Development and application of an EGFP reporter system for identifying N4BP1 endoribonuclease activity

Abstract

The invention provides the development and application of an EGFP reporter system for identifying N4BP1 endoribonuclease activity, relates to the field of biomedical technology, and uses a modified EGFP fluorescent protein to identify the endoribonuclease activity of N4BP1. Through experiments, it is found that the core sequence of the target gene FosC degraded by N4BP1 is determined, and this sequence is connected to the C-terminus of the mRNA of EGFP to construct a new EGFP-mRNA sequence. The N4BP1 overexpression plasmid is co-expressed in HEK293T cells, and then the endoribonuclease activity of N4BP1 can be determined by comparing the intensity of the fluorescence signal. The modified EGFP, due to the connection of the target sequence recognized by N4BP1, the expression amount of EGFP will change according to the amount of N4BP1 enzyme activity. By detecting the intensity of EGFP, the amount and enzyme activity of N4BP1 protein can be determined. Therefore, the GFP reporter system of N4BP1 can be used to detect the enzyme activity of N4BP1. Since the N4BP1 gene plays a key role in immunity, inflammation and tumors. The cell can detect the endoribonuclease activity of N4BP1 in related disease cells.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, and in particular to the development and application of an EGFP reporter system for identifying N4BP1 endoribonuclease activity. Background Art

[0002] NEDD4-binding protein 1 (N4BP1) was originally identified as a substrate of the E3 ubiquitin ligase NEDD4. To date, N4BP1 homologous sequences have been identified in mammals, birds, amphibians, and fish. The human N4BP1 gene is located on chromosome 16, encodes 896 amino acids, and is expressed in many tissues and organs, with higher expression in lymphoid tissues, brain, and skin [2]. N4BP1 has no specific localization in cells and is scattered in the cytoplasm, nucleus, and nucleolus. Mice deficient in N4BP1 develop normally with no obvious morphological changes.

[0003] In addition to two RNA binding domains (KH domains), the N4BP1 protein also contains a NYN functional domain, which is similar to the PIN (RNase catalytic activity) domain of Regnase-1 and shares a common protein fold, suggesting an important role for N4BP1 in regulating mRNA metabolism. The first RNase activity of N4BP1 was proposed in inhibiting viral RNA replication, and it has been confirmed that N4BP1 can reduce HIV mRNA and protein expression levels. Further studies have found that the nuclease domain of N4BP1 directly degrades viral mRNA, rather than affecting viral transcription through long terminal repeat sequences (LTR). However, the specific sequence features that mediate N4BP1 degradation have not yet been determined.

[0004] Previous studies have not clarified the specificity of the target sequence of N4BP1 when it performs the endoribonuclease function, which limits researchers' understanding of the function of N4BP1. At the same time, seeking a technology to detect the endoribonuclease activity of N4BP1 can provide important support for better exploring the mechanism of action of N4BP1 in the body's tissues and organs. Summary of the invention

[0005] The purpose of the present invention is to provide a technology for detecting the endonuclease activity of N4BP1, so as to provide important support for better exploring the mechanism of action of N4BP1 in body tissues and organs.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for constructing a target for detecting N4BP1 endonuclease activity comprises the following steps:

[0008] S1: construct plasmid;

[0009] S2: Design the experiment:

[0010] Specifically, a psoriasis model was constructed in N4BP1-deficient and wild-type mice, and transcriptome sequencing and RT-PCR analysis of lesion skin were performed to obtain potential targets for N4BP1 in regulating psoriasis;

[0011] Then, by co-cloning the potential targets into an expression vector, RT-PCR and Western blot analysis were used to confirm that N4BP1 could effectively inhibit the potential targets;

[0012] Then, potential targets were judged to respond to fragments of N4BP1;

[0013] S3: Verify the results: Use a fragment of potential target responsive to N4BP1 to co-express with N4BP1. If N4BP1 inhibits the expression of the fragment, further shorten the target region and repeat steps S1 and S2 until the final target sequence is obtained;

[0014] If N4BP1 fails to repress expression of the fragment, a conclusion is reached.

[0015] Preferably, the S1 comprises the following steps:

[0016] Obtain the target sequence through the NCBI website and design cloning primers for PCR amplification;

[0017] Then the PCR product and the vector were double-digested, and then the target gene fragment was connected to the vector overnight, and finally sequenced and verified to ensure that the plasmid was successfully constructed.

[0018] The present application also provides a response RNA fragment of N4BP1, and the sequence of the RNA fragment is shown in SEQ ID NO.1.

[0019] The present application also provides the use of an EGFP reporter system as a detection target in the preparation of a product for detecting N4BP1 endonuclease activity. The sequence of the EGFP reporter system is shown in SEQ ID NO.2.

[0020] The present application also provides a method for identifying the endonuclease activity of N4BP1, by detecting the intensity of EGFP of a sequence such as SEQ ID NO.2, to determine the amount and enzyme activity of N4BP1 protein.

[0021] In summary, the reporter group constructed in this application encodes EGFP fluorescent protein, which is easy to detect; it is not expressed in receptor cells and tissues, and has no cytotoxicity. This reporter group can be widely used in the field of life sciences, and can be used to detect the biological activity of endogenous N4BP1 protein in different tissues and individuals (experimental animals), and can also be used to determine the endonuclease activity of artificially synthesized N4BP1 polypeptides, as well as the endonuclease activity of different mutants or domain-deficient truncations of N4BP1. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A schematic diagram of a process for constructing a detection target for detecting N4BP1 endonuclease activity in one embodiment of the present invention;

[0023] Figure 2 In one embodiment of the present invention, N4BP1 and h-FosC-CDS-3'UTR were overexpressed in HEK293 cells, and the mRNA and protein levels of N4BP1 and FosC were detected by RT-PCR and Western blot;

[0024] Figure 3 In one embodiment of the present invention, N4BP1 and h-FosC-CDS were overexpressed in HEK293 cells, and the mRNA and protein levels of N4BP1 and FosC were detected by RT-PCR and Western blot;

[0025] Figure 4 To construct a FosC CDS 3' end fragment deletion plasmid in one embodiment of the present invention, and to analyze the expression of FosC transcripts by RT-PCR;

[0026] Figure 5 In one embodiment of the present invention, a GFP expression plasmid carrying a FosC CDS nucleic acid fragment was constructed and co-expressed with N4BP1 to detect GFP expression;

[0027] Figure 6 The EGFP reporter gene constructed by the present invention is used to verify the N4BP1 endonuclease activity. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with specific embodiments.

[0029] This application provides a method for constructing a target for detecting N4BP1 endonuclease activity. Figure 1 , including the following steps:

[0030] S1: Construction of plasmid:

[0031] Specifically, in one embodiment, the plasmid construction comprises the following specific steps:

[0032] First, the target sequence was obtained from the NCBI website, and cloning primers were designed for PCR amplification;

[0033] Then the PCR product and the vector were double-digested, and then the target gene fragment was connected to the vector overnight, and finally sequenced and verified to ensure that the plasmid was successfully constructed.

[0034] S2: Designing the Experiment

[0035] In one embodiment, a psoriasis model is first constructed in N4BP1-deficient and wild-type mice, and potential targets for N4BP1 regulating psoriasis are obtained by transcriptome sequencing and RT-PCR analysis of lesion skin;

[0036] Then, by co-cloning the potential targets into an expression vector, RT-PCR and Western blot analysis were used to confirm that N4BP1 could effectively inhibit the potential targets.

[0037] Then, potential targets were judged to respond to fragments of N4BP1;

[0038] S3: Verification results:

[0039] Specifically, in one embodiment, a fragment of a potential target responsive N4BP1 is co-expressed with N4BP1, and if N4BP1 inhibits expression of the fragment, the target region is further shortened, and steps S1 and S2 are repeated until the final target sequence is obtained;

[0040] If N4BP1 fails to repress expression of the fragment, a conclusion is reached.

[0041] A method for identifying N4BP1 endonuclease activity determines the amount and enzyme activity of N4BP1 protein by detecting the intensity of EGFP.

[0042] The above contents are described below in conjunction with specific embodiments:

[0043] First, the present application constructed a psoriasis model in N4BP1-deficient and wild-type mice. Through transcriptome sequencing and RT-PCR analysis of the diseased skin, the results showed that AP-1 family members including FosC, FosB, JunB and chemokine CXCL1 are potential targets for N4BP1 to regulate psoriasis.

[0044] To further verify the inhibitory effect of N4BP1 on FosC, Figure 2As shown, the FosC coding sequence (CDS) and 3' untranslated region (3'UTR) were co-cloned into an expression vector, and RT-PCR and Western blot analysis confirmed that overexpression of N4BP1 could effectively inhibit the expression of FosC mRNA and protein.

[0045] Multiple studies have shown that 3'UTR is rich in sequence elements that regulate mRNA stability. Therefore, in this application, we next determined whether the important region of FosC response to N4BP1 occurs in the 3'UTR. Here, in this application, only FosC-CDS (1-1143) was overexpressed, and the results confirmed that the lack of 3'UTR did not affect the inhibition of FosC CDS by N4BP1.

[0046] The results are as follows Figure 3 As shown, it was preliminarily determined that the site where N4BP1 degrades FosC mRNA occurs in the CDS region.

[0047] In order to explore the specific location of FosC CDS recognition and degradation by N4BP1, a series of expression plasmids with 3' end fragment deletion of FosC CDS were constructed in this application. When the full-length FosC CDS was co-expressed with N4BP1, FosC expression was downregulated. Further overexpression of N4BP1 reduced the transcript expression of CDS-501, but the expression of N4BP1 did not inhibit the transcript expression of CDS-300 and CDS-225. The results are shown in Figure 2. Figure 4 As shown, there is a sequence recognized and degraded by N4BP1 in the FosC CDS (301-500).

[0048] Next, we continued to shorten the region to find the conserved elements for N4BP1 degradation;

[0049] First, two GFP expression plasmids were constructed, carrying the base sequences of FosC CDS (232-502) and FosC (322-502), respectively. Figure 5 As shown, the experiment confirmed that GFP containing FosC (232-502) was affected by N4BP1 and its expression was reduced, but GFP containing 322-502 did not respond to the degradation of N4BP1. Based on this, it was determined that the sequence shown in SEQ ID NO.1 was the necessary RNA sequence for the N4BP1 degradation target.

[0050] Based on the above findings, the present application inserts the sequence shown in SEQ ID NO.1 into the EGFP overexpression plasmid to construct the EGFP reporter system shown in SEQ ID NO.2. Figure 6 , Experiments confirmed that overexpression of N4BP1 could inhibit the fluorescence intensity and protein expression of EGFP.

[0051] In this application, a reporter group encoding EGFP fluorescent protein is constructed, which is easy to detect; it is not expressed in receptor cells and tissues, and has no cytotoxicity. This reporter group can be widely used in the field of life sciences, and can be used to detect the biological activity of endogenous N4BP1 protein in different tissues and individuals (experimental animals), and can also be used to determine the endonuclease activity of artificially synthesized N4BP1 polypeptides, as well as the endonuclease activity of different mutants or domain-deficient truncations of N4BP1.

[0052] The following are some experimental steps:

[0053] Plasmid construction

[0054] First, obtain the target sequence through the NCBI website, and design cloning primers for PCR amplification. Then double-digest the PCR product and the vector, then connect the target gene fragment to the vector overnight, and finally sequence and verify to ensure the successful construction of the plasmid. For the construction of FosC-CDS (232-502), FosC-CDS (322-502) and reporter gene, the above method is used to fuse the target sequence to the GFP or EGFP expression vector. For the N4BP1 overexpression plasmid, first fuse the 3xFlag tag to the N-terminus of the target sequence before constructing the plasmid.

[0055] Cell culture

[0056] The HEK293T cells used in the experiment were cultured in a complete medium containing 10% fetal bovine serum and 1% double antibody (a mixture of penicillin and streptomycin), and 5% CO2 was given at a temperature of 37°C.

[0057] Cell transfection

[0058] Use trypsin to digest the cells. After the complete medium stops digestion, inoculate them into a 6-well plate. When the cell density is about 70%, use serum-containing medium to culture for 24 hours. Take a sterilized 1.5ep tube to mark the group number, add 80ul opti-mem serum-reduced medium to dilute the plasmid content according to the experimental design, let it stand for 5 minutes, and then add 1ul of low-toxic polymer transfection reagent from Novizan. The concentration of transfection reagent and plasmid can be made in a 1:1 ratio. Let it stand at room temperature for 20 minutes, and then add it drop by drop to each well. There is no need to change the culture medium before transfection. Discard the old culture medium 24 hours after transfection and replace it with fresh culture medium. 48 hours after transfection, collect the cells and extract protein or RNA according to experimental requirements.

[0059] Real-time quantitative PCR

[0060] RNA extraction using Trizol (guanidine thiocyanate-phenol) method

[0061] (1) After trypsin digestion of cells, transfer to RNase-free EP tubes, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, collect the cell pellet, add 1 ml of Trizol and mix by pipetting;

[0062] (2) Incubate at room temperature for 5 min, add 200 μl of chloroform, vortex vigorously for 15 s, incubate at room temperature for 3 min, and then centrifuge at 12,000 rpm at 4°C for 15 min.

[0063] (3) After centrifugation, the sample can be observed to be divided into three layers. Take the upper colorless aqueous phase

[0064] (4) Add an equal amount of isopropanol, mix by inverting, and let stand at room temperature for 20 minutes. To obtain more RNA precipitation, you can let it stand at -20℃ for 1 hour. Centrifuge at 12000rpm and 4℃ for 10 minutes. You can see a tiny precipitate on the side of the EP tube, and then carefully aspirate the supernatant from the opposite side of the precipitate.

[0065] (5) Wash twice with 1 ml of 75% ethanol (made from anhydrous ethanol and DEPC water), centrifuge at 4°C, 7500 rpm for 5 minutes each time. Drain the ethanol for the last time, dry the remaining alcohol in the tube, add 30-50 ul of DEPC water and mix thoroughly, place in a 55°C water bath for 10 minutes, and finally measure the RNA concentration using a spectrophotometer. Use RNase-free pipette tips throughout the entire step.

[0066] Reverse transcription and RT-PCR

[0067] (1) Place 8 RNase-free tubes on ice, add 4ul 4x gDNAwiper Mix, 1ug ​​RNA sample, and 16ul RNase-free water, mix gently with a pipette, and bathe at 42°C for 2 minutes, then add 4ul 5xHiScriptⅡ, and perform reverse reaction on a PCR instrument. Set the program to cycle at 37°C for 15 minutes, 85°C for 5 seconds, and 4°C. The obtained product can be used directly for qPCR or stored at -20°C.

[0068] (2) The cDNA product obtained by the above reversal was diluted 10 times, and 0.2ul of upstream and downstream primers, 5ul of 5xSYBRGreen, 2.6ul of ddH2O, and 2ul of cDNA were added to a 96-well plate.

[0069] (3) Place the 96-well plate with the sample loaded into a low-speed centrifuge and centrifuge at 3500 rpm for 3 minutes to mix the sample thoroughly. Load the sample into the centrifuge and set the reaction conditions.

[0070]

[0071] Primers

[0072]

[0073] Western blotting

[0074] Protein extraction

[0075] After trypsin digestion of cells, place in a 15ml centrifuge tube, centrifuge at 1000rpm for 3 minutes, discard the upper culture medium, add PBS, pipette to mix, transfer to a 1.5mlep tube using a pipette gun, centrifuge at 1500rpm for 5 minutes, discard the supernatant, add an appropriate amount of RIPA lysis buffer according to the amount of cells, add PMSF proteasome inhibitor before use, pipette to mix, and shake on ice for 45 minutes.

[0076] Centrifuge at 12000rpm and 4℃ for 15 minutes, take the supernatant and add 4x protein loading buffer, boil in a metal bath at 105℃ for 5 minutes, or heat at 95℃ for 10 minutes.

[0077] Western blotting

[0078] Prepare SDS-PAGE gel: Before preparing the gel, the glass plate needs to be cleaned and set up, and ddH2O is used to check for leaks. Use it only after ensuring that it is correct. Prepare the corresponding separation gel according to the molecular weight of the protein. The separation gel components include ddH2O, 30% polyacrylamide, Tris-HCl (ph8.8), 10% SDS, 10% ammonium persulfate, and TEMED. According to the principle that the larger the protein molecule, the smaller the concentration of the separation gel, the required separation gel should be reasonably prepared. Add the above solutions to a 50ml centrifuge tube in sequence according to the formula, mix thoroughly, and transfer to a pre-set glass plate with a pipette. Take 1ml of isopropanol to flatten the liquid surface to isolate the air. After the lower separation gel solidifies, prepare the upper concentrated gel. The concentrated gel components include ddH2O, 30% polyacrylamide, Tris-HCl (ph6.8), 10% SDS, 10% ammonium persulfate, and TEMED. Add the prepared upper gel solution to the glass plate until it overflows, insert a comb, and wait for solidification at room temperature.

[0079] Prepare 1x electrophoresis buffer: weigh 3.01 g Tris, 18.8 g glycine, and 1 g SDS, dissolve in 1 L ddH2O, stir to dissolve, and store at room temperature.

[0080] Prepare 1x transfer buffer: weigh 3.03g Tris, 14.4g glycine, 800ml dd water, stir to dissolve, then add 200ml methanol, seal with plastic wrap to prevent methanol from evaporating, and place in a 4℃ refrigerator to precool.

[0081] Prepare 1x TBST membrane washing solution: weigh 2.42g Tris, 8g NaCl, add 2ml Tween, dissolve in 1L ddH2O to make up to volume, stir to mix, and store at room temperature.

[0082] Prepare 5% skim milk: weigh 5 g skim milk powder, add 1xTBST to make up to 100 ml, and store in a 4°C refrigerator.

[0083] Loading electrophoresis

[0084] Place the prepared gel in the electrophoresis tank, add the electrophoresis solution, unplug the comb, add the protein sample to each well in turn, turn on the electrophoresis tank power supply, set the starting voltage to 80V, and slowly compress the protein sample in the concentrated gel to form a horizontal line. When the protein marker begins to separate clear bands in the separation gel, increase the voltage to 120V, or 80V to the bottom. End the electrophoresis when the protein marker is fully separated or bromophenol blue migrates to 1cm from the bottom of the gel.

[0085] Transfer

[0086] Adopt a "sandwich" structure. Place the transfer clip in the water tank with the transfer solution, so that the black side of the clip is at the bottom and the white side is at the top. Then place a sponge pad on the blackboard, and then place the filter paper on the sponge floor. Then knock the glass plate open, let the glue fall off and place it on the filter paper, and place the gel to dry. Put the 0.45um PVDF membrane in methanol to activate for 15 seconds, then put it in water to deactivate, and finally wet it in the transfer solution and carefully place it on the gel. Gently pour the transfer solution on the membrane to drive away bubbles, cover the filter paper and sponge pad on the membrane in turn, and finally gently close the clip. The sponge pad or filter paper in the whole structure needs to be moistened with the transfer solution. Put it in the transfer tank, black against black, red against white, pour the transfer solution, turn on the power, 300mA current, and set the transfer time according to the molecular weight. Generally, 1kDa is equal to one minute. Heat will be generated during transfer, and proteins are easy to diffuse, so ice cubes should be placed around the electrophoresis tank to cool it down.

[0087] Closed

[0088] The PVDF membrane after transfer was placed in 5% skim milk and placed on a shaker at room temperature for 2 hours for blocking.

[0089] Antibody application

[0090] Apply primary antibody at 4°C overnight. The dilution concentration of primary antibody should be determined according to the quality of different antibodies. The next day, recover the primary antibody and wash the membrane three times with 1xTBST, 5 minutes each time. Prepare secondary antibody, dilute the secondary antibody with 5% skim milk at 1:2000, apply secondary antibody at room temperature, and wash the membrane three times with 1xTBST, 20 minutes each time.

[0091] development

[0092] Mix ECL luminescent developer A and B in a ratio of 1:1, prepare and use immediately, drop evenly on the membrane, and use a chemiluminescence instrument for development. The development time and number of images can be determined according to the differences in different protein antibodies.

[0093] Statistical analysis

[0094] The research data in this paper were statistically analyzed using sigmaplot software, and the measurement data that conformed to the normal distribution were described in the form of mean ± standard deviation; the comparison between groups of measurement data that conformed to the normal distribution was performed using independent sample t test or one-way ANOVA. P<0.05 is represented by *, P<0.01 is represented by **, and P<0.001 is represented by ***.

Claims

1. An EGFP reporter system for identifying N4BP1 endoribonuclease activity, characterized in that: The sequence number of the reporter system is shown as SEQ ID NO.2.

Images