Application of RNF213 carboxyl terminal E3 structural domain or coding gene thereof, or biological material containing coding gene thereof in product preparation

Abstract

The invention relates to the technical field of gene engineering, in particular to application of an RNF213 carboxyl terminal E3 structural domain or a coding gene thereof or a biological material containing the coding gene thereof in product preparation. The application of the RNF213 E3 structural domain in regulating the innate immune pathway is disclosed for the first time, and a powerful scientific basis and a tool are provided for prevention, control and treatment of bacteria such as salmonella and the like. The constructed vector containing the RNF213 E3 structural domain can significantly improve the expression level of immune factors, promote the activity of an NF-kappa B pathway and reduce the cell bacterium loading amount, provides a thought for scientific research and industrial application related to prevention and control of gram-negative bacteria such as salmonella, and lays a scientific foundation.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to the application of the carboxyl-terminal E3 domain of RNF213, or its encoding gene, or a biological material containing its encoding gene in the preparation of a product. Background Art

[0002] Salmonellosis is a zoonotic disease caused by Salmonella infection and is one of the main bacterial diseases in the poultry industry worldwide. Salmonella belongs to the family Enterobacteriaceae, the genus Salmonella, and is a Gram-negative bacterium. The bacterial cells are oval or rod-shaped, without a capsule or spore, scattered singly or arranged in pairs, with a cell size of (0.5-1) μm × (1-3) μm, and most of them have flagella.

[0003] Salmonella pullorum, Salmonella gallinarum, Salmonella enteritidis, Salmonella typhimurium, etc. can all cause diseases in chicken flocks, reducing the production performance of poultry. Moreover, this disease occurs throughout the year, generally showing sporadic or endemic distribution, and can infect poultry of different species and ages. Salmonella can be horizontally transmitted through feed, water, litter, etc. contaminated by feces, and can also be vertically transmitted through infected ovaries, oviducts, etc., seriously endangering the development of the poultry breeding industry.

[0004] At present, due to the unreasonable use of antibacterial drugs, the drug resistance of Salmonella is constantly developing, and it has become resistant to a variety of antibiotics. Preventive measures, vaccination, and the use of antibiotics are not sufficient to eradicate Salmonellosis in poultry. Therefore, selecting more resistant chickens is considered an alternative solution to reduce the occurrence of the disease.

[0005] Therefore, exploring resistance genes that can effectively regulate the innate immune activity of poultry and regulate the antibacterial response has become a technical problem urgently to be solved in this field. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides the application of the carboxyl-terminal E3 domain of RNF213, or its encoding gene, or a biological material containing its encoding gene in the preparation of a product; the product is used for at least one of the following aspects: (1) Regulating the innate immune activity of poultry; (2) Regulating the antibacterial response; (3) Preventing and / or treating bacterial infections.

[0007] Preferably, the amino acid sequence of the carboxyl-terminal E3 domain of RNF213 is as shown in SEQ ID No.1.

[0008] RNF213 is the largest E3 ubiquitin ligase in the human proteome. It utilizes an as-yet-undisclosed E3 scaffold to perform ubiquitin ligase function in a RING-independent manner. The host resists bacterial infection by producing a bacterial ubiquitin coat through ubiquitination of lipopolysaccharide to restrict bacterial proliferation. The RNF213 protein contains an N-terminal stem, a dynein-like core with six ATPase units, and a multi-domain E3 module. The present invention discovers that only the E3 domain can also regulate avian innate immune activity and regulate the antibacterial response.

[0009] Preferably, the biomaterial is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector or engineered bacterium.

[0010] Furthermore, the present invention also provides the application of a reagent for regulating the expression level or activity of the carboxyl-terminal E3 domain of RNF213 in the preparation of a product; the product is used for at least one of the following aspects: (1) Regulating avian innate immune activity; (2) Regulating the antibacterial response; (3) Preventing and / or treating bacterial infections.

[0011] Preferably, the product is a drug, feed or feed additive.

[0012] Preferably, the reagent is an agonist, promoter, antagonist or inhibitor.

[0013] Preferably, the reagent includes but is not limited to small molecule activators, oligonucleotides, antibodies, polypeptides or fusion proteins.

[0014] By using an agonist, promoter, antagonist or inhibitor targeting the carboxyl-terminal E3 domain of RNF213, the expression level or activity of the carboxyl-terminal E3 domain of RNF213 can be indirectly regulated, thereby achieving the above applications.

[0015] Preferably, the regulation of avian innate immune activity is to regulate the avian's perception of Gram-negative bacterial infection and initiate an immune response.

[0016] Preferably, by increasing the expression level or activity of the carboxyl-terminal E3 domain of RNF213, the avian innate immune activity is enhanced.

[0017] Preferably, the enhancement of avian innate immune activity is manifested as an increase in the level of cellular immune factors.

[0018] Preferably, the cellular immune factors include IL-1β or IL-8.

[0019] Preferably, by increasing the expression level or activity of the carboxyl-terminal E3 domain of RNF213, the activity of NF-κB is increased, thereby increasing the level of cellular immune factors and enhancing the avian innate immune activity.

[0020] Preferably, by increasing the expression level or activity of the carboxyl-terminal E3 domain of RNF213, Gram-negative bacteria are inhibited or killed.

[0021] Preferably, the Gram-negative bacteria are Salmonella, more preferably Salmonella typhi.

[0022] Preferably, by increasing the expression level or activity of the carboxyl-terminal E3 domain of RNF213, the resistance of the body to Gram-negative bacteria is enhanced.

[0023] Preferably, the enhancement of the body's resistance to Gram-negative bacteria is manifested as an increase in the resistance of monocytes and macrophages to Gram-negative bacteria.

[0024] Preferably, the poultry are chickens, ducks, geese, etc.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: By overexpressing the carboxyl-terminal E3 domain of RNF213 in the present invention, it is found that the resistance of monocytes and macrophages to Salmonella can be enhanced, the activity of NF-κB can be increased, and further the level of cellular immune factors can be increased, significantly enhancing the innate immune activity of poultry. The present invention provides a strong scientific basis and tool for the prevention, control and treatment of Gram-negative bacteria such as Salmonella, provides a scientific means for scientific research and industrial application related to the prevention and control of Gram-negative bacteria, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the result of Western blot of the protein successfully expressed by the vector containing the RNF213 E3 domain.

[0027] Figure 2 is the result diagram of the influence of overexpressing the RNF213 E3 domain on cellular immune factors detected by fluorescence quantitative PCR; among them, RNF213 is the experimental group, FLAG is the control group, and * indicates P <0.05.

[0028] Figure 3 is the result of the anti-infection ability of chicken monocytes and macrophages to Salmonella after overexpressing the RNF213 E3 domain; among them, RNF213 is the experimental group, and FLAG is the control group.

[0029] Figure 4 is the result of detecting the fluorescence intensity of the NF-κB reporter vector by the dual-luciferase reporter assay; among them, RNF213 is the experimental group, FLAG is the control group, and * indicates P <0.05. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts belong to the scope of protection of the present invention.

[0031] Unless otherwise specified, the embodiments are all carried out under conventional experimental conditions, such as those in the Molecular Cloning experimental manual by Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or under the conditions recommended in the manufacturer's instructions. Reagents or instruments without indicating the manufacturer can all be obtained as conventional products through regular channels.

[0032] Example 1 In this example, the carboxyl-terminal E3 domain of human and murine RNF213 was referred to, the E3 fragment of the chicken RNF213 gene was determined according to homology, and a vector was constructed. The specific steps are as follows: 1. Determine the E3 domain of chicken RNF213 The amino acid region corresponding to the murine RNF213 E3 domain fragment is 3589 - 4926. The amino acid sequences of murine (accession number: NM_001040005.3), human (accession number: NM_001256071.3), and chicken (accession number: NM_001389479.2) RNF213 were downloaded from NCBI and aligned using SnapGene software. The amino acid sequence of murine RNF213 3589 - 4926 corresponds to the human 3643 - 4983 and chicken 3643 - 4980 amino acid sequences. According to the amino acid sequence of the chicken E3 domain, the gene was synthesized by Beijing Tsingke Biotechnology Co., Ltd. and ligated into the pcDNA3.1 - 3×Flag C vector. The amino acid sequence of the RNF213 E3 domain is shown in SEQ ID No.1, and its protein molecular weight is 152.73 kDa.

[0033] 2. Culture of chicken monocyte - macrophage cell line HD11 (1) Cell resuscitation: Take out the cryopreserved cells from liquid nitrogen, immediately place them in a 37°C water bath and shake. After the cell solution is completely dissolved, add the cell solution to 5 mL of complete medium and mix well. Centrifuge at 1200 rpm for 5 minutes, discard the supernatant, resuspend and mix the cell pellet with 1 mL of complete medium, add it to a T25 cell culture flask, shake well, and place it in a 37°C, 5% CO2 incubator for culture; (2)Cell medium replacement: Observe the cell density and morphology under the microscope. Generally, replace the medium every 24 hours. Pre-warm the cell culture medium, pour out the old medium, and add the new medium. (3)Cell passage: Observe under the microscope. When the cell confluence reaches about 90%, passage the cells. Transfer the old medium to a 15 mL centrifuge tube. Add 2 mL of 0.25% trypsin to the T25 flask and incubate in a 37°C incubator for 1 minute. Observe the cells under the microscope until the cells detach from the wall and form a single cell suspension. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, resuspend the cell pellet thoroughly with 1 mL of complete medium, add it to the T25 flask containing 5 mL of medium, mix well, and place it in a 37°C, 5% CO2 incubator for culture.

[0034] 3. Plasmid transfection Cell transfection is carried out according to the instructions of the TransIT-X2® Dynamic Delivery System reagent (Mirus, MIR6000): (1)Transfer the cells to a 6-well plate for culture. When the cell density ≥ 80%, perform transfection. (2)Equilibrate the TransIT-X2 to room temperature and gently vortex it before use. (3)Add 250 μL of Opti-MEM® medium to an empty tube. (4)Add 2.5 μg of plasmid and mix gently and thoroughly. (5)Add 7.5 μL of TransIT-X2, mix gently and thoroughly, and let it stand at room temperature for 15 - 30 minutes. (6)Add the prepared transfection reagent evenly to the cultured cells and continue to culture in the incubator for 24 - 72 hours.

[0035] 4. Protein extraction (1)Collect the original medium of the transfected cells into a 15 mL centrifuge tube. (2)Add 1 mL of trypsin to the cells and incubate in a 37°C incubator for 1 minute. Then observe the cell digestion under the microscope. Transfer the cells to a 15 mL centrifuge tube, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant. (3)Resuspend the cell pellet with 2 mL of phosphate buffered saline and centrifuge at 1200 rpm for 5 minutes. Discard the supernatant. (4)Add pre-cooled IP lysis buffer and 10× protease inhibitor. After resuspending the cell pellet, transfer it from the 15 mL centrifuge tube to a new 1.5 mL centrifuge tube (which is conducive to high-speed low-temperature centrifugation). (5)Place the cell pellet on ice for 5 minutes and centrifuge at 4°C, 13000g for 10 minutes. Take the supernatant.

[0036] 5. Western Blot (1)Protein denaturation: Take a certain amount of protein supernatant, add 6×protein buffer, centrifuge, vortex, and centrifuge again, and heat-denature at 95°C in a metal bath for 10 min; (2)Loading: Place the PAGE gel in the electrophoresis tank, pour in the electrophoresis buffer, pull out the comb, slowly add a certain amount of protein denaturation solution into the sample wells, and reserve one well to add 6 μL of protein marker; (3)Electrophoresis: At a voltage of 80 V, after the protein sample is compressed into a line, increase the voltage to 120 V and perform electrophoresis for 40 min; (4)Transfer: Transfer the protein to a PVDF membrane, and the transfer conditions are a current of 350 mA for 70 min; (5)Blocking: Transfer the PVDF membrane to a blocking box, immediately add milk powder, place it on a transfer shaker, and block for 61 h; (6)Primary antibody incubation: Add DDDDK-Tag Rabbit mAb (AE063, ABclonal) at a ratio of 1:5000 and incubate for 1 h. After completion, pour out the blocking solution, add TBST, place it on a shaker for 5 min, replace TBST, repeat washing three times, 5 min each time, and finally add 5 mL of blocking solution; (7)Secondary antibody incubation: Add HRP Goat Anti-Rabbit IgG (A5014, ABclonal) at a ratio of 1:2000 and incubate for 1 h. Wash three times according to the above primary antibody washing method; (8)Development: Evenly drop the developing solution onto the PVDF membrane, and place the PVDF membrane in a pre-cooled chemiluminescence instrument for development and observation.

[0037] 6. Results As Figure 1 shown, the results showed that the vector containing the RNF213 E3 domain was successfully expressed in HD11.

[0038] Example 2 In this example, only the RNF213 E3 domain was overexpressed. After stimulation with Salmonella typhimurium (ST), the levels of cellular immune factors increased. The specific steps are as follows: 1. Experimental grouping and plating The experiment was divided into four groups: FLAG empty vector (ST stimulation for 0 h), FLAG empty vector (ST stimulation for 12 h), RNF213 (ST stimulation for 0 h), RNF213 (ST stimulation for 12 h), and three biological replicates were set for each group; According to the plasmid transfection method of Example 1, FLAG empty vector and constructed RNF213 E3 fragment vector with FLAG tag were transfected respectively. 24 hours after transfection, Salmonella typhimurium with MOI=100 and PBS were added respectively. 12 hours after infection, cells were collected to extract RNA.

[0039] 2. Fluorescence quantitative PCR experiment RNA extraction was performed according to the instructions of the total RNA extraction kit (Tiangen, DP419), reverse transcription was performed according to the FastQuantcDNA first-strand synthesis kit (Tiangen, KR106), and the level of cellular immune factors was detected by fluorescent quantitative PCR. The qPCR reaction solution used the One Step SYBR® PrimeScript™ RT-PCR Kit II (Takara) to configure the reaction system, and the primer sequences and reaction system are shown in Tables 1 and 2 respectively: Table 1 Primer sequences for fluorescence quantitative PCR

[0040] Table 2 Fluorescence quantitative PCR reaction system

[0041] The expression levels of IL-1β and IL-8 were analyzed by ABI Q7 Flex system (Applied Biosystems, USA), with β-Actin as the internal reference gene. -ΔΔct Method calculation.

[0042] 3. Results like Figure 2 As shown, fluorescence quantitative PCR detected that after overexpression of RNF213 E3 domain, the expression levels of cellular immune factors IL-1β and IL-8 were significantly increased after stimulation with Salmonella typhimurium (ST), indicating that the expression of RNF213 E3 domain alone can increase the level of cellular immune factors.

[0043] Example 3 In this example, only the RNF213 E3 domain was overexpressed, and the cell bacterial load decreased after stimulation with Salmonella typhimurium (ST). The specific steps are as follows: 1. Experimental grouping and layout The experiment was divided into two groups, control group (FLAG empty vector) and experimental group (RNF213), and three biological replicates were set in each group; According to the plasmid transfection method of Example 1, 24 hours after transfection of FLAG empty vector and RNF213 plasmid, Salmonella typhimurium with MOI=100 was added respectively.

[0044] 2. Cell bacterial load detection After 4 h of Salmonella typhimurium infection of cells, cell pellets were collected, resuspended in 1 mL of PBS, placed at -80 °C, and repeatedly frozen and thawed 3 times. Then, they were serially diluted. 100 μL was taken from each dilution and spread on an agarose plate, which was then placed in an inverted position in a 37 °C water bath thermostatic incubator overnight. Three biological replicates were set up. Colony counts were performed after 12 h.

[0045] 3. Results As Figure 3 shown, after 4 h of Salmonella typhimurium infection, overexpression of the RNF213 E3 domain reduced the bacterial load in cells, indicating that the RNF213 E3 domain can promote cells to kill bacteria.

[0046] Example 4 In this example, only the RNF213 E3 domain was overexpressed. After stimulation with lipopolysaccharide (LPS), the activity of the NF-κB transcription factor increased significantly. The specific steps are as follows: 1. Experimental grouping and plating The experiment was divided into five groups: a control group (empty FLAG), an experimental group (RNF213), a Control group (control vector for the dual-luciferase experiment), a Promoter group (promoter vector), and a Basic group (Basic vector). Three biological replicates were set up for each group. An appropriate amount of HD11 cell line was inoculated into a 24-well plate according to the experimental design and cultured in a 37 °C, 5% CO2 incubator.

[0047] 2. Transfection of the HD11 cell line and LPS treatment When the cell seeding density reached about 80%, plasmid transfection was performed according to the method of Example 1. After 24 h of transfection, LPS with a final concentration of 100 ng / mL was added to the culture dish to treat the cells for 4 h. Immediately after the treatment, luciferase chemiluminescence was detected.

[0048] 3. Dual-luciferase reporter gene detection experiment The dual-luciferase reporter gene detection was performed according to the instructions of the Dual-Luciferase® Reporter Assay System (Promega, E1910): (1) Prepare a lysis buffer to lyse the cells: Prepare the lysis buffer by mixing 1 volume of 5× Passive Lysis Buffer and 4 volumes of distilled water, and mix well. Aspirate the culture medium, add 100 μL of cell lysis buffer to the cell culture plate, lyse the cells thoroughly for 15 min, and aspirate the supernatant for subsequent determination; (2)Prepare Luciferase Assay Reagent II: Calculate the required detection buffer according to a volume of 100 µL per sample, and resuspend the lyophilized Luciferase Assay Substrate (provided in the kit) with 10 mL of Luciferase Assay Buffer II provided in the kit to prepare the working solution of Luciferase Assay Reagent II (LARII); (3)Determination of firefly luciferase: Add 100 µL of cell lysate to the microplate, then add 100 µL of firefly luciferase detection reagent, pipette and mix well, place it in the microplate reader, and the measured chemiluminescence value is the firefly luciferase value; (4)Prepare Stop&Glo® reagent: Add 1 volume of 50X Stop&Glo® substrate to 50 volumes of Stop&Glo® buffer, mix well and set aside; (5)Determination of Renilla luciferase: After the above steps are completed, add 100 µL of Renilla luciferase detection buffer, pipette and mix well, and place it in the microplate reader to measure the Renilla luciferase value; (6)Calculation: The ratio of the firefly luciferase value to the Renilla luciferase value is the activity of the relevant reporter gene.

[0049] 4. Results As Figure 4 shown, after HD11 cells were treated with LPS, when only the E3 domain of RNF213 was overexpressed, the chemiluminescence intensity showed a significant increase in the activity of the transcription factor NF-κB, indicating that the E3 domain of RNF213 regulates the expression of downstream immune factors by affecting the activity of the transcription factor NF-κB.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of the carboxyl-terminal E3 domain of RNF213, or its coding gene, or a biological material containing its coding gene in the preparation of a product; the product is used for at least one of the following aspects: (1) Regulating the innate immune activity of poultry; (2) Regulating the antibacterial response; (3) Preventing and / or treating bacterial infections.

2. Use of a reagent for regulating the expression level or activity of the carboxyl-terminal E3 domain of RNF213 in the preparation of a product; the product is used for at least one of the following aspects: (1) Regulating the innate immune activity of poultry; (2) Regulating the antibacterial response; (3) Preventing and / or treating bacterial infections.

3. The application according to claim 1 or 2, characterized in that, The product is a drug, feed or feed additive.

4. The application according to claim 2, characterized in that, The reagent is an agonist, promoter, antagonist or inhibitor.

5. The application according to claim 1 or 2, characterized in that, The regulation of the innate immune activity of poultry is to regulate the perception of Gram-negative bacterial infection by poultry and initiate an immune response.

6. The application according to claim 1 or 2, characterized in that, By increasing the expression level or activity of the carboxyl-terminal E3 domain of RNF213, the innate immune activity of poultry is enhanced.

7. The application according to claim 6, wherein By increasing the expression level or activity of the carboxyl-terminal E3 domain of RNF213, the activity of NF-κB is increased, and then the level of cellular immune factors is increased, enhancing the innate immune activity of poultry.

8. The application according to claim 1 or 2, characterized in that, By increasing the expression level or activity of the carboxyl-terminal E3 domain of RNF213, Gram-negative bacteria are inhibited or killed.

9. The application according to claim 1 or 2, characterized in that, By increasing the expression level or activity of the carboxyl-terminal E3 domain of RNF213, the resistance of the body to Gram-negative bacteria is increased.

10. The application according to claim 9, characterized in that, The increase in the resistance of the body to Gram-negative bacteria is manifested as an increase in the resistance of monocytes and macrophages to Gram-negative bacteria.

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