 of the autophagy receptor protein NDP52, thereby accelerating the elimination of Salmonella and providing new ideas for the prevention and treatment of bacterial infectious diseases such as Salmonella.

[0010] The technical solution of the present invention is:

[0011] Provided is a protein, which is a dephosphorylated variant of NDP52 protein, wherein the serine at position 162 of NDP52 protein (codon is AGC) is mutated to alanine (codon is GCC), which is referred to in this application as: NDP52 S162A A protein encoding an amino acid sequence of 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 dephosphorylation of the protein.

[0012] Furthermore, the coding NDP52 is also provided. S162A The gene encoding the protein NDP52 S162A The nucleotide sequence of the protein gene is SEQ ID No. 2. This application also covers nucleotide sequences with a homology of more than 90% to SEQ ID NO: 2.

[0013] Furthermore, a biomaterial is provided, which is a recombinant vector, transgenic cell line or recombinant virus containing a gene encoding a dephosphorylated variant of the NDP52 protein, or a substance that can achieve the dephosphorylated expression of the gene, that is, a substance that can dephosphorylate the NDP52 protein.

[0014] Furthermore, the substance capable of dephosphorylating the NDP52 protein is a substance capable of dephosphorylating serine at position 162 of the NDP52 protein.

[0015] The present application also provides an application, providing the following four applications in the preparation of drugs for regulating Salmonella infection:

[0016] (1) Protein: dephosphorylated variant of NDP52 protein;

[0017] (2) Gene: encoding NDP52 S162A genes;

[0018] (3) a recombinant vector, transgenic cell line, or recombinant virus containing a gene encoding a dephosphorylated variant of NDP52 protein;

[0019] (4) Substances that achieve dephosphorylation of NDP52 protein.

[0020] Specifically, the control of Salmonella infection in an organism is achieved by expressing or overexpressing at least one of the above four items.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] In the specific implementation, C57BL / 6J mice were used as experimental materials, and AAV virus was injected into the tail vein to specifically express NDP52 in the mouse liver. WT 、NDP52 S162A (162-site dephosphorylated variant) protein, NDP52 S162D (162-site phosphorylated variant) protein, a bacterial infection model was constructed, and plate counting, fluorescence imaging and HE staining showed that after overexpression of NDP52 wild-type protein, the number of bacteria in the mouse liver tissue was significantly reduced, and the liver necrosis was significantly reduced; overexpression of NDP52 dephosphorylated variant NDP52 S162A The number of bacteria in the liver tissue of mice was further reduced, and the liver necrosis was further reduced. S162D The number of bacteria in the liver tissue of mice increased significantly, and the part of liver necrosis increased significantly, indicating that the dephosphorylation of NDP52 protein and its 162-site serine promoted the clearance of bacteria in the mouse liver and had a therapeutic effect on bacterial-induced liver necrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the AAV9-TBG-NDP52-GFP plasmid map;

[0024] Figure 2 AAV9-TBG-NDP52 S162A -GFP plasmid map;

[0025] Figure 3 AAV9-TBG-NDP52 S162D -GFP plasmid map;

[0026] Figure 4 Flowchart for the implementation of the embodiment;

[0027] Figure 5 Fluorescence imaging of liver sections was used to verify the expression of NDP52;

[0028] Figure 6 This is the result of observing Salmonella infection in the liver by fluorescence imaging;

[0029] Figure 7 The results of colony counts in mouse liver are shown;

[0030] Figure 8 The results of HE staining of mouse liver. DETAILED DESCRIPTION

[0031] In this application:

[0032] The gene sequence encoding NDP52 is: NCBI ID: 10241. The amino acid sequence of the NDP52 protein is from: UniProt website Q13137. The amino acid sequence of the NDP52 protein is shown in SEQ ID No. 3, which is:

[0033] MEETIKDPPTSAVLLDHCHFSQVIFNSVEKFYIPGGDVTCHYTFTQHFIPRRKDWIGIFRVGWKTTREYYTFMWVTLPIDLNNKSAKQQEVQFKAYYLPKDDEYYQFCYVD EDGVVRGASIPFQFRPENEEDILVVTTQGEVEEIEQHNKELCKENQELKDSCISLQKQNSDMQAELQKKQEELETLQSINKKLELKVKEQKDYWETELLQLKEQNQKMSSEN EKMGIRVDQLQAQLSTQEKEMEKLVQGDQDKTEQLEQLKKENDHLFLSLTEQRKDQKKLEQTVEQMKQNETTAMKKQQELMDENFDLSKRLSENEIICNALQRQKERLEGEN DLLKRENSRLLSYMGLDFNSLPYQVPTSDEGGARQNPGLAYGNPYSGIQESSSPSPLSIKKCPICKADDICDHTLEQQQMQPLCFNCPICDKIFPATEKQIFEDHVFCHSL.

[0034] The wild-type WT gene sequence of NDP52 is from NCBI website NM_005831.4. The wild-type WT nucleotide sequence of NDP52 is shown in SEQ ID No. 4. SEQ ID No. 4 is:

[0035]

[0036] NDP52 S162A The amino acid sequence of the protein is SEQ ID No. 1, which is:

[0037] MEETIKDPPTSAVLLDHCHFSQVIFNSVEKFYIPGGDVTCHYTFTQHFIPRRKDWIGIFRVGWKTTREYYTFMWVTLPIDLNNKSAKQQEVQFKAYYLPKDDEYYQFCYVD EDGVVRGASIPFQFRPENEEDILVVTTQGEVEEIEQHNKELCKENQELKDACISLQKQNSDMQAELQKKQEELETLQSINKKLELKVKEQKDYWETELLQLKEQNQKMSSEN EKMGIRVDQLQAQLSTQEKEMEKLVQGDQDKTEQLEQLKKENDHLFLSLTEQRKDQKKLEQTVEQMKQNETTAMKKQQELMDENFDLSKRLSENEIICNALQRQKERLEGEN DLLKRENSRLLSYMGLDFNSLPYQVPTSDEGGARQNPGLAYGNPYSGIQESSSPSPLSIKKCPICKADDICDHTLEQQQMQPLCFNCPICDKIFPATEKQIFEDHVFCHSL.

[0038] Encoding NDP52 S162A The nucleotide sequence of the protein gene is SEQ ID No. 2, which is:

[0039]

[0040] NDP52 S162D The amino acid sequence of the protein is SEQ ID No. 5, which is:

[0041] MEETIKDPPTSAVLLDHCHFSQVIFNSVEKFYIPGGDVTCHYTFTQHFIPRRKDWIGIFRVGWKTTREYYTFMWVTLPIDLNNKSAKQQEVQFKAYYLPKDDEYYQFCYVD EDGVVRGASIPFQFRPENEEDILVVTTQGEVEEIEQHNKELCKENQELKDDCISLQKQNSDMQAELQKKQEELETLQSINKKLELKVKEQKDYWETELLQLKEQNQKMSSEN EKMGIRVDQLQAQLSTQEKEMEKLVQGDQDKTEQLEQLKKENDHLFLSLTEQRKDQKKLEQTVEQMKQNETTAMKKQQELMDENFDLSKRLSENEIICNALQRQKERLEGEN DLLKRENSRLLSYMGLDFNSLPYQVPTSDEGGARQNPGLAYGNPYSGIQESSSPSPLSIKKCPICKADDICDHTLEQQQMQPLCFNCPICDKIFPATEKQIFEDHVFCHSL.

[0042] Encoding NDP52 S162D The nucleotide sequence of the protein gene is SEQ ID No.6, which is:

[0043]

[0044] Example 1 NDP52 WT 、NDP52 S162A 、NDP52 S162D Plasmid construction

[0045] The specific experimental steps are as follows:

[0046] (1) Polymerase chain reaction (PCR)

[0047] AAV9-TBG-GFP was used as a vector template, pcDNA5-NDP52-Flag, pcDNA5-NDP52 S162A -Flag, pcDNA5-NDP52 S162D -Flag was used as the fragment template in a 50 μL reaction volume. Add 25 μL of PureSTARMax enzyme, 1 μL of template plasmid, 0.2 μM upstream primer, 0.2 μM downstream primer, and ddH2O to a PCR tube. Gently flick the tube to mix thoroughly. Centrifuge and place in a PCR instrument. Perform a pre-denaturation at 95°C for 5 minutes. Denature at 95°C for 15 seconds, anneal at 55°C for 20 seconds, and extend at 72°C at 0.2 kb / s for 30 cycles. Extend at 72°C for 5 minutes, then store at 4°C.

[0048] The primer series in this reaction system are shown in Table 1:

[0049] Table 1 Primer sequences:

[0050] Primers Nucleotide sequence Fragment upstream primer (SEQ ID No.7) ATGGAGGAGACCATCAAAGATCCCCCCAC Fragment downstream primer (SEQ ID No.8) CGGTCCAGGATTCTCTTCGACATCTCCGGCTTG Vector upstream primer (SEQ ID No.9) GAGAATCCTGGACCGATGGTGAGCAAGGGCGAGGAGC Vector downstream primer (SEQ ID No.10) GATGTCTCCTCCATGGCGGCCGCCTGGACACC

[0051] (2) Agarose gel electrophoresis

[0052] Prepare 50× TAE electrophoresis buffer and dilute it to 1× TAE. Place the base plate onto the gel plate, insert a suitable comb, weigh 0.6 g of agarose, add 60 mL of 1× TAE, and heat in a microwave until completely dissolved. Add YeaGreen Nucleic Acid Gel Stain and shake to mix thoroughly. Pour the solution into the gel plate and allow the gel to solidify for 30 minutes. Prepare for sample loading by adding a small amount of 6× DNA loading buffer to the PCR product. Add 10 μL of marker based on fragment size (15K for large fragments, 2K for small fragments). Turn on the electrophoresis instrument, set a constant voltage of 125 V, and run for 30 minutes. After electrophoresis, remove the gel block from the mold and place it under a UV analyzer. Use the marker to check for bright bands. Use a clean blade to cut the desired band and place it into a 2 mL EP tube for gel recovery.

[0053] (3) Glue recovery and infusion connection

[0054] Add 300 μL of binding buffer II to the excised agarose gel and place in a 55°C metal bath for 10 minutes to completely dissolve the gel. After dissolution, remove from the metal bath, cool, and prepare for column loading. Place the EZ Spin Column in a 2 mL collection tube, apply the dissolved gel to the column, and let it sit for 1 minute to improve recovery. Centrifuge at 12,000 rpm for 1 minute. Re-apply the liquid from the collection tube to the same column and centrifuge again at 12,000 rpm for 1 minute. Discard the waste liquid. Remove 500 μL of wash buffer containing anhydrous ethanol and centrifuge again at 12,000 rpm for 3 minutes to ensure removal of any residual wash buffer. Remove the cap from the column and place it in a sterile 1.5 mL EP tube. Place in a 50-60°C metal bath for 10 minutes until the alcohol evaporates. Add 30 μL of preheated ddH2O and centrifuge at 12,000 rpm for 3 minutes to elute the DNA. Add the gel recovery product, basic mix, fragments and vector into a PCR tube and incubate in a PCR machine at 50°C for 20 min.

[0055] (4) Plasmid transformation and bacterial culture

[0056] Add an appropriate amount of competent cells to the ligation product and place on ice for 30 minutes. Heat shock in a 42°C water bath for 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. Place the plate upside down in a 37°C constant temperature incubator and culture overnight. The next day, pick a single clone from the plate, add it to 500μL of culture 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 culture medium with the corresponding resistance and culture it on a 37°C shaker at 220rpm for about 15 hours.

[0057] (5) Endotoxin-free plasmid extraction

[0058] Transfer the overnight culture to a centrifuge tube and centrifuge at 4000 rpm for 15 minutes at room temperature to collect the bacteria. Aspirate the supernatant. Add 8 mL of Solution P1 (to which RNase A has been added) to the centrifuge tube containing the bacterial pellet. Vortex and thoroughly resuspend the bacterial cell pellet. Add 8 mL of Solution P2 to the centrifuge tube and gently invert the tube 6-8 times to fully lyse the cells. Let it stand at room temperature for 5 minutes. Add 8 mL of Solution P4 to the centrifuge tube and gently invert the tube 6-8 times to thoroughly mix until a white, dispersed, flocculent precipitate appears. Let it stand at room temperature for approximately 10 minutes. Centrifuge at 8000 rpm for 5-10 minutes to allow the white precipitate to settle to the bottom of the tube. Pour the entire solution into a filter CS1 and filter by pushing the handle. Collect the filtrate in a clean 50 mL tube. Add 0.3 times the volume of isopropanol to the filtrate, mix thoroughly by inversion, and transfer to an adsorption column CP6. Centrifuge at 8000 rpm for 2 minutes. Discard the waste liquid and repeat the column transfer until all the liquid has passed through the column. Add 10 mL of rinse solution PW to the adsorption column CP6, centrifuge at 8000 rpm for 2 minutes, discard the waste liquid, and repeat once. Add 3 mL of anhydrous ethanol to the adsorption column CP6, centrifuge at 8000 rpm for 2 minutes, and discard the waste liquid. Return the adsorption column CP6 to the collection tube and centrifuge at 8000 rpm for 5 minutes. Place the adsorption column CP6 in a clean 50 mL collection tube, add 1 mL of sterile ddH2O to the middle part of the adsorption membrane, let it stand at room temperature for 5 minutes, and centrifuge at 8000 rpm for 2 minutes. Transfer all the eluate in the 50 mL centrifuge tube to a clean 1.5 mL centrifuge tube and store at -20°C.

[0059] Finally, wild-type AAV-TBG-NDP52 was obtained WT -GFP, dephosphorylated variant AAV-TBG-NDP52 S162A -GFP, phosphorylated variant AAV-TBG-NDP52 S162D -GFP three plasmids encoding NDP52 WT The nucleotide sequence of the protein gene is shown in SEQ ID No. 4, and the map is shown in Figure 1 Shown; encoding NDP52 S162A The nucleotide sequence of the protein gene is shown in SEQ ID No. 2, and the map is shown in Figure 2 Shown; encoding NDP52 S162D The nucleotide sequence of the protein gene is shown in SE Q ID No.6, and the map is shown in Figure 3 shown.

[0060] Example 2 Construction of AAV adeno-associated virus

[0061] Obtain control AAV-TBG-GFP, wild-type AAV-TBG-NDP52 WT-GFP, dephosphorylated variant AAV-TBG-NDP52 S162A -GFP, phosphorylated variant AAV-TBG-NDP52 S162D -GFP four adeno-associated viruses (customized by Hangzhou Guannan Biotechnology Co., Ltd.), with virus titers of 5.73×10 13 , 3.57×10 13 , 1.31×10 13 , 6.69×10 13 μg / mL.

[0062] Example 3 NDP52 in mouse liver WT 、NDP52 S162A 、NDP52 S162D Expression

[0063] 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 Vector group (injection of empty virus), NDP52 WT Group (injected with NDP52 wild-type virus), NDP52 S162A Group (injected with NDP52 dephosphorylated variant virus), NDP52 S162D Group (injected with NDP52 phosphorylation variant virus), 10 mice in each group.

[0064] The livers of the four groups of mice injected with adeno-associated virus were removed, frozen sections were made, and imaging was performed after DAPI staining. The expression of adeno-associated virus in the mouse liver was observed by observing the green fluorescence excited at 488 nm. Figure 5 As shown (scale bar 50 μm): Vector group is the empty vector group, NDP52 WT For the group injected with wild-type NDP52, NDP52 S162A NDP52 protein 162 serine site dephosphorylation mutant group was injected. S162D The NDP52 protein 162 serine site phosphorylation mutant group was injected, and the results showed that the AAV virus was successfully expressed in the liver.

[0065] Example 4: Fluorescence Imaging Observation of Salmonella Infection in Mouse Liver

[0066] After verifying that the wild-type, dephosphorylated, and phosphorylated NDP52 proteins were successfully expressed in the mouse liver, the mice were intraperitoneally infected with Salmonella typhimurium at 1×10 6 CFU / mouse, 8 mice were infected in each group, and mice were killed and dissected after 2 days, and the liver was removed for histopathological examination. The experimental process is as follows Figure 4shown.

[0067] Vector group, NDP52 WT group, NDP52 S162A group, NDP52 S162D The livers of the four groups of mice infected with bacteria were frozen and sectioned, and fluorescence imaging was performed after DAPI staining. Green GFP represents the expression of AAV virus, red RFP represents Salmonella, and blue represents the cell nucleus. The fluorescence imaging results were used to observe and analyze the Salmonella infection in the liver. Figure 6 shown.

[0068] In the fluorescence imaging diagram on the left, green GFP represents the expression of AAV virus, red RFP represents Salmonella, Merge is the merged image of the three channels of green, red and blue (DAPI staining), image scale: 50μm; on the right is the statistical result diagram, the vertical axis represents the number of red Salmonella in each microscopic field of view, a total of 30 fields of view of 3 mice were counted, **P < 0.001.

[0069] Example 5 Colony Count Experiment to Observe Salmonella Infection in Mouse Liver

[0070] Pathological analysis of mouse liver after bacterial infection: Figure 4 The mouse liver was crushed and centrifuged at 250×g and 4°C for 5 min. The supernatant was diluted to 10 3 or 10 4 times, and then take 50 μL to plate and count. After incubation at 37℃ overnight, observe the growth of colonies. Figure 7 shown.

[0071] The left side shows the representative plate results, and the right side shows the statistical results (n=6). As shown in the results: NDP52 is overexpressed in mouse liver WT After the overexpression of dephosphorylated variant NDP52, the bacterial count in the liver decreased significantly compared with the control group (P < 0.05). S162A After NDP52 was overexpressed, the number of liver bacteria decreased significantly compared with the wild-type group (P < 0.05). S162D After that, compared with the expression of NDP52 S162A In the control group, the number of bacteria in the liver increased significantly (P < 0.01), indicating that the dephosphorylation of NDP52 protein and its 162 site can significantly promote the clearance of bacteria in the liver and have a certain therapeutic effect on Salmonella infection.

[0072] Example 6 Observation of liver necrosis in mice by hematoxylin-eosin staining

[0073] Take the injection virus Vector and NDP52 respectively WT 、NDP52S162A 、NDP52 S162D The livers of the four groups of mice that were not infected with bacteria and those that were infected with bacteria were embedded in paraffin and sectioned. They were then stained with hematoxylin and eosin (HE) to observe the necrosis of the mouse livers. The experimental results are as follows: Figure 8 shown.

[0074] Figure 8 S.Tm in the middle is S.Typhimurium (Salmonella typhimurium), the left side shows the HE staining results and magnified images of the livers of uninfected and infected mice, image scale: 200 μm; the right side shows the statistical results of the number of liver necrosis (n=10).

[0075] like Figure 8 The results showed that the liver staining of the mice in the non-Salmonella infection group was normal; in the Salmonella infection group, the liver necrosis area was more in the control group, and the NDP52 overexpression WT The liver necrosis area was relatively reduced in the group overexpressing the dephosphorylated variant NDP52 S162A The necrotic area in the liver was further reduced in the group overexpressing the phosphorylated variant NDP52. S162D The liver necrosis area in group NDP52 was higher than that in group S162A The expression of NDP52 in the liver was significantly increased in the control group, indicating that the dephosphorylation of NDP52 protein and its 162 site can protect the liver infected by Salmonella and play a certain therapeutic role in Salmonella infection.

Claims

1. A protein, characterized in that: is (a) or (b) as follows: (a) NDP52 S162A The protein is a dephosphorylated variant of the NDP52 protein, wherein the serine at position 162 of the NDP52 protein is mutated to alanine, and the amino acid sequence is 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 dephosphorylation of the protein.

2. A gene encoding the protein according to claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence is SEQ ID No. 2, or a nucleotide sequence with a homology of more than 90% with SEQ ID NO:

2.

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 dephosphorylate the NDP52 protein.

5. Use of at least one of the following in the preparation of a drug for controlling Salmonella infection: (1) The protein according to claim 1; (2) The gene according to claim 2 or 3; (3) A recombinant vector, a transgenic cell line or a recombinant virus containing the gene of claim 2 or 3; (4) A substance that achieves dephosphorylation of NDP52 protein.

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