Use of PBLD or its agonist as an anti-RNA virus active ingredient

Abstract

The present invention relates to the use of PBLD or its agonist as an anti-RNA virus active ingredient. The present invention for the first time provides the application of PBLD in inhibiting virus replication: when cells are infected with BEFV and VSV, overexpression of PBLD can significantly promote the protein expression of MAVS and the expression of its downstream p-TBK1 and p-IRF3, while after silencing PBLD, it is significantly downregulated; and PBLD activates the I-IFN signaling pathway by promoting the expression of MAVS, thereby inhibiting the replication of BEFV and VSV. The present invention also transfected poly I:C into transient overexpression / silencing PBLD cells respectively, and confirmed that PBLD also shows the same inhibitory effect on double-stranded RNA viruses, providing the application of PBLD as an anti-RNA virus active ingredient.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-RNA virus, and particularly relates to the application of a phenazine biosynthesis-like domain protein (PBLD) or an agonist thereof as an active ingredient for anti-RNA virus activity. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Bovine Ephemeral Fever Virus (BEFV) is a single-stranded negative-strand RNA virus, belonging to the genus Ephemerovirus of the family Rhabdoviridae. It can be transmitted through the respiratory tract and can cause bovine ephemeral fever (also known as three-day fever). This disease is characterized by a wide epidemic area, rapid transmission, and high incidence rate. Its main clinical symptoms include high fever, dyspnea, photophobia and lacrimation, and motor dysfunction, which significantly reduces the milk production of dairy cows. Moreover, some diseased cows are often culled due to paralysis, posing a serious threat to the cattle industry.

[0004] Vesicular stomatitis virus (VSV) belongs to the genus Vesiculovirus of the family Rhabdoviridae. It is an enveloped single-stranded negative-strand RNA virus that can infect various animals such as pigs, cattle, horses, mules, and deer, causing a zoonotic disease - vesicular stomatitis. This disease is characterized by the appearance of vesicles and erosions on the oral mucosa, teats, and skin of the coronary band of the diseased animals, and symptoms such as fever, anorexia, and depression, which reduce the production capacity of the infected animals and cause serious economic losses, and have a serious impact on international trade. It has important socio-economic and public health significance and is listed as a class II infectious disease by the World Organization for Animal Health.

[0005] Currently, the research on the above diseases mainly focuses on epidemiological investigations, genetic evolution analysis of virus strains, establishment of diagnostic methods, and preliminary exploration of immune vaccines. The molecular mechanism of the interaction between the virus and the host remains to be further studied. Therefore, studying the interaction mechanism between the virus and the host, especially discovering the key genes that regulate virus replication, can provide a theoretical basis for the research and development of antiviral drugs and treatment, and also provide new ideas for the prevention and control of infectious diseases.

[0006] Phenazine biosynthesis like protein domain containing (PBLD) is a serine-threonine kinase receptor-related isomerase protein with high affinity. Studies have found that PBLD has anti-tumor activity and can inhibit multiple signaling pathways related to tumor progression, such as TGF-β and MAPK. Screening studies of the genomes and proteomes of relevant tissues have found that the expression of PBLD changes significantly in tumor tissues and it is identified as a tumor suppressor in gastric cancer, liver cancer and breast cancer. The expression of PBLD is down-regulated in gastric cancer tissues. Co-overexpression of PBLD and MAWD in gastric cancer cells can inhibit TGF-β-induced EMT by weakening the phosphorylation of Smad3 and reducing the nuclear translocation of Smad3, that is, PBLD negatively regulates the growth and invasion of gastric cancer cells by inhibiting TGF-β-induced EMT, thereby playing an anti-tumor role. After overexpression of PBLD, the expressions of key molecules p-ERK, p-P38 and p-JNK in the MAPK signaling pathway are significantly inhibited, thereby inhibiting the proliferation, invasion and migration of liver cancer cells. Studies on ulcerative colitis have shown that there is a negative correlation between the expression of PBLD and MAPK, and the angiogenesis dependent on vascular endothelial growth factor requires MAPK activation, indicating that PBLD may become a target for tumor treatment through the MAPK pathway and VEGF. So far, there is still a lack of systematic research on the expression pattern, clinical significance and biological function of PBLD in viruses, and there is no report on its regulation of the IFN-I signaling pathway and its participation in virus replication. Summary of the Invention

[0007] PBLD is an isomerase protein widely distributed in various cells and participates in regulating anti-tumor effects. There has been no study on the relationship between PBLD and viruses. In this invention, BEFV was infected respectively after overexpressing and silencing PBLD, and it was found that the mRNA level of I-IFN and the protein level of ISGs were significantly up-regulated after overexpressing PBLD, and significantly down-regulated after silencing PBLD, providing for the first time a new function of PBLD in participating in regulating the innate immunity induced by BEFV and affecting virus replication.

[0008] Furthermore, the present invention designed experiments to verify the nodal protein of PBLD regulating the expression of IFN-I and ISGs: MAVS, as a key regulatory molecule in the signal transduction cascade, participates in the host's antiviral innate immune response. After RNA virus infection, the pattern recognition receptors RIG-I or MDA-5 can recognize and bind to the viral RNA. Subsequently, the conformation of RIG-I or MDA-5 itself changes and binds to MAVS, causing MAVS to form prion-like aggregates and bind to TRAF3, and then further activate the kinases TBK1 (TANK-binding kinase 1) and IKKε (I kappaB kinase) to form a complex, phosphorylate IRF-3 and IRF-7, and form active IRF-3 and IRF-7 homodimers and / or heterodimers, which then enter the nucleus to initiate the expression of molecules such as interferons. The present invention further studies found that after infecting cells with BEFV and VSV respectively, overexpressing PBLD can significantly promote the protein expression of MAVS and the expression of its downstream p-TBK1 and p-IRF3, while silencing PBLD significantly downregulates them. Therefore, PBLD regulates the expression of IFN-I and ISGs by targeting MAVS.

[0009] Based on the above research, the present invention first confirmed that PBLD activates the IFN-I signaling pathway by promoting the expression of MAVS, thereby inhibiting the replication of BEFV and VSV. To verify whether PBLD has the same effect on other RNA viruses. The present invention used transient overexpression / silencing of PBLD cells to transfect poly I:C respectively. Poly I:C is an analog of double-stranded RNA that can mimic the activation function of RNA viruses in the RLRs signaling pathway. The results showed that in the case of transfecting poly I:C, overexpressing PBLD promoted the expression of MAVS, thereby promoting the expression of p-TBK1, p-IRF3, IFN-β, and ISG15; while silencing PBLD inhibited the expression of MAVS, p-TBK1, and p-IRF3, and inhibited the expression of IFN-β and ISG15. Therefore, PBLD has a new function of promoting IFN-β to inhibit RNA virus replication by regulating MAVS.

[0010] The research of the present invention has confirmed that overexpression of PBLD can inhibit the replication of BEFV and VSV, and provided a clear regulatory mechanism. According to the above research results, those skilled in the art can expect that increasing the dose of PBLD in cattle or increasing the expression of PBLD in the bovine body through stimulation can effectively inhibit the replication of BEFV or VSV viruses, control the degree of virus infection, and inhibit the disease process. Since the above-mentioned BEFV or VSV viruses are both single-stranded negative-strand viruses, the present invention uses poly I:C as the research object and has confirmed that PBLD can inhibit the key nodes in the process of double-stranded virus infection, suggesting that PBLD is also expected to achieve an inhibitory effect on double-stranded RNA viruses.

[0011] Therefore, based on the above research conclusions, the main technical objectives of the present invention are as follows:

[0012] In the first aspect: to provide the use of phenazine biosynthesis-like domain protein (PBLD) or its agonist as an anti-RNA virus active ingredient.

[0013] In the second aspect: to provide the use of phenazine biosynthesis-like domain protein (PBLD), its agonist or its composition form in anti-RNA virus preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0015] Figure 1 : Identification of the expression of siRNA silencing the PBLD gene;

[0016] Figure 2 : Effect of PBLD on the replication of BEFV;

[0017] Among them, the left figure is the result of overexpressing PBLD to inhibit the replication of BEFV, and the right figure is the result of silencing PBLD to promote the replication of BEFV;

[0018] Figure 3 : Effect of PBLD on the expression of IFN-β mRNA after BEFV infection;

[0019] Among them, the left figure is the result of overexpressing PBLD to promote the expression of IFN-β mRNA, and the right figure is the result of silencing PBLD to inhibit the expression of IFN-β mRNA;

[0020] Figure 4 : Effect of PBLD on the expression of ISG15 after BEFV infection;

[0021] Among them, the left figure shows the effect of overexpressing PBLD on the protein levels of downstream IFN-β and interferon-stimulated gene ISG15;

[0022] The right figure shows the effect of silencing PBLD on the protein levels of downstream IFN-β and interferon-stimulated gene ISG15;

[0023] Figure 5 : The effect of PBLD on the expression of key regulatory molecules upstream of IFN-I after BEFV infection;

[0024] Among them, the left figure shows the promoting results of overexpressing PBLD on MAVS, p-TBK1, and p-IRF3;

[0025] The right figure shows the inhibitory results of silencing PBLD on MAVS, p-TBK1, and p-IRF3;

[0026] Figure 6 : The effect of PBLD on the expression of key genes in the IFN-I signaling pathway after VSV infection;

[0027] Among them, the left figure shows the promoting results of overexpressing PBLD on MAVS and its downstream signals mediated by VSV infection;

[0028] The right figure shows the inhibitory results of silencing PBLD on MAVS and its downstream signals mediated by VSV infection;

[0029] Figure 7 : The effect of PBLD on the expression of IFN-β after VSV infection;

[0030] Among them, the left figure shows the promoting results of overexpressing PBLD on the expression of IFN-β, and the right figure shows the inhibitory results of silencing PBLD on the expression of IFN-β;

[0031] Figure 8 : The effect of PBLD on the expression of key genes in the IFN-I signaling pathway under the action of poly I:C;

[0032] Among them, the left figure shows the promoting results of overexpressing PBLD on MAVS and its downstream signals, and the right figure shows the inhibitory results of silencing PBLD on MAVS and its downstream signals;

[0033] Figure 9 : The effect of PBLD on the expression of IFN-β gene under the action of poly I:C;

[0034] Among them, the left figure shows the promoting results of overexpressing PBLD on the expression of IFN-β, and the right figure shows the inhibitory results of silencing PBLD on the expression of IFN-β. Specific implementation manners

[0035] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention pertains.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0037] The present invention first reveals the function of PBLD in regulating the replication of BEFV and VSV viruses, and is expected to inhibit different types of RNA viruses by inhibiting the key proteins in the activation process of double-stranded RNA viruses. Based on the above research results, the present invention specifically provides the following technical solutions:

[0038] In a first aspect of the present invention, there is provided the use of PBLD or its agonist as an anti-RNA virus active ingredient.

[0039] The PBLD described in the above first aspect can adopt a commercially available recombinant PBLD protein preparation, and the preferred purity range is 95% or above; the source of the recombinant PBLD protein preparation is not limited to expression by engineering strains such as Escherichia coli or liquid-phase synthesis, solid-phase synthesis, etc.

[0040] The agonists of PBLD include, but are not limited to, compound entities, polymers, polypeptides or nucleic acid substances that can stimulate the increased expression content of PBLD in the body of a subject, and also include related reagents for overexpressing PBLD in the body of a subject by genetic engineering methods, such as plasmids, lentiviruses, etc.

[0041] Preferably, the RNA virus includes double-stranded RNA and single-stranded RNA; more preferably, the RNA virus is a single-stranded negative-strand RNA virus, and specific examples are bovine ephemeral fever virus (BEFV) and vesicular stomatitis virus (VSV).

[0042] Preferably, the use of PBLD or its agonist as an anti-RNA virus active ingredient includes, but is not limited to, any one of the following:

[0043] (1) For preparing an anti-RNA virus preparation;

[0044] (2) Administering to an individual in need of treatment to inhibit virus infection symptoms;

[0045] (3) For the preparation of cattle feed or feed additives.

[0046] In the application of the above aspect (1), the anti-RNA virus agent is one of a drug, a vaccine, and a model agent for preventing and treating cattle from being infected with bovine ephemeral fever virus (BEFV) and vesicular stomatitis virus (VSV); further, the model agent is applicable to mammals, tissues or cells, and is used to construct a bovine ephemeral fever virus (BEFV) and vesicular stomatitis virus (VSV) inhibition model, and the construction purpose includes screening of active therapeutic drugs, etc.

[0047] In the application of the above aspect (2), the administration method includes, but is not limited to, delivering PBLD or its agonist to the virus infection site through diet, drinking water, oral administration, injection or intervention means.

[0048] In the above aspect (3), an example of the cattle feed is a premix.

[0049] In the second aspect of the present invention, a pharmaceutical composition is provided. In the pharmaceutical composition, it includes an active dose of PBLD or its agonist, and also includes a pharmaceutically necessary carrier.

[0050] The added dose of PBLD or its agonist in the above pharmaceutical composition is technical content that those skilled in the art can routinely determine according to factors such as the administration purpose of the pharmaceutical composition, the test subject, and the administration method. The administration purpose of the pharmaceutical composition includes preventing, improving or inhibiting the disease symptoms caused by RNA virus infection; the test subject is preferably cattle, and further, is dairy cows.

[0051] There is no particular limitation on the dosage form of the pharmaceutical composition. The preferred form is a solid preparation or a liquid preparation; in the case of a solid preparation, the carrier includes, but is not limited to, excipients, lubricants, binders, disintegrants, etc.; in the case of a liquid preparation, the carrier includes, but is not limited to, solvents, solubilizers, suspending agents, isotonizing agents, buffers, soothing agents, etc., and preservatives, antioxidants, colorants, sweeteners and other formulation additives can also be appropriately added as needed.

[0052] As a preferred example of an excipient, for example, lactose, sucrose, D-mannitol, starch, crystalline cellulose, light anhydrous silicic acid, etc. can be used.

[0053] As a preferred example of a lubricant, for example, magnesium stearate, calcium stearate, talc powder, colloidal silica, etc. can be used.

[0054] As a preferred example of a binder, for example, crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, etc. can be used.

[0055] As preferred examples of disintegrants, for example, starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, sodium carboxymethyl starch, etc. can be used.

[0056] As preferred examples of solvents, water for injection, alcohols, propylene glycol, polyethylene glycol, sesame oil, corn oil, etc. can be used.

[0057] As preferred examples of solubilizers, for example, polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, etc. are used.

[0058] Suitable examples of suspending agents include, for example, surfactants (stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, etc.), and hydrophilic polymers (polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, etc.) can be used.

[0059] As suitable examples of tonicity agents, for example, sodium chloride, glycerol, D-mannitol, etc. can be used.

[0060] As preferred examples of buffers, for example, buffer solutions of phosphates, acetates, carbonates, citrates, etc. can be used.

[0061] As preferred examples of soothing agents, for example, benzyl alcohol, etc. can be used.

[0062] As preferred examples of preservatives, for example, parabens, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, etc. can be used.

[0063] As preferred examples of antioxidants, for example, sulfites, ascorbic acid, etc. can be used.

[0064] Preferably, in the pharmaceutical composition, other antiviral active ingredients or auxiliary antiviral ingredients are further included. The antiviral active ingredient is preferably an RNA polymerase inhibitor such as remdesivir and favipiravir; the auxiliary antiviral ingredients include but are not limited to immunomodulatory drugs, antibiotics, antipyretic and respiratory distress relieving drugs, and specific examples are compound aminopyrine, amidopyrine, penicillin, streptomycin, nikethamide, aminophylline, etc.

[0065] In the third aspect of the present invention, a drug for preventing and treating bovine epizootic fever is provided, and the drug includes an active dose of a phenazine biosynthesis-like domain protein, an agonist thereof, or the pharmaceutical composition described in the second aspect.

[0066] In the fourth aspect of the present invention, there is provided a drug for preventing and treating vesicular stomatitis, which comprises an active dose of a phenazine biosynthesis-like domain protein, an agonist thereof or the drug composition described in the second aspect.

[0067] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0068] Example 1

[0069] 1. Materials

[0070] BHK-21, Hela, 293T cells, bovine epizootic fever virus (BEFV), and vesicular stomatitis virus (VSV) are all preserved by the Ruminant Disease Research Center of Shandong Normal University. rTaq enzyme, PrimeSTAR GXL DNA polymerase, dNTPs, DNA Marker DL2000, T4 DNA ligase, PrimeScript TM RT Master Mix reverse transcription kit, Premix Ex Taq TM Ⅱ (Tli RNaseH Plus) and restriction enzymes and other reagents are all purchased from Takara Bio Inc. (Dalian); DH5α competent cells are purchased from Shanghai Weidi Biotechnology Co., Ltd.; Total RNA extraction kit, plasmid extraction kit, and gel extraction kit are purchased from Beijing TransGen Biotech Co., Ltd.; Primary antibodies such as β-actin, PBLD, RIG-I, MAVS, TBK1, p-TBK1, IRF3, p-IRF-3, ISG15 and HRP-labeled secondary antibodies are purchased from Santa Cruz Biotechnology, Inc. (USA); PVDF membrane and ECL developing luminescent solution are purchased from Solarbio Science & Technology Co., Ltd.

[0071] 2. Methods and Results

[0072] 2.1 Construction and expression detection of PBLD gene overexpression vector

[0073] According to the PBLD gene sequence (Gene ID: 64081) published in GenBank, primers for amplifying the CDS region were designed using Primer Premier 5.0 software,

[0074] pLVX-Flag-PBLD-EcoR I-F:

[0075] 5′- CGGAATTCCG ATGAAGCTTCCTATTTTCATAG-3′;

[0076] pLVX-Flag-PBLD-KpnI-R:

[0077]

[0078] Among them, the underlined part is the restriction enzyme cleavage site sequence, and the italic part is the Flag tag sequence. Total RNA of Hela cells was extracted according to the instructions of the total cell RNA extraction kit, reverse transcribed to obtain cDNA. PCR amplification was carried out. The amplification system was 20 μL: 0.5 μL of rTaq I, 1 μL each of the upstream primer and the downstream primer, 8 μL of dNTPs, 2 μL of 10× buffer, and 7.5 μL of ddH 2 O. The reaction conditions were pre-denaturation at 95 °C for 5 min, and then 30 cycles were carried out according to 95 °C denaturation for 20 s, 55 °C for 20 s, and 72 °C for 1 min; 72 °C for 7 min, and the reaction ended at 4 °C for 10 min.

[0079] The PCR product was detected by 1% agarose gel electrophoresis to obtain the target fragment with the expected size. The PCR product and the pLVX-IRES-puro vector were digested with the restriction enzymes EcoR I and Kpn I respectively, recovered by gel extraction, ligated with T4 DNA ligase, transformed into Escherichia coli DH5α, screened by Amp, white colonies were picked, and the positive recombinant bacteria identified by PCR and double digestion were subjected to DNA sequencing. The recombinant vector with correct sequencing was named pLVX-Flag-PBLD.

[0080] The pLVX-Flag-PBLD plasmid was extracted using an endotoxin-free plasmid miniprep kit and its concentration was measured. The pLVX-Flag-PBLD plasmid and the empty pLVX-IRES-puro were transfected into 293T cells respectively. After 48 h, the cells were collected, and the expression of PBLD was identified by Western blot technology. The transient expression cells of PBLD were PBLD-Flag, and the control cells were NC.

[0081] 2.2 Screening of siRNA for silencing PBLD gene

[0082] Three pairs of siRNA primer sequences for the PBLD gene were designed and synthesized using BLOCK-iT™ RNAi Designer (see Table 1). After being transfected into 293T cells for 24 h respectively with the control siNC, the samples were collected, and the protein expression of the PBLD gene in the cells was identified by Western Blot. The results showed that compared with the control group, the protein expressions of siPBLD-1, siPBLD-2, and siPBLD-3 were all down-regulated, and the silencing effect of siPBLD-3 was better ( Figure 1 ), and this silencing vector was named siPBLD for subsequent experiments.

[0083] Table 1 siPBLD sequences

[0084]

[0085]

[0086] 2.3 Effect of PBLD on BEFV replication

[0087] The pLVX-Flag-PBLD plasmid and the empty vector pLVX-IRES-puro, and the siPBLD and the control siNC were transfected into BHK-21 cells respectively. After 24 h, BEFV at 0.1 MOI was inoculated. The virus was harvested at 12 h and 24 h respectively, frozen and thawed 3 times repeatedly, centrifuged, and the supernatant was taken to measure the TCID of the virus 50 . This experiment was repeated 3 times. The results showed that overexpression of PBLD inhibited the replication of BEFV, while silencing of PBLD promoted the replication of BEFV( Figure 2 ).

[0088] 2.4 Effect of PBLD on the expression of type I IFN and interferon-stimulated genes

[0089] To explore the molecular mechanism by which PBLD affects the replication of BEFV, the present invention detected the effect of PBLD on the expression of type I interferon (IFN-I). BHK-21 cells were cultured in 6-well cell culture plates until they grew into a monolayer. The pLVX-Flag-PBLD plasmid and the empty vector pLVX-IRES-puro, and the siPBLD and the control siNC were transfected into BHK-21 cells respectively. After 24 h, BEFV at 0.1 MOI was inoculated. After inoculation for 12 h and 24 h, the supernatant was discarded, the cells were scraped and harvested with a cell scraper, washed with PBS, divided into two parts, and the cell precipitate was taken by centrifugation. One part of the cell precipitate was used for the extraction of total RNA, reverse transcription into cDNA, and fluorescence quantitative PCR analysis to detect the mRNA expression of the IFN-β gene. The other part of the cells was lysed with RIPA reagent to detect the protein expression of the key regulatory genes upstream of IFN-I

[0090] Fluorescence quantitative PCR was used to detect the mRNA expression of the IFN-β gene

[0091] Based on the gene sequences of golden hamster IFN-β and the internal reference gene GAPDH published in GenBank, primers were designed using Primer5 software

[0092] IFN-β-F: 5’-AGATGCCTATGGAGGTGAA-3’

[0093] IFN-β-R: 5’-AGTGCTGGAGAAAGTGTTG-3’

[0094] GAPDH-F: 5’-TCATGACCACAGTCCATGCC-3’;

[0095] GAPDH-R: 5’-GGATGACCTTGCCCACAGCC-3’.

[0096] Prepare a 20 μL PCR reaction system: 10 μL of 2×SYBR Premix Ex Taq II, 2 μL of cDNA, 0.5 μL each of the upstream primer and the downstream primer IFN-β-F / R, 7 μL of RNase Free ddH 2 O, add it to a 96-well plate, and repeat each sample three times. Amplify using a Roche LightCycler 96 real-time fluorescence quantitative PCR instrument. The reaction conditions are pre-denaturation at 95°C for 30 s, and then 40 cycles are carried out according to denaturation at 95°C for 5 s and annealing extension at 60°C for 30 s; the melting curve is 95°C for 10 s, 60°C for 60 s, 95°C continuous; finally, the reaction ends at 50°C for 30 s. The temperature conversion rate is 20°C / s, and the fluorescence signal is detected at the end of the extension of each cycle. Plot the data using GraphPad Prism 8.0 software. The results show that after BEFV infection, overexpression of PBLD promotes the mRNA expression of IFN-β, while silencing of PBLD has the opposite result, inhibiting the mRNA expression of IFN-β( Figure 3 ).

[0097] Detect the protein expression of the interferon-stimulated gene ISG15 downstream of the IFN-β signal by Western blot analysis.

[0098] The protein sample was added with 6×SDS Loading Buffer and boiled in boiling water for 10 min, followed by SDS-PAGE gel electrophoresis. After transferring the membrane, it was blocked with 5% non-fat milk powder. The membrane was placed in an incubation box containing 5% non-fat milk powder blocking solution and blocked at room temperature on a shaker for 2 h. After the blocking was completed, the original blocking solution was discarded, and new blocking solution was added to the incubation box. The preservative was added at a ratio of 1:100, and the corresponding primary antibody was diluted at a ratio of 1:2000, and incubated overnight on a shaker at 4°C. After the incubation was completed, the primary antibody was recovered, and then the membrane was washed with 1×TBST buffer on a shaker three times, 5 min each time. After the membrane washing was completed, new blocking solution was added to the incubation box, and the corresponding secondary antibody diluted at 1:2000 was added, and incubated on a shaker at room temperature in the dark for 1 h. After the incubation was completed, the membrane was washed with 1×TBST buffer three times, 5 min each time. The ECL developing luminescent solution was prepared by mixing equal amounts of luminescent developing A and B solutions, and developed under the 5200 of the automatic chemiluminescence image analysis system. The results showed that after infection with BEFV, overexpression of PBLD promoted the protein level expression of the interferon-stimulated gene ISG15 downstream of IFN-β, while silencing of PBLD had the opposite result, inhibiting the protein level expression of ISG15( Figure 4 ).

[0099] 2.5 Effect of PBLD on the expression of key genes regulating the upstream of I-IFN

[0100] To explore the key regulatory molecules affecting the expression of I-IFN by PBLD, the present invention screened and verified the key molecules regulating the expression of I-IFN by PBLD. BHK-21 cells were cultured in 6-well cell culture plates until they grew into a monolayer. Two groups of pLVX-Flag-N-PBLD plasmid and empty pLVX-IRES-puro, siPBLD and control siNC were transfected into BHK-21 cells respectively. After 24 h, 0.1 MOI of BEFV was inoculated. At 12 h and 24 h after virus inoculation, the supernatant was discarded, the cells were scraped with a cell scraper, harvested, washed with PBS, and the cell pellet was obtained by centrifugation. The cells were lysed with RIPA reagent, and the protein expression of key regulatory genes upstream of I-IFN was detected by Western blot. The results showed that after infection with BEFV, overexpression of PBLD promoted the expression of MAVS and phosphorylated TBK1 (p-TBK1), and further promoted the expression of phosphorylated IRF3 (p-IRF3); the result was opposite after silencing of PBLD, and silencing of PBLD inhibited the expression of p-TBK1, and further inhibited the expression of p-IRF3( Figure 5 ).

[0101] 2.6 PBLD inhibits the I-IFN signaling pathway mediated by VSV

[0102] To explore whether the new function of PBLD activating the IFN-I signaling pathway to inhibit BEFV replication has a similar function in other viruses, the present invention detected the expression of key molecules in the IFN-I signaling pathway mediated by PBLD through VSV infection. Hela cells were cultured in 6-well cell culture plates until they grew into a monolayer. The pLVX-Flag-N-PBLD plasmid and the empty vector pLVX-IRES-puro, and the siPBLD and the control siNC were transfected into Hela cells respectively. After 24 h, 0.1 MOI of VSV was inoculated respectively, and the transfected cells without virus inoculation were harvested. After 12 h of virus inoculation, the supernatant was discarded, and the cells were harvested by scraping with a cell scraper, and the cell precipitate was obtained by centrifugation. One part of the precipitate was used for total RNA extraction, and fluorescence quantitative PCR was performed using the primers in Table 2 to analyze the mRNA expression of IFN-β. Another part of the precipitate was lysed with RIPA to detect the protein expression of RIG-I, MAVS, TBK1, p-TBK1, IRF3, p-IRF3, and ISG15 by Western blot. The results showed that overexpression of PBLD promoted the expression of MAVS and its downstream p-TBK1 and p-IRF3 mediated by VSV infection (Figure 6), thereby promoting the expression of β-IFN ( Figure 7 ), and promoting the expression of ISG15 ( Figure 6 ); the results were opposite after silencing PBLD. After silencing PBLD, the expression of MAVS and its downstream p-TBK1 and p-IRF3 mediated by VSV infection ( Figure 6 ) and the expression of β-IFN ( Figure 7 ) were inhibited, and the expression of ISG15 ( Figure 6 ) was inhibited.

[0103] Table 2 Primer sequences for fluorescence quantitative PCR detection of IFN-β gene and internal reference gene GAPDH in Hela cells

[0104]

[0105] 2.7 PBLD inhibits the IFN-I signaling pathway mediated by RNA viruses

[0106] To explore whether the new function of PBLD activating the IFN-I signaling pathway to inhibit BEFV replication is widespread in RNA virus replication. Hela cells were cultured in 6-well cell culture plates until they grew into a monolayer. Two groups, namely the pCMV-Flag-N-PBLD plasmid and the empty vector pLVX-IRES-puro, and siPBLD and the control siNC, were transfected into Hela cells respectively. After 24 h, the transfected cells were inoculated with the RNA virus mimic poly I:C. After 4 h, the supernatant was discarded, and the cells were scraped with a cell scraper, harvested, and centrifuged to obtain cell pellets. One part of the pellet was used for total RNA extraction, and the mRNA expression of IFN-β was detected by fluorescence quantitative PCR; another part of the pellet was lysed with RIPA to detect the protein expression of RIG-I, MAVS, TBK1, p-TBK1, IRF3, p-IRF3, and ISG15 by Western blot. The results showed that in the case of transfection with the RNA virus mimic poly I:C, overexpression of PBLD promoted the expression of MAVS and its downstream p-TBK1 and p-IRF3 ( Figure 8 ), thus promoting the expression of β-IFN ( Figure 9 ), and promoting the expression of ISG15 ( Figure 8 ); the results were opposite after silencing PBLD. After silencing PBLD, the expression of MAVS and its downstream p-TBK1 and p-IRF3 was inhibited ( Figure 8 ), thus inhibiting the expression of β-IFN ( Figure 9 ), and inhibiting the expression of ISG15 ( Figure 8 ).

[0107] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of PBLD in the preparation of an anti-RNA virus preparation, wherein the RNA virus is bovine ephemeral fever virus or vesicular stomatitis virus.

2. The use of PBLD in the preparation of an anti-RNA virus preparation as claimed in claim 1, characterized in that the PBLD uses a commercially available recombinant PBLD protein preparation with a purity range of 95% or more; the recombinant PBLD protein preparation is derived from expression by engineering strains or liquid-phase synthesis and solid-phase synthesis methods.

3. The use of PBLD in the preparation of an anti-RNA virus preparation as claimed in claim 1, characterized in that the anti-RNA virus preparation is a drug for preventing and treating cattle infected with bovine ephemeral fever virus and vesicular stomatitis virus.

4. The use of PBLD in the preparation of an anti-RNA virus preparation as claimed in claim 3, characterized in that the drug is a vaccine.

5. The use of PBLD in the preparation of an anti-RNA virus preparation as claimed in claim 3, characterized in that the drug is a model agent, and the model agent is applicable to mammals, tissues or cells and is used to construct an inhibition model of bovine ephemeral fever virus and vesicular stomatitis virus.

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