Application of recombinant RASA3 in preparation of medicine for preventing and treating viral pneumonia

Abstract

The invention belongs to the technical field of biotechnology and genetic engineering, and particularly relates to application of recombinant RASA3 in preparation of a medicine for preventing and treating viral pneumonia. The recombinant RASA3 is used for promoting autophagy degradation of a transcription factor CEBP / beta protein and reducing the expression level of the transcription factor CEBP / beta protein, so that generation of inflammatory factors such as IL-6 is reduced, and inflammatory damage caused by excessive antiviral innate immune response in the virus infection process is improved. The invention provides a novel treatment strategy for inflammatory diseases caused by RNA viruses such as influenza and respiratory syncytial virus infection.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and genetic engineering technology, and specifically relates to an application of recombinant RASA3 in the preparation of a drug for the prevention and treatment of viral pneumonia. Background Art

[0002] Lung inflammation caused by viral infection is usually called viral pneumonia. Viral infections such as influenza virus, respiratory syncytial virus, adenovirus, and new coronavirus can all cause viral pneumonia. After the virus infects the host cell, its genetic material such as viral RNA is recognized by intracellular pattern recognition receptors such as TLR3, TLR7, TLR8, RIG-I, MDA5, etc., thereby activating the host's antiviral innate immune response. For example, after the viral RNA in the cytoplasm is recognized by RIG-I, it can activate MAVS (also known as VISA, IPS-1, Cardif) on the mitochondria to cause it to oligomerize, and then recruit TRAF, TBK1, IKK and other proteins to form a signal transducer (signalosome), further causing the activation of downstream transcription factors IRF3 and NF-κB, thereby producing type I interferons such as IFN-α / β, proinflammatory cytokines such as IL-6, TNF-α, IL-1β and other inflammatory mediators to resist viral invasion and protect the body. However, excessive activation of innate immunity can trigger a cytokine storm. [1] . This uncontrolled immune response can not only lead to Acute Respiratory Distress Syndrome (ARDS), manifested as severe lung inflammation and respiratory failure, but may also induce pulmonary fibrosis, causing lung tissue hardening and long-term functional damage. In addition, cytokine storms may also lead to multiple organ dysfunction or failure, affecting important organs such as the heart, kidneys, and liver, and ultimately increasing the risk of death in patients with viral infection. In viral pneumonia, the IL-6-STAT3 and NF-κB signaling pathways play a key role in the formation of cytokine storms, among which IL-6 is at the core of this process and has a very important impact on the occurrence and development of the disease.

[0003] During SARS-CoV-2 infection-induced lung inflammation, the massive production of IL-6 is closely related to members of the transcription factor CEBP family. [2] Studies have shown that CEBP / β can directly interact with the Rel homology domain between NF-κB subunits p50, p65 and C-Rel, thereby stabilizing NF-κB and synergistically activating the expression of multiple pro-inflammatory factors including IL-6. [3]This mechanism not only enhances local inflammatory responses but may also accelerate disease progression. In patients with severe COVID-19, CEBP / δ in peripheral immune cells is highly activated, while CEBP / β is mainly active in respiratory epithelial cells. [4] This suggests that when the body faces viral infection, different types of cells will use different CEBP family members to regulate inflammatory responses. Especially in respiratory epithelial cells, the activation of CEBP / β may be one of the key factors that trigger local inflammation. In addition, CEBP / β is important for the replication and proliferation of HIV-1 in monocytes. [5] This suggests that it may play a similar role in other viral infections, exacerbating the inflammatory response by promoting viral proliferation. Lipopolysaccharide (LPS) can also activate CEBP / β, prompting it to enter the cell nucleus and initiate the expression of a series of inflammatory factors such as IL-6. [6] In addition to its role in viral infections, CEBP / β also plays an important role in other pathological conditions. For example, in microglia, the loss of the E3 ubiquitinase COP1 leads to abnormal accumulation of CEBP / β in the brain, which may lead to the occurrence of neurodegenerative diseases. However, when COP1 is present, it can promote the ubiquitination of CEBP / β and its degradation through the proteasome pathway, thereby maintaining normal levels of CEBP / β. [7] .

[0004] The RAS superfamily is a large family of small GTPase proteins whose main functions involve multiple key cellular processes, including intracellular signal transduction, regulation of cell growth, differentiation, cytoskeleton reorganization, intracellular transport, and nuclear-cytoplasmic material exchange. RasGTPase activating protein (RasGAP) family members, such as RASA3, can inhibit R-RAS activity and promote tumor cell invasion. [8] In addition to its regulatory functions in cell growth and differentiation, RASA3 has a close relationship with the hematopoietic system. For example, Scat mice with an autosomal recessive missense mutation in RASA3 exhibit symptoms such as severe anemia, leukopenia, splenomegaly, bleeding, and thrombocytopenia. Approximately 94% of homozygous mice die within 30 days of birth. [9] Iwashita et al. used a mouse model constructed by replacing exons 11 and 12 of RASA3 with a neomycin resistance gene to observe massive bleeding in embryos.

[10] In addition, many studies in recent years have shown that RASA3 plays an important role in regulating T cell function. For example, T cells lacking RASA3 have enhanced adhesion ability, which makes it difficult for them to effectively enter and exit lymph nodes.

[11] knocking out RASA2 gene homologous to RASA3 in T cells can enhance the activity of T cells in various immunosuppressive environments, improve their sensitivity to antigens, and thus enhance the anti-tumor immune response

[12] RASA3 can also promote the degradation of transcription factor IRF4 by recruiting E3 ubiquitin ligase Cbl-b, thereby inhibiting Th2-related programs and promoting the generation of pathogenic Th17 to drive systemic inflammation

[13] To date, there has been no report of a treatment method or drug targeting RASA3 to inhibit the CEBP / β-IL-6 signaling pathway for treating viral pneumonia.

[0005] In response to the excessive production of IL-6 and the resulting inflammatory response caused by viral infection, the medical community has adopted a variety of comprehensive treatment strategies, including the use of anti-IL-6 receptor blocking antibodies such as tocilizumab to directly inhibit IL-6 signaling and thus alleviate inflammatory symptoms

[14] ; in certain cases, the use of glucocorticoids to control excessive inflammatory response

[15] ; the use of other immunomodulators and biologics, such as JAK inhibitors, to regulate immune response, JAK inhibitors Tofacitinib and Baricitinib, which are small molecule drugs that inhibit JAK kinase to prevent downstream signaling of IL-6 and other cytokines [16,17]; combined with supportive care measures such as fluid resuscitation, mechanical ventilation, and antibiotic therapy to maintain vital functions and support the body's ability to fight infection. These approaches work together to effectively manage and alleviate inflammatory diseases mediated by IL-6. However, existing technologies for treating IL-6-related inflammatory diseases still face some challenges and problems. For example, tocilizumab has high production costs due to its complex biomanufacturing process; its administration is inconvenient, usually requiring injection and not suitable for oral administration; it may cause immune reactions, as patients may develop anti-drug antibodies, affecting efficacy; its penetrance is limited, making it difficult for large molecules to cross physiological barriers such as the blood-brain barrier; and it also has some potentially serious side effects and disadvantages, particularly the risk of infection, liver damage, and gastrointestinal perforation. Long-term use of glucocorticoids such as prednisone may lead to serious side effects such as osteoporosis, immunosuppression, and hypertension. While it reduces inflammation by broadly suppressing the immune system, it can also weaken the body's defenses and increase the risk of infection and other complications. Small molecule inhibitors such as tofacitinib and baricitinib, in addition to the common drawbacks of small molecule drugs, can also lead to a variety of health risks with long-term use. These include increased risk of infection and certain types of tumors, particularly lymphoma; cardiovascular risks, including myocardial infarction and stroke; thrombotic risk, potentially increasing the risk of venous thrombosis and pulmonary embolism; liver damage, leading to elevated liver enzymes; and dyslipidemia, leading to elevated cholesterol and triglyceride levels. Furthermore, there's the high cost: tofacitinib is a new drug with a relatively high price, which may impose a financial burden on patients.

[0006] Therefore, new strategies and new drugs are urgently needed to prevent and treat viral pneumonia by specifically inhibiting IL-6 production.

[0007] References

[0008] 1. Hirano T, Murakami M. COVID-19: A New Virus, but a Familiar Receptor and Cytokine Release Syndrome. Immunity 2020,52:731-733.

[0009] 2.Guillermo B,Elena CM,Manuel MB,Silvia RR,Beatriz S, P-P,etal.Whole blood DNA methylation analysis reveals respiratory environmentaltraits involved in COVID-19severity following SARS-CoV-2infection.Nat Commun2022,13:4597.

[0010] 3.Matsusaka T,Fujikawa K,Nishio Y,Mukaida N,Matsushima K,Kishimoto T,et al.Transcription factors NF-IL6 and NF-kappa B synergistically activatetranscription of the inflammatory cytokines,interleukin 6and interleukin8.Proc Natl Acad Sci USA 1993,90:10193-10197.

[0011] 4.Zhang L,Nishi H,Kinoshita K.Single-cell RNA-seq public data revealthe gene regulatory network landscape of respiratory epithelial andperipheral immune cells in COVID-19patients.Front Immunol 2023,14:1194614.

[0012] 5.Henderson AJ,Connor RI,Calame KL.C / EBP activators are required forHIV-1replication and proviral induction in monocytic cell lines.Immunity1996,5:91-101.

[0013] 6. CT, CC, P vdG, TH, MK, MC. Transactivation by NF-IL6 / LAP is enhanced by phosphorylation of its activation domain. Nature 1993, 364: 544-547.

[0014] 7. Ndoja A, Reja R, Lee SH, Webster JD, Ngu H, Rose CM, et al. Ubiquitin Ligase COPl Suppresses Neuroinflammation by Degrading c / EBPbeta in Microglia. Cell 2020, 182: 1156-1169

[0015] 8. Li HA-O, Prever L, Hsu MY, Lo WT, Margaria JP, De Santis MC, et al. Phosphoinositide Conversion Inactivates R-RAS and Drives Metastases in Breast Cancer. Adv Sci 2022, 9::e2103249.9. Peters LL, McFarland-Starr EC, Wood BG, Barker JE. Heritable severe combined anemia and thrombocytopenia in the mouse: description of the disease and successful therapy. Blood 1990, 76: 745-754.

[0016] 10. Iwashita S, Kobayashi M, Kubo Y, Hinohara Y, Sezaki M, Nakamura K, et al. Versatile roles of R-Ras GAP in neurite formation of PC12 cells and embryonic vascular development. J Biol Chem 2007, 282: 3413-3417.

[0017] 11.Johansen KA-O,Golec DA-O,Huang BA-O,Park CA-O,Thomsen JA-O,PreiteS,et al.A CRISPR screen targeting PI3K effectors identifies RASA3 as anegative regulator of LFA-1-mediated adhesion in T cells.Sci Signal 2022 15:eabl9169.

[0018] 12.Carnevale J,Shifrut E,Kale N,Nyberg WA,Blaeschke F,Chen YY,etal.RASA2 ablation in T cells boosts antigen sensitivity and long-termfunction.Nature 2022,609:174-182.

[0019] 13.Wu B,Zhang S,Guo Z,Wang G,Zhang G,Xie L,et al.RAS P21 ProteinActivator 3(RASA3)Specifically Promotes Pathogenic T Helper 17Cell Generationby Repressing T-Helper-2-Cell-Biased Programs.Immunity 2018,49:886-898e885.

[0020] 14.Hermine O,Mariette X,Tharaux PL,Resche-Rigon M,Porcher R,Ravaud P,et al.Effect of Tocilizumab vs Usual Care in Adults Hospitalized With COVID-19and Moderate or Severe Pneumonia:A Randomized Clinical Trial.JAMA InternMed 2021,181:32-40.

[0021] 15.Moreno-González G,Mussetti A,Albasanz-Puig A,Salvador I,Sureda A,Gudiol C,et al.A Phase I / IIClinical Trial to evaluate the efficacy ofbaricitinib to prevent respiratory insufficiency progression in onco-hematological patients affected with COVID19:A structured summary of a studyprotocol for a randomised controlled trial.Trials 2021,22:116.

[0022] 16.Hashemian SMR,Farhadi T.A narrative review on tofacitinib:Theproperties,function,and usefulness to treat coronavirus disease 2019.Int JCrit Illn Inj Sci 2023,13:192-198.

[0023] 17.Wei C,Yin W,Hu T,Zhang J,Dan H,Wu B.Agranulocytosis and secondaryinfection related to JAK inhibitors and IL-6receptor blockers:adisproportionality analysis using the US Food and drug administration adverseevent reporting system.Front Pharmacol 2023,14:1323240. 发明内容

[0024] Problems to be solved by the inventionIn view of the problems existing in the prior drugs and treatment schemes, the present application discloses a recombinant RASA3, which comprises a Δ1-329 truncated body of RASA3, and is capable of improving inflammation caused by RNA virus infection by specifically inhibiting the production of IL-6. The inhibitory effect of the recombinant RASA3 and the RASA3 truncated body thereof on inflammation caused by RNA virus infection is detected by constructing a recombinant RASA3 and a RASA3 truncated body overexpression vector, and using a RASA3 myeloid knockout cell and a myeloid knockout mouse.

[0025] Solutions for solving problems

[0026] [1]. Use of a recombinant RASA3 in the preparation of a product for preventing and / or treating a disease and / or a symptom associated with viral infection, wherein the recombinant RASA3 comprises a Δ1-329 truncated body interacting with a CEBP / β protein;

[0027] The Δ1-329 truncated body promotes degradation of the CEBP / β protein by interacting with the CEBP / β protein; preferably, the degradation of the CEBP / β protein comprises degradation by a protein autophagy-lysosome pathway;

[0028] The Δ1-329 truncated body comprises a PH / Btk domain of RASA3, and optionally a GAP domain of RASA3;

[0029] Optionally, the GAP domain comprises a sequence as shown in SEQ ID NO. 2, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the sequence as shown in SEQ ID NO. 2;

[0030] Optionally, the PH / Btk domain comprises a sequence as shown in SEQ ID NO. 3, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the sequence as shown in SEQ ID NO. 3;

[0031] Preferably, the Δ1-329 truncated body comprises a sequence as shown in SEQ ID NO. 1, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the sequence as shown in SEQ ID NO. 1;

[0032] The virus is an RNA virus.

[0033] [2]. The use according to [1], wherein the recombinant RASA3 further comprises at least one of a C2A domain and a C2B domain;

[0034] Optionally, the C2A domain comprises the sequence shown in SEQ ID NO. 4, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity to the sequence shown in SEQ ID NO. 4;

[0035] Optionally, the C2B domain comprises the sequence shown in SEQ ID NO.5, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the sequence shown in SEQ ID NO.5.

[0036] [3] The use according to [1] or [2], wherein the virus comprises at least one of influenza virus, coronavirus, novel coronavirus, adenovirus, respiratory syncytial virus and vesicular stomatitis virus;

[0037] Preferably, the virus is vesicular stomatitis virus, influenza virus and / or respiratory syncytial virus;

[0038] More preferably, the influenza virus is influenza A virus A / PR / 8 / 34 strain.

[0039] [4] The use according to any one of [1] to [3], wherein the symptoms of the disease and / or condition associated with the viral infection include at least one of the following (i) to (iv):

[0040] (i) Increased expression of inflammatory factors in the body;

[0041] (ii) increased immune cell infiltration;

[0042] (iii) inflammatory damage caused by infection;

[0043] (iv) inflammatory diseases caused by viral infection;

[0044] Optionally, the inflammatory factors include IL-6, IL-1β, IP-10, and TNF-α;

[0045] Optionally, the inflammatory damage includes inflammatory damage to the respiratory system, especially inflammatory damage to the lungs;

[0046] Optionally, the inflammatory disease comprises pneumonia, preferably acute inflammatory pneumonia;

[0047] Optionally, the organism is an individual who is likely to suffer from a disease and / or symptom associated with viral infection; the individual includes mice, rats, guinea pigs, cows, sheep, cats, dogs, horses, rabbits, pigs, monkeys and humans.

[0048] [5]. The use of any one of [1]-[4], wherein the product comprises a recombinant RASA3 protein, a polynucleotide encoding a recombinant RASA3, a vector carrying a recombinant RASA3, and a vector carrying a polynucleotide encoding a recombinant RASA3;

[0049] The vector comprises any one or more of a plasmid, an adeno-associated virus, a retrovirus, a liposome, a lipid nanoparticle, N-acetylgalactosamine, and an extracellular vesicle.

[0050] [6]. The use of any one of [1]-[5], wherein the product further comprises a drug that promotes autophagy of CEBP / β protein;

[0051] Optionally, the drug that promotes autophagy degradation of CEBP / β protein comprises an mTOR inhibitor, an AMPK activator, and / or a natural compound;

[0052] The natural compound comprises curcumin and / or resveratrol.

[0053] [7]. The use of any one of [1]-[6], wherein the product for preventing and / or treating a disease and / or a symptom associated with a viral infection comprises a drug and a pharmaceutical composition;

[0054] Optionally, the pharmaceutical composition further comprises one or more drugs that are clinically used to treat a disease and / or a symptom associated with a viral infection and / or one or more pharmaceutically acceptable carriers;

[0055] Optionally, the pharmaceutically acceptable carrier comprises a combination of one or more of a solvent, a solubilizer, a cosolvent, an emulsifier, a flavoring agent, an odorant, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a pH regulator, a stabilizer, a surfactant, and a preservative.

[0056] [8]. Use of a recombinant RASA3 in the preparation of a CEBP / β inhibitor, wherein the recombinant RASA3 comprises a Δ1-329 truncation that interacts with CEBP / β protein;

[0057] The Δ1-329 truncation promotes degradation of CEBP / β protein through interaction with CEBP / β protein; preferably, the degradation of CEBP / β protein comprises degradation through a proteoautophagy-lysosomal pathway;

[0058] The Δ1-329 truncation comprises a PH / Btk domain of RASA3, and, optionally, a GAP domain of RASA3;

[0059] Optionally, the GAP domain comprises a sequence as set forth in SEQ ID NO. 2, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the sequence as set forth in SEQ ID NO. 2;

[0060] Optionally, the PH / Btk domain comprises a sequence as set forth in SEQ ID NO. 3, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the sequence as set forth in SEQ ID NO. 3;

[0061] Preferably, the Δ1-329 truncation comprises a sequence as set forth in SEQ ID NO. 1, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the sequence as set forth in SEQ ID NO. 1.

[0062] [9]. The use according to [8], wherein the recombinant RASA3 further comprises at least one of a C2A domain, a C2B domain;

[0063] Optionally, the C2A domain comprises a sequence as set forth in SEQ ID NO. 4, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the sequence as set forth in SEQ ID NO. 4;

[0064] Optionally, the C2B domain comprises a sequence as set forth in SEQ ID NO. 5, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the sequence as set forth in SEQ ID NO. 5.

[0065]

[10] . The use according to [8] or [9], wherein the CEBP / β inhibitor further comprises a polynucleotide encoding the recombinant RASA3, a vector carrying the recombinant RASA3 and / or a vector carrying a polynucleotide encoding the recombinant RASA3;

[0066] The vector comprises any one or more of a plasmid, an adeno-associated virus, a retrovirus, a liposome, a lipid nanoparticle, N-acetylgalactosamine, and an extracellular vesicle.

[0067] Effects of the Invention

[0068] The present invention discovered for the first time that RASA3 inhibits the production of inflammatory factor IL-6 caused by RNA virus infection by promoting the degradation of CEBP / β, thereby having the function of broadly inhibiting the production of inflammatory factors such as IL-6 caused by RNA virus infection.

[0069] In some aspects of the present invention, the amino acid sequence of RASA3, and the use of overexpression vectors containing RASA3 and truncated mutants thereof for inhibiting the production of inflammatory factors such as IL-6 caused by RNA viral infection are provided. In some embodiments, overexpression vectors containing RASA3 and truncated mutants thereof are used to inhibit the production of IL-6, an inflammatory factor caused by RNA viral infection, thereby suppressing the generation of an inflammatory cytokine storm. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Figure 3 is the vector map (pcDNA3.1) for overexpression of RASA3 and its truncated mutants (△1-329 and PH / Btk).

[0071] Figure 2 Screening and identification of Rasa3-cKO mice. Figure 2 A is the use of CRISPR / Cas9 system to insert flox into Rasa3 gene to obtain Rasa3 fl / + mice; Figure 2 B is Rasa3 fl / + After mating with LysM-Cre mice, Rasa3 fl / + ;LysM-Cre mice, and then Rasa3 fl / + ; LysM-Cre offspring were mated to obtain myeloid cell conditional knockout mice Rasa3 fl / fl ;LysM-Cre, i.e. Rasa3-cKO, WT mice are Rasa3 fl / fl ; Figure 2 C is the electrophoresis diagram of nucleic acid identification in mice.

[0072] Figure 3 RASA3 inhibits IL-6 transcription independently of the NF-κB and MAPK signaling pathways. Figure 3 A is the effect of RASA3 on IL-6 and TNF-α in mouse primary BMDM cells detected by RT-qPCR under VSV infection or Poly(I:C) stimulation; Figure 3 B is RT-qPCR detection of the effect of overexpression of RASA3 on IL-6 and TNF-α in A549 cells under VSV infection or Poly(I:C) stimulation; Figure 3 C is the effect of RASA3 on IL-6 and TNF-α in mouse primary BMDM cells under VSV infection or Poly(I:C) stimulation by ELISA; Figure 3 D is the effect of overexpression of RASA3 on IL-6 and TNF-α in A549 cells under VSV infection or Poly(I:C) stimulation detected by ELISA; Figure 3 E- Figure 3 F is the effect of knockout / overexpression of RASA3 on the activation of NF-κB, MAPK, AKT and STAT3 signaling pathways detected by Western Blot.

[0073] Figure 4 RASA3 overexpression suppressed the protein level of CEBP / β. Figure 4 A: Western Blot analysis of the effect of RASA3 on CEBP / β protein levels; Figure 4 B is the effect of RASA3 on CEBP / β mRNA level detected by RT-qPCR; Figure 4 C is the effect of RASA3 on CEBP / β protein level in A549 cell lines of FGEH (control group) and FGEH-RASA3 (overexpression group) detected by Western Blot. Figure 4 D is RT-qPCR detection of the effect of RASA3 on CEBP / β mRNA levels in A549 cell lines with FGEH (control group) and FGEH-RASA3 (overexpression group).

[0074] Figure 5 Protein interaction between RASA3 and CEBP / β. Figure 5 A- Figure 5 B is Co-IP detection of the interaction between RASA3 and CEBP / β; Figure 5 C is a schematic diagram of different truncated forms of RASA3; Figure 5 D is Co-IP detection of the interaction between different truncations of RASA3 and CEBP / β; Figure 5 E is Co-IP detection of the interaction between RASA3 domain GAP and PH / Btk and CEBP / β, with the truncated form △1-329 as a positive reference; Figure 5 F is the effect of RASA3-flag, truncated △PH / Btk, PH / Btk and △1-329 overexpression on CEBP / β protein level detected by Western Blot.

[0075] Figure 6 Determination of the half-life of RASA3-promoted CEBP / β degradation. Figure 6 A: Western Blot analysis of the effect of RASA3 on CEBP / β protein levels; Figure 6 B is Figure 6 Quantification and half-life calculation of A.

[0076] Figure 7RASA3 promotes autophagic degradation of CEBP / β. Figure 7 A shows how RASA3 promotes CEBP / β degradation under different inhibitor treatments as detected by Western Blot; Figure 7 B is the Western Blot analysis of how RASA3 promotes CEBP / β degradation under treatment with different concentrations of 3-MA.

[0077] Figure 8 RASA3 colocalizes with CEBP / β in the cytoplasm. Figure 8 A: Immunofluorescence detection of co-localization of RASA3 and CEBP / β; Figure 8 B is the use of software ImageJ Figure 8 A is quantified.

[0078] Figure 9 Interaction between RASA3 and CEBP / β in primary cells. Figure 9 A: The interaction between RASA3 and CEBP / β was detected using PLA assay in primary mouse cells; Figure 9 B is Figure 9 Quantitative statistics of A.

[0079] Figure 10 After knocking out RASA3 in bone marrow-derived macrophages (BMDM), CEBP / β protein levels were significantly increased.

[0080] Figure 11 RASA3 knockout exacerbated lung inflammation in the presence of VSV infection. Figure 11 A- Figure 11 B: Female wild-type (WT) mice and Rasa3-cKO mice were intraperitoneally infected with 5×10 7 Plaque-forming units (PFU) of VSV were added, and 6 hours later, the levels of Il-6, Tnfα, and viral RNA in lung tissue were detected by RT-qPCR, and the levels of IL-6, TNF-α, and IP-10 in serum were detected by ELISA. Mock was the uninfected group. Figure 11 C: Female WT mice and Rasa3-cKO mice were intraperitoneally infected with 5×10 7 HE staining results of lung tissue sections after 6 hours of infection with PFU of VSV. Mock is the uninfected group. Figure 11 D: WT mice and Rasa3-cKO mice were infected by tail vein with 1×10 8 PFU of VSV, and the survival status of the two groups of mice were statistically analyzed at different time points.

[0081] Figure 12RASA3 knockout aggravated lung inflammation in PR8 infection. Female WT mice and Rasa3-cKO mice were intranasally infected with 200 PFU of PR8, and 3 days later, the expression levels of IL-6 and TNF-a in lung tissues were detected by ELISA Figure 12 A), TCID 50 The viral titers in lung tissues were detected Figure 12 B), and the HE staining results of lung tissue sections Figure 12 C).

[0082] Figure 13 RASA3 knockout aggravated lung inflammation in RSV infection. Female WT mice and Rasa3-cKO mice were intranasally infected with 1 x 10 6 plaque forming units (PFU) of RSV, and 5 days later, the contents of Il-6, Tnf a and viral RNA in lung tissues were detected by RT-qPCR Figure 13 A), the contents of IL-6 and TNF-a in serum were detected by ELISA Figure 13 B), and the HE staining results of lung tissue sections Figure 13 C). DETAILED DESCRIPTION

[0083] Various illustrative embodiments, features and aspects of the present application are described below in detail. The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0084] In addition, for the purpose of convenience and brevity, detailed descriptions of well-known methods, apparatus, materials and steps are omitted in the following detailed description. The skilled person will appreciate that the present application can be practiced in the absence of these specific details. In other instances, methods, apparatus, materials and steps that would be

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The meaning of "in" and "d" should be understood as including the systematic errors that are inevitable in industrial production.

[0086] As used herein, the terms "comprises", "comprising", "includes", "including" and the like are specifically intended to be open-ended and do not exclude additional, unrecited elements or method steps.

[0087] Throughout this application the term "about" is used to indicate that a value includes the standard deviation of the error of the apparatus or method being used to determine the value.

[0088] Although the disclosure supports a definition of the term "or" as only alternatives as well as "and / or," the term "or" in the claims means "and / or" unless explicitly stated as only alternatives or as mutually exclusive between alternatives.

[0089] When used in the claims or description, a selected / optional / preferred "numerical range" includes both the numerical endpoints at both ends of the range and all natural numbers covered between the numerical endpoints relative to the aforementioned numerical endpoints.

[0090] As used in the present invention, the term "viral infection" refers to the process in which viruses invade the body through various pathways and proliferate in susceptible host cells. Viral pathogenicity begins by invading the host and infecting cells, and the pathogenic effects are manifested in both the body as a whole and the cells.

[0091] As used in the present invention, the term "inflammation caused by viral infection" refers to a pathological process in which, after the virus invades, the host immune system recognizes viral components (such as viral nucleic acids or proteins) and activates an inflammatory response by releasing mediators such as cytokines and chemokines to eliminate the virus, but at the same time may cause tissue damage due to excessive reaction.

[0092] As used herein, the term "inflammatory cytokine" refers to a class of bioactive molecules secreted by immune cells (e.g., macrophages, neutrophils, lymphocytes) or non-immune cells (e.g., endothelial cells, fibroblasts) in response to infection, tissue damage, or immune abnormalities. In some specific embodiments, the inflammatory cytokine is IL-6 and / or TNF-α.

[0093] As used herein, the term "RASA3" refers to RAS p21 protein activator 3. The RASA3 protein can be of human (Gene ID: 22821) or murine (Gene ID: 19414). However, those skilled in the art will appreciate that both murine and human RASA3 possess a C2A domain (positions 1-112), a C2B domain (positions 123-263), a GAP domain (positions 330-524), and a PH / Btk domain (positions 576-715). Among them, mouse RASA3 and human RASA3 have greater than 90% identity in the above-mentioned domains (C2A domain identity 97.3%; C2B domain identity 96.5%; GAP domain identity 95.4%; PH / Btk domain identity 92.1%). More specifically, the identity between mouse RASA3 and human RASA3 proteins is no less than 94%, for example, 94.84%. Those skilled in the art can reasonably expect that human RASA3 can achieve the same or substantially similar functions and effects as mouse RASA3 in the specific applications of the present invention.

[0094] As used in the present invention, the term "identity" means that if two sequences (proteins or nucleic acids) are the same length, then their identity is defined as the percentage of the number of identical residues (one letter, amino acid or base) at their corresponding positions in the total length, that is, identity = (number of identical characters / global alignment length) * 100%.

[0095] As used herein, the term "CEBP / β" refers to CCAAT / enhancer-binding protein beta (CEBP / β), a member of the CCAAT family of enhancer-binding proteins. It is a bZIP transcription factor with DNA binding and dimerization capabilities, capable of binding to specific DNA regulatory regions to regulate gene expression. CEBP / β plays a key role in immune and inflammatory responses, regulating the expression of proinflammatory cytokines such as IL-6 and IL-1β, and is a key regulator of IL-6 production.

[0096] As used herein, the term "treating" refers to exposing a subject to (e.g., administering) a recombinant RASA3, a polynucleotide encoding recombinant RASA3, a pharmaceutical composition, or the like of the present invention after the subject has contracted a disease, thereby partially or completely alleviating, ameliorating, alleviating, suppressing, delaying the onset of, reducing the severity of, and / or reducing the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition, compared to the absence of such exposure. It does not necessarily mean that the symptoms of the disease are necessarily suppressed. Suffering from a disease means that symptoms of the disease appear in the body.

[0097] As used in the present invention, the term "prevention" means that before a subject develops a disease, the subject is exposed to (e.g., administered) the recombinant RASA3, polynucleotides encoding recombinant RASA3, pharmaceutical compositions, etc. disclosed herein, thereby reducing the symptoms after developing the disease compared to when the subject does not develop the disease. It does not necessarily mean that the disease must be completely suppressed.

[0098] As used in the present invention, the term "pneumonia" refers to an inflammatory pathological process of the terminal airways, alveoli and lung interstitium caused by factors such as viral infection, which is characterized by congestion and edema of alveolar capillaries, fibrin exudation and inflammatory cell infiltration, accompanied by clinical symptoms such as respiratory dysfunction.

[0099] As used herein, the term "protein autophagy" refers to the promotion of protein degradation by activating or regulating the autophagic process in cells. This process can promote protein autophagy by acting on key regulatory proteins of protein autophagy (such as ULK1, Atg4B, and Beclin 1), or indirectly by regulating upstream signaling pathways (such as AMPK and SIRT3).

[0100] In the present invention, the "degradation of CEBP / β protein" includes degradation through the protein autophagy-lysosome pathway. Specifically, CEBP / β protein is first encapsulated into an autophagosome formed by a double-layer membrane structure. As the autophagosome fuses with the lysosome, CEBP / β is finally degraded into small molecules, thereby achieving the degradation of CEBP / β protein. The technical solution of the present invention is described in detail below:

[0101] The present invention reveals the regulatory role of RASA3 on the inflammatory response during RNA virus infection and its molecular mechanism through systematic in vitro cell experiments and in vivo animal model studies. Specifically, in primary macrophages of the RASA3-overexpressing A549 cell line and myeloid RASA3 knockout mice (Rasa3-cKO), RASA3 was found to significantly inhibit the transcription of IL-6 through VSV infection or Poly (I:C) stimulation, and this effect was independent of the NF-κB and MAPK signaling pathways. Further studies have shown that RASA3 interacts with the transcription factor CEBP / β through its △1-329 truncation and promotes the degradation of CEBP / β through the autophagy-lysosomal pathway, thereby inhibiting the transcriptional activation of IL-6 and STAT3 phosphorylation. In the myeloid RASA3 knockout mouse model, after VSV, PR8 or RSV infection, the IL-6 level in the mouse lungs or serum was significantly increased, accompanied by severe immune cell infiltration and tissue damage, and the survival rate of Rasa3-cKO mice was significantly reduced under VSV infection, while the viral load was not affected. Based on this, the present invention reveals that RASA3 exerts a broad-spectrum inhibitory effect on RNA virus-associated inflammation by targeting the CEBP / β-IL-6 axis, providing a new target and intervention strategy for the treatment of inflammatory diseases such as viral pneumonia. Given the central role of IL-6 in diseases such as viral pneumonia and cytokine release syndrome, the RASA3 regulatory network proposed in this invention provides a theoretical basis and potential drug targets for the development of broad-spectrum anti-inflammatory therapies, and has significant application prospects in the prevention and treatment of infectious inflammatory diseases.

[0102] <Recombinant RASA3>

[0103] In some embodiments, the present invention provides a recombinant RASA3, which can be obtained by conventional methods in the art, such as by biosynthetic preparation. The "recombinant RASA3" described in the present invention refers to a non-naturally occurring RASA3 protein, including but not limited to a protein prepared by biosynthetic means having the same amino acid sequence as a RASA3 protein, a protein prepared by biosynthetic means having an amino acid sequence obtained by substituting, deleting, and / or adding one or more amino acids to the amino acid sequence of a natural (e.g., human or mouse) RASA3 protein, a protein prepared by biosynthetic means having a functionally active amino acid sequence fragment of a natural (e.g., human or mouse) RASA3 protein, etc.

[0104] In some specific embodiments, the Δ1-329 truncation promotes the degradation of CEBP / β protein through the interaction of CEBP / β protein. For example, the degradation of CEBP / β protein can be inhibited by the autophagy inhibitors CQ or 3-MA.

[0105] In some preferred embodiments, the degradation of the CEBP / β protein comprises degradation via the autophagy-lysosome pathway.

[0106] In some embodiments, the recombinant RASA3 comprises a Δ1-329 truncation that interacts with the CEBP / β protein; the Δ1-329 truncation promotes the degradation of the CEBP / β protein by interacting with the CEBP / β protein; preferably, the degradation of the CEBP / β protein includes degradation through the protein autophagy-lysosomal pathway; the Δ1-329 truncation comprises the PH / Btk domain of RASA3, and optionally the GAP domain of RASA3.

[0107] In some optional embodiments, the GAP domain comprises a sequence as shown in SEQ ID NO.2, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the sequence shown in SEQ ID NO.2.

[0108] In some optional embodiments, the PH / Btk domain comprises a sequence as shown in SEQ ID NO. 3, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity to the sequence shown in SEQ ID NO. 3.

[0109] In some embodiments, the Δ1-329 truncation comprises a sequence as shown in SEQ ID NO.1, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the sequence shown in SEQ ID NO.1.

[0110] Furthermore, the recombinant RASA3 may also contain at least one of the C2A domain and the C2B domain.

[0111] In some optional embodiments, the C2A domain comprises a sequence as shown in SEQ ID NO.4, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the sequence shown in SEQ ID NO.4.

[0112] In some optional embodiments, the C2B domain comprises a sequence as shown in SEQ ID NO.5, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the sequence shown in SEQ ID NO.5.

[0113] In some specific embodiments, the recombinant RASA3 comprises a sequence as set forth in SEQ ID NO. 6.

[0114] In some alternative embodiments, the recombinant RASA3 comprises a sequence as set forth in SEQ ID NO. 7.

[0115] <Products for preventing and / or treating diseases and / or symptoms associated with viral infection>

[0116] The present application provides products prepared by using recombinant RASA3, which can inhibit the level of inflammatory factors in the body by promoting the degradation of CEBP / β, and can be used for preventing and / or treating diseases and / or symptoms associated with viral infection.

[0117] (Products)

[0118] In some embodiments of the present application, the products comprise the above-mentioned <recombinant RASA3>, polynucleotides encoding the recombinant RASA3, vectors carrying the recombinant RASA3, and vectors carrying polynucleotides encoding the recombinant RASA3.

[0119] The vectors include any one or more of plasmids, adeno-associated viruses, retroviruses, liposomes, lipid nanoparticles, N-acetylgalactosamine, and extracellular vesicles; in some alternative embodiments, the plasmids include eukaryotic expression plasmids, shuttle plasmids; the retroviruses include lentiviruses, alpha-retroviruses, foamy viruses; the adeno-associated viruses include natural serotype adeno-associated viruses, recombinant serotype adeno-associated viruses, tissue-specific adeno-associated viruses; the liposomes include single-layer liposomes, multi-layer liposomes; the lipid nanoparticles include solid lipid nanoparticles, cationic lipid nanoparticles.

[0120] In some specific embodiments, the plasmids include pcDNA3.1, pFGEH.

[0121] In some embodiments, the products can inhibit the expression of inflammatory factors.

[0122] In some embodiments, the products comprise drugs that promote the degradation of CEBP / β protein through the autophagy-lysosome pathway.

[0123] In some alternative embodiments, the drugs that promote the degradation of CEBP / β protein through the autophagy-lysosome pathway include mTOR inhibitors, AMPK activators, natural compounds; illustratively, the natural compounds include curcumin and / or resveratrol.

[0124] In some embodiments, the products include drugs or pharmaceutical compositions.

[0125] In some optional embodiments, the pharmaceutical composition further includes one or more pharmaceutically acceptable carriers; further, the pharmaceutically acceptable carrier includes one or a combination of two or more of solvents, solubilizers, cosolvents, emulsifiers, flavoring agents, olfactory agents, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, surfactants and preservatives.

[0126] In some preferred embodiments, the pharmaceutically acceptable carrier is a stabilizer. By combining the stabilizer with the polynucleotide having antiviral activity, the polynucleotide can be made to have a drug stability suitable for clinical use.

[0127] In some embodiments, the polynucleotide having antiviral activity is mixed with one or more pharmaceutically acceptable carriers and then formulated into a solid, semi-solid, or liquid preparation. Suitable solid preparations include tablets, capsules, granules, and pills; suitable semi-solid preparations include gels, suppositories, and ointments; and suitable liquid preparations include emulsions, mixtures, suspensions, solutions, and injections.

[0128] (Diseases and / or symptoms associated with viral infection)

[0129] In the present invention, the virus is an RNA virus. In some embodiments, the virus includes at least one of influenza virus, coronavirus, novel coronavirus, adenovirus, respiratory syncytial virus and vesicular stomatitis virus.

[0130] In some preferred embodiments, the virus is vesicular stomatitis virus and / or influenza virus. Further, the vesicular stomatitis virus includes New Jersey vesicular stomatitis virus and Indiana vesicular stomatitis virus; the influenza virus includes influenza A virus, influenza B virus, influenza C virus, and influenza D virus. In some more preferred embodiments, the influenza virus is influenza A virus A / PR / 8 / 34 strain.

[0131] In some specific embodiments, the viral infection-related diseases and / or symptoms include at least one of the following (i) to (iv):

[0132] (i) Increased expression of inflammatory factors in the body;

[0133] (ii) increased immune cell infiltration;

[0134] (iii) inflammatory damage caused by infection;

[0135] (iv) Inflammatory diseases caused by viral infection.

[0136] In some alternative embodiments, the inflammatory factors include IL-6, IL-1β, IP-10, TNF-α.

[0137] In some alternative embodiments, the inflammatory injury refers to tissue / cell injury caused by over-activation or dysregulation of the immune system. Further, the inflammatory injury includes inflammatory injury of the respiratory system, particularly the lung; the inflammatory injury of the lung includes acute inflammatory injury, such as acute lung injury, pulmonary inflammatory edema, acute respiratory distress syndrome, etc.

[0138] In some alternative embodiments, the inflammatory disease includes pneumonia, particularly acute inflammatory pneumonia.

[0139] In some alternative embodiments, the organism is an individual having the potential to suffer from a disease and / or symptoms associated with viral infection; the individual includes mouse, rat, guinea pig, cow, sheep, cat, dog, horse, rabbit, pig, monkey and human.

[0140] <Use of recombinant RASA3 in preparing CEBP / β inhibitor>

[0141] According to some embodiments of the present application, a CEBP / β inhibitor is provided, which comprises one or more of the <recombinant RASA3>, polynucleotide encoding the recombinant RASA3, vector carrying the recombinant RASA3 and vector carrying the polynucleotide encoding the recombinant RASA3.

[0142] In some embodiments, the vector includes any one or more of plasmid, adeno-associated virus, retrovirus, liposome, lipid nanoparticle, N-acetylgalactosamine and extracellular vesicle; in some alternative embodiments, the plasmid includes eukaryotic expression plasmid, shuttle plasmid; the retrovirus includes lentivirus, alpha-retrovirus, foamy virus; the adeno-associated virus includes natural serotype adeno-associated virus, recombinant serotype adeno-associated virus, tissue-specific adeno-associated virus; the liposome includes single-layer liposome, multi-layer liposome; the lipid nanoparticle includes solid lipid nanoparticle, cationic lipid nanoparticle.

[0143] In some alternative embodiments, the drug promoting autophagic degradation of CEBP / β protein includes mTOR inhibitor, AMPK activator, natural compound; illustratively, the natural compound includes curcumin and / or resveratrol.

[0144] In some embodiments, the CEBP / β inhibitor is effective in inhibiting the protein activity of CEBP / β, reducing the expression of pro-inflammatory factors such as IL-6, IL-1β, etc., thereby alleviating the immune response and combating viral infection.

[0145] In some specific embodiments, the virus is an RNA virus. In some embodiments, the virus includes at least one of influenza virus, coronavirus, novel coronavirus, adenovirus, respiratory syncytial virus and vesicular stomatitis virus.

[0146] In some preferred embodiments, the virus is vesicular stomatitis virus and / or influenza virus. Further, the vesicular stomatitis virus includes New Jersey vesicular stomatitis virus and Indiana vesicular stomatitis virus; the influenza virus includes influenza A virus, influenza B virus, influenza C virus, and influenza D virus. In some more preferred embodiments, the influenza virus is influenza A virus A / PR / 8 / 34 strain.

[0147] Example

[0148] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples (although indicating specific embodiments of the present invention) are given for illustrative purposes only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art after reading this detailed description.

[0149] The following is described in conjunction with specific embodiments. Reagents and samples, etc. are commercially available or available to the public in other ways. These are merely examples and are not exclusive to the present invention. Other suitable tools and biomaterials can be used instead. The experimental procedures involved can be performed according to the conditions and methods described in "Molecular Cloning Experimental Guide (3rd Edition)" (Science Press, 2002), and can be performed according to the manufacturer's instructions for commercial enzymes and test kits. Other test methods not described in detail are conventional methods well known to those skilled in the art unless otherwise specified.

[0150] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0151] Example 1: Experimental methods involved in Examples 2 to 8

[0152] 1.1 Construction of pFGEH-RASA3 plasmid

[0153] RASA3 gene primer design: The RASA3 gene (Gene ID: 22821) was searched on NCBI. Based on the coding sequence (CDS) region, approximately 20 bp of DNA sequences at the 5' and 3' ends of the coding sequence were selected as upstream and downstream primer seed sequences. Approximately 15 bp of vector homology arms and restriction sites were introduced at the 5' ends of the upstream and downstream primers, and a flag tag was inserted into the downstream primer. The primer information is as follows:

[0154] pFGEH-RASA3-F: 5'-ACCGCCGCGATCGCCGGCGCGCCACCATGGCGGTGGAGGACGA-3';

[0155] pFGEH-RASA3-R: 5'-CCCCTACCCGGTAGATTAATTAATCACTTATCGTCGTCATCCTTGTAATCACGCGTAATGGAATGAGTGGAGGTCT-3'

[0156] Target fragment amplification: freshly prepared A549 cDNA was used as template. PCR amplification was performed using High-Fidelity 2× Master Mix. Prepare the reaction solution according to the instructions for High-Fidelity 2× Master Mix. Mix the above PCR components and place in a PCR instrument for PCR reaction. After the PCR reaction is completed, perform nucleic acid electrophoresis to recover the target fragment.

[0157] Vector linearization: pFGEH vector (sequence shown in SEQ ID NO. 11) was double-digested with restriction endonucleases AscI and PacI at 37°C for 2 hours. After digestion, the target fragment was recovered by nucleic acid gel electrophoresis.

[0158] Recombination reaction: Use the Norwegian homologous recombination cloning kit to perform homologous recombination reaction between the target fragment and the linearized plasmid. After the reaction, immediately place it on ice or store it at 4°C.

[0159] The ligation products were transformed and plated using DH5α competent cells, positive clones were identified, sequenced, and plasmids were extracted.

[0160] 1.2 Construction and identification of Rasa3-cKO mice

[0161] (1) Rasa3 flox / +Mice (C57BL / 6J background) were constructed by Nanjing Biotech Co., Ltd. Based on the genomic fragment where Rasa3 (Gene ID: 19414) is located, using the principle of homologous recombination, the fertilized egg homologous recombination method was used to insert flox into the Rasa3 gene. First, Cas9 mRNA and sgRNA were obtained by in vitro transcription, and then a homologous recombination vector (Donor vector) was constructed by In-Fusion cloning method, which contains 5'-2.9kb homologous arm, 0.6kb flox region and 3'-3.0kb homologous arm. Finally, Cas9 mRNA (the corresponding DNA template sequence is shown in SEQ ID NO: 12), sgRNA and Donor vector were microinjected into the fertilized eggs of C57BL / 6J mice to obtain F0 mice. PCR amplification and sequencing were used to identify positive F0 mice, which were mated with C57BL / 6J mice to obtain positive F1 mice. The pair of guide RNAs (small guide RNA, sgRNA) used in the experiment are as follows: sgRNA1: 5'-CCCACAAACCTGCCTGCCCT-3'; sgRNA2: 5'-TTCGTGAGGCTACAACCATC-3'.

[0162] (2) After receiving the mice, the mouse genomic DNA was extracted using the tissue DNA rapid extraction kit, and PCR identification was performed.

[0163] (3) Rasa3 flox / + Mouse PCR identification primer.

[0164] Table 1 Rasa3 flox / + Mouse identification primer

[0165]

[0166] (4) According to the 2x Taq enzyme instruction manual of Novozyme, the PCR reaction system was configured, and PCR amplification was performed:

[0167] Table 2 PCR reaction system

[0168]

[0169] The above PCR reaction system was mixed and centrifuged briefly and placed in a PCR instrument for PCR amplification,

[0170] Table 3 PCR amplification program

[0171]

[0172] After PCR amplification, nucleic acid electrophoresis was performed for verification. WT: one band, 306 bp in length; heterozygote: two bands, 306 bp and 372 bp in length, respectively; homozygote: one band, 372 bp in length.

[0173] (5) Screening and identification of Rasa3-cKO mice

[0174] Rasa3 identified by (4) flox / + The heterozygous mice were bred with homozygous LysM-Cre mice in the laboratory, and PCR identified the Rasa3 flox / + ; LysM-Cre (i.e. Figure 2 Rasa3 in fl / + ; LysM-Cre), named F0.

[0175] Table 4 LysM-Cre mouse identification primers

[0176]

[0177] The F0 mice were mated and the second generation of mice Rasa3 was identified by PCR. flox / flox ; LysM-Cre, named F1, the F1 generation mice were further bred and identified, and positive F2 generation mice were obtained for subsequent experiments ( Figure 2 ).

[0178] 1.3 Cell extraction and culture

[0179] 1.3.1 BMDM extraction and culture

[0180] (1) Prepare RPMI-1640 complete medium (containing 10% FBS, 1% P / S, and 1% GMG14-12 supernatant) for culturing BMDM in advance. Prepare sterile PBS, a 1ml syringe, sterile surgical instruments (scissors, forceps), a 70μm sterile filter, red blood cell lysis buffer, several centrifuge tubes, and several cell culture dishes.

[0181] (2) Eight-week-old WT mice (C57BL / 6J background) or Rasa3-cKO mice were sacrificed by cervical dislocation and disinfected by soaking in 75% ethanol for 5 minutes. In a biosafety cabinet, the legs of the mice were cut and the femur and tibia were removed. The meat was removed and soaked in PBS in a cell culture dish.

[0182] (3) Slightly cut a small section at each end of the femur and tibia to expose the bone marrow cavity. Use a 1 mL syringe to draw RPMI-1640 complete medium and blow into the bone marrow cavity from one end. Repeat the flushing several times until the bone cavity turns white. Collect the cell suspension into a 15 mL centrifuge tube and centrifuge at 1500 rpm at room temperature for 5 minutes to collect the cell pellet.

[0183] (4) Discard the supernatant, add 1 mL of red blood cell lysis buffer to resuspend the cells, let it stand at room temperature for 3-5 minutes, add 3 mL of RPMI-1640 complete medium and pipette to mix thoroughly. Filter the cell suspension through a filter and centrifuge at 1500 rpm for 5 minutes at room temperature to obtain a cell pellet.

[0184] (5) Resuspend the cells in the pre-prepared RPMI-1640 complete medium, count and plate the cells, and then culture them in a 37°C, 5% CO2 incubator. On the third day, add an equal volume of RPMI-1640 complete medium, and on the fifth day, change the medium. After the BMDM mature on the seventh day, subsequent experiments can be performed.

[0185] 1.3.2 A549 cell culture

[0186] (1) Preheat a water bath to 37°C in advance. Prepare complete DMEM culture medium and preheat it in a 37°C water bath.

[0187] (2) Take a new 15ml centrifuge tube in advance and add 3mL of preheated culture medium.

[0188] (3) Remove the stored A549 cells from the liquid nitrogen tank and quickly transfer them to a water bath. Gently shake them to thaw them quickly. After the cells are thawed, spray them with alcohol and transfer them to a biosafety cabinet.

[0189] (4) Wipe the cryopreservation tube with an alcohol cotton ball, unscrew the bottle cap, aspirate the frozen cells into the centrifuge tube in step (2), centrifuge at 800 rpm at room temperature for 3 minutes, discard the supernatant and add fresh DMEM complete medium to resuspend the cells.

[0190] (5) The cell suspension was transferred to a new cell culture dish and cultured in a 37°C, 5% CO2 incubator.

[0191] 1.4 Plasmid construction

[0192] (1) Plasmids are as follows: RASA3-flag, RASA3-ΔC2A-flag, RASA3-ΔC2B-flag, RASA3-ΔGAP-flag, RASA3-ΔPH / Btk-flag, RASA3-Δ1-329-flag, RASA3-Δ525-834-flag, GAP-flag, PH / Btk-flag, CEBP / β-HA.

[0193] PCR primers are as follows

[0194] Table 5 PCR primers

[0195]

[0196] (2) RASA3 truncated plasmid primer design: search RASA3 gene (Gene ID: 19414) on NCBI, and manually design RASA3 PCR amplification primers according to the primer design principle. The RASA3 domain is shown in C, and the amplification primers of truncated plasmid RASA3-flag, RASA3-ΔC2A-flag, RASA3-ΔC2B-flag, RASA3-ΔGAP-flag, RASA3-ΔPH / Btk-flag, RASA3-Δ1-329-flag, RASA3-Δ525-834-flag, GAP-flag and PH / Btk-flag are designed according to different domains. Figure 5

[0197] (3) Cebpb amplification primer design: according to the sequence of CEBP / β-HA vector and the sequence of Cebpb gene, the amplification primers are designed.

[0198] (4) Target gene fragment amplification: Rasa3 gene and Cebpb gene are amplified using cDNA of BMDM cells as template.

[0199] (5) Vector construction and subcloning: the amplified Rasa3 gene is connected to the multiple cloning enzyme cutting site of pcDNA3.1 vector, and the recombinant plasmid containing RASA3-flag fragment is constructed (RASA3-flag), and after the recombinant plasmid is sequenced correctly, the subcloning is carried out by using the primers in Table 5 and the recombinant plasmid containing RASA3-flag fragment as template, and the recombinant plasmid containing different truncated bodies is obtained by sequencing and plasmid extraction. Figure 1

[0200] The amplified Cebpb gene is connected to the multiple cloning enzyme cutting site of pcDNA3.1 vector, and the recombinant plasmid containing Cebpb gene is constructed, and the CEBP / β overexpression plasmid is obtained by sequencing and plasmid extraction.

[0201] 1.5 Co-Immunoprecipitation (Co-IP)

[0202] 1.5.1 Sample preparation

[0203] (1) The cells were plated in 6 cm culture dishes, and when the cell density reached 70%-85%, the corresponding treatment and plasmid transfection were carried out, and the medium was changed after 8 hours of transfection.

[0204] ​​(2) After 24 hours of transfection, wash the cells with PBS, discard the PBS, add 500 μL of IP lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA and 1% NP-40), and add phosphatase and protease inhibitors, and lyse for 30 minutes on ice.

[0205] (3) Scrape the cells with a cell scraper, and transfer them to a 1.5 mL centrifuge tube using a pipette, and centrifuge at 15000 rpm for 10 minutes at 4°C, and transfer the supernatant to a new 1.5 mL centrifuge tube for subsequent experiments, and reserve 30 μL for later WCL (whole cell lysis) detection.

[0206] 1.5.2 Co-IP

[0207] (1) Wash the magnetic beads three times with IP lysis buffer, and then add the lysate of step (3) of 1.5.1, and incubate overnight at 4°C on a rotary shaker.

[0208] (2) The next day, use a magnetic stand to adsorb the magnetic beads, discard the supernatant, and add 1 mL of cold IP lysis buffer, and wash for 5 minutes at 4°C on a rotary shaker.

[0209] (3) Repeat step (2) three times.

[0210] (4) After washing, add 1x SDS loading buffer, and denature by boiling at 100°C for 10 minutes. Add 10 μL of 4x SDS loading buffer to the WCL, and denature by boiling for Western Blot detection.

[0211] 1.6 Cell immunofluorescence detection

[0212] 1.6.1 Cell preparation

[0213] (1) Uniformly spread mouse BMDM cells or HEK293T cells in a confocal special cell culture dish, and incubate in a cell incubator at 37°C and 5% CO2.

[0214] (2) The next day, when the cell density reaches about 60-70%, perform plasmid transfection (BMDM cells are not treated), and after 8 hours of transfection, replace the fresh culture medium and continue to culture, and perform experiments 24 hours after plasmid transfection.

[0215] 1.6.2 Fixation, permeabilization and blocking

[0216] (1) Fixation: remove the confocal dish, discard the culture medium, add 1 mL of PBS, and wash twice on a shaker at the lowest speed for 5 minutes each time, discard the PBS, and add 1 mL of 4% paraformaldehyde solution, and fix at room temperature for 15-20 minutes.

[0217] (2) Permeabilization: Discard the paraformaldehyde, add 1 mL of PBS and place on a shaker at the lowest speed to wash three times, each time for 5 minutes. Discard the PBS and add 1 mL of 0.2% Triton X-100 solution. Permeabilize at room temperature for 10-15 minutes.

[0218] (3) Blocking: Discard Triton X-100, add 1 mL of PBS and place on a shaker at the lowest speed to wash three times, 5 minutes each time, discard the PBS, add 100 μL of 10% goat serum to the central chamber of the confocal dish, and block in a 37°C incubator for 1 hour.

[0219] 1.6.3 Antibody incubation

[0220] (1) Primary antibody incubation: After blocking, remove the blocking solution with a pipette, add the pre-prepared primary antibody diluent (diluted in 5% goat serum according to the antibody instructions) into the central chamber, place in a humidified chamber, and incubate overnight at 4°C.

[0221] (2) Secondary antibody incubation: Use a pipette to recover the primary antibody, add 1 mL of PBS and place on a shaker at the lowest speed to wash three times, 5 minutes each time. Discard the PBS and add fluorescent secondary antibody (fluorescent secondary antibody: 5% goat serum = 1:100) to the central chamber. Place in a humidified box and incubate in a 37°C incubator in the dark for 2 hours.

[0222] 1.6.4 Nuclear staining

[0223] Remove the secondary antibody with a pipette, add 1 mL of PBS and wash three times on a shaker at the lowest speed for 5 minutes each time. Discard the PBS and add the pre-prepared DAPI staining solution (DAPI:PBS=1:200) to the central chamber and place it in a wet box. Incubate at room temperature in the dark for 10-15 minutes.

[0224] 1.6.5 Fluorescence Image Acquisition

[0225] Use a pipette to remove the DAPI stain, add 1 mL of PBS, and wash three times on a shaker at the lowest speed, each time for 5 minutes. Discard the PBS, add 1 mL of PBS, and use a Leica TCA SP8 laser confocal microscope to collect fluorescence images.

[0226] 1.7. Proximity ligation assay (PLA)

[0227] In the present invention, the kit used for PLA is derived from Sigma's Duolink in situ PLA probe anti-rabbit PLUSDUO92002-100RUN and Sigma's Duolink in situ PLA probe anti-mouse MINUSDUO92004-100RUN.

[0228] (1) Cell preparation: Mouse BMDM cells were prepared at 2 x 10 5 Inoculate into the confocal special small dish, the third day to supplement fresh culture medium, the 5th day to replace fresh culture medium, the 7th day to carry out experiment.

[0229] (2) Fixation: After the BMDM matures on the 7th day, discard the culture medium, add 1 mL PBS and place in the shaker at the lowest speed for 3 times, 5 minutes each time, discard the PBS, add 1 mL of 4% paraformaldehyde solution and fix at room temperature for 15-20 minutes.

[0230] (3) Permeation: After the fixation is completed, discard the 4% paraformaldehyde solution, add 1 mL PBS and place in the shaker at the lowest speed for 3 times, 5 minutes each time, discard the PBS, add 1 mL of 0.2% Triton X-100 and permeate the membrane at room temperature for 10-15 min.

[0231] (4) Blocking: After the permeation is completed, discard the 4% paraformaldehyde solution, add 1 mL PBS and place in the shaker at the lowest speed for 3 times, 5 minutes each time, discard the PBS, add two drops of Blocking buffer in the central chamber groove and block at 37°C for 0.5 hours.

[0232] (7) Primary antibody incubation: After the blocking is completed, use a pipette to suck off the blocking liquid, add the prepared primary antibody (antibody: special antibody diluent = 1:50) in the central chamber groove, place in a wet box and incubate at 4°C overnight.

[0233] (8) Secondary antibody incubation: After the primary antibody incubation is completed, recover the primary antibody, add 1 mL of Buffer A, place in the shaker at the lowest speed for 3 times, 5 minutes each time, discard the Buffer A, add special secondary antibody (antibody: special antibody diluent = 1:5), place in a wet box and incubate at 37°C for 1 hour.

[0234] (9) Ligation: According to the requirements of the kit, the ligation reaction system is as follows:

[0235] Table 6 Ligation reaction system

[0236]

[0237] Place the above system in a 37°C incubator for 30 minutes. After the reaction is completed, wash with Buffer A for 2 times, 2 min each time.

[0238] (10) PCR amplification: The PCR amplification system is as follows:

[0239] Table 7 PCR amplification system

[0240]

[0241] The PCR system was placed in a 37°C incubator for 100 minutes. After the reaction, the cells were washed once with Buffer B for 10 minutes.

[0242] (11) Nuclear staining: Remove Buffer B with a pipette, add 1 mL of PBS and wash three times on a shaker at the lowest speed, 5 minutes each time. Discard the PBS and add the pre-prepared DAPI staining solution (DAPI:PBS=1:200) to the groove of the central chamber and place it in a wet box. Incubate at room temperature in the dark for 10-15 minutes.

[0243] (12) Fluorescence image acquisition: Use a pipette to remove the DAPI staining solution, add 1 mL of PBS, and place on a shaker at the lowest speed to wash three times, each time for 5 minutes. Discard the PBS, add 1 mL of PBS, and use a Leica TCASP8 laser confocal microscope to collect fluorescence images.

[0244] Example 2: RASA3 inhibits IL-6 transcription independently of NF-κB and MAPK signaling pathways

[0245] BMDM cells from 8-week-old WT mice (C57BL / 6J background) and Rasa3-cKO mice were obtained. After the BMDM matured on the seventh day, they were infected with VSV (MOI 0.5) for 4 and 8 hours and stimulated with poly(I:C) (1μg / mL) for 8 hours. The expression levels of IL-6 and TNF-α were measured by RT-qPCR and ELISA. A549 cells containing FGEH (control group) and FGEH-RASA3 (overexpression group) were plated and, when the cell density reached approximately 70%, infected with VSV (MOI 0.2) for 4 and 8 hours and stimulated with poly(I:C) (1μg / mL) for 8 hours. The expression levels of IL-6 and TNF-α were measured by RT-qPCR and ELISA. WT and Rasa3-cKO BMDMs were infected with VSV (MOI 0.5), and the control group and RASA3 overexpression group cell lines were infected with VSV (MOI 0.2). Western Blot was performed 4h and 8h later to detect the total protein levels and phosphorylation levels of NF-κB, MAPK, AKT signaling pathways and STAT3.

[0246] The results showed that RASA3 knockout promoted the expression of IL-6 under VSV infection or poly(I:C) stimulation, but had no effect on TNF-α ( Figure 3 A, Figure 3 C), overexpression of RASA3 can inhibit the expression of IL-6 without affecting the expression of TNF-α ( Figure 3 B, Figure 3D), RASA3 knockout or overexpression had no significant effect on NF-κB, MAPK and AKT signaling pathways, but as expected, RASA3 significantly affected the phosphorylation level of STAT3 ( Figure 3 E- Figure 3 F).

[0247] These results indicate that RASA3 inhibits IL-6 transcription independently of the NF-κB and MAPK signaling pathways.

[0248] Example 3: RASA3 overexpression inhibits CEBP / β protein levels

[0249] A549 cells were co-transfected with RASA3-flag and CEBP / β-HA overexpression plasmids. The transfection amount of CEBP / β-HA was 500 ng, and the transfection amount of RASA3-flag was 0, 500, 1000, and 2000 ng, respectively. CEBP / β transcript and protein levels were determined by RT-qPCR and Western blot. FGEH (control) and FGEH-RASA3 (overexpression) cell lines were infected with VSV (MOI 0.2). CEBP / β transcript and protein levels were determined by RT-qPCR and Western blot 4 and 8 hours later.

[0250] The results showed that the gradient overexpression of RASA3 did not affect the transcription level of CEBP / β ( Figure 4 B), but significantly inhibited the protein level of CEBP / β ( Figure 4 A), Under VSV infection conditions, overexpression of RASA3 still does not affect the transcription level of CEBP / β ( Figure 4 D), but significantly inhibited the protein level of CEBP / β ( Figure 4 B).

[0251] The results showed that RASA3 overexpression inhibited the protein level of CEBP / β.

[0252] Example 4: Protein Interaction between RASA3 and CEBP / β

[0253] HEK293T cells were transfected with CEBP / β-HA (500 ng) and the cells were Figure 5 The cells were transfected with or without RASA3-flag (1 μg) as shown in A. After 24 hours, the cell protein samples were collected with IP lysis buffer and incubated with flag magnetic beads. The interaction between RASA3 and CEBP / β in the cells was analyzed by Western Blot. Figure 5Cells were transfected or not with CEBP / β-HA (500 ng) as shown in B. After 24 hours, cell protein samples were collected using IP lysis buffer and incubated with HA magnetic beads. The interaction between RASA3 and CEBP / β in cells was analyzed by Western blot. HEK293T cells were plated in 6 cm culture dishes. When the cell density reached 70%-85%, the full-length and truncated mutants of RASA3-flag (1 μg) were co-transfected with CEBP / β-HA (1 μg) for 18 hours. The cells were then treated with the lysosomal inhibitor CQ (5 μM) for 6 hours. Cell protein samples were collected using IP lysis buffer and incubated with flag magnetic beads. The interaction domains of CEBP / β and RASA3 in cells were analyzed by Western blot. A549 cells were co-transfected with RASA3-flag, ΔPH / Btk, PH / Btk (as shown in SEQ ID NO. 10), Δ1-329 (as shown in SEQ ID NO. 9), and a CEBP / β-HA overexpression plasmid. The amount of CEBP / β-HA overexpression plasmid was 500 ng, and the amount of RASA3-flag and its truncated form overexpression plasmid was 2000 ng. CEBP / β protein levels were detected by Western Blot. The results showed: Figure 5 A- Figure 5 B is the Co-IP result of RASA3 and CEBP / β; Figure 5 C is a schematic diagram of different truncated forms of RASA3; Figure 5 D is the Co-IP results of different truncations of RASA3 and CEBP / β; Figure 5 E is the protein Co-IP result of the GAP and PH / Btk domains of RASA3 with CEBP / β, in which the truncated form of RASA3 △1-329 and CEBP / β were used as positive references. Figure 5 F is the truncated form of RASA3△1-329, which significantly inhibited the protein level of CEBP / β.

[0254] The results showed that RASA3 △1-329 and PH / Btk truncation interacted with CEBP / β protein, and overexpression of truncated △1-329 inhibited the protein level of CEBP / β.

[0255] Example 5: RASA3 promotes CEBP / β degradation

[0256] A549 cells were plated in 6 cm culture dishes and transfected with plasmid CEBP / β-HA (500 ng) when the cell density reached 70%-85%. Figure 6Plasmid RASA3-flag (2 μg) was transfected or not as shown. 24 h after transfection, CHX (100 ng / mL) was treated at specific time points, and CEBP / β protein levels were detected by Western Blot, and statistical analysis of CEBP / β protein half-life was performed.

[0257] The results show: Figure 6 A shows the effect of RASA3 on the protein level of CEBP / β at different time points after CHX treatment; Figure 6 B is the use of ImageJ software Figure 6 Western Blot results of A were quantified and statistically analyzed, and half-life was calculated.

[0258] The results indicate that RASA3 promotes CEBP / β degradation.

[0259] Example 6: RASA3 promotes autophagic degradation of CEBP / β

[0260] A549 cells were plated in 6 cm culture dishes and transfected with CEBP / β-HA (500 ng) when the cell density reached 70%-85%. Figure 7 The cells were transfected with or without RASA3-flag (2 μg) as shown in the figure. 24 h after transfection, the cells were treated with the nascent protein synthesis inhibitor CHX (100 ng / mL) and the proteasome inhibitors MG132 (10 μM), Ps-341 (0.5 μM), the lysosomal inhibitor PepA (10 μM), and CQ (5 μM), respectively, for 8 h, or co-treated with the nascent protein synthesis inhibitor CHX (100 ng / mL) and the autophagy inhibitor 3-MA (1 mM or 3 mM; MCE, Cat. No. HY-19312) for 8 h. CEBP / β-HA protein expression was detected by Western Blot.

[0261] The results show: Figure 7 A is Western Blot analysis of the effect of RASA3 on promoting CEBP / β degradation under different inhibitor treatments; Figure 7 B is Western Blot detection of the effect of RASA3 in promoting CEBP / β degradation under different concentrations of 3-MA treatment.

[0262] These results indicate that RASA3 promotes CEBP / β degradation through the autophagy-lysosome pathway.

[0263] Example 7: RASA3 and CEBP / β Cytoplasmic Colocalization

[0264] A549 cells were plated on a confocal microplate and the RASA3 and CEBP / β overexpression plasmids (such as Figure 8) were transfected and treated with lysosomal inhibitor CQ (5 μM) 24 hours later (as shown in Figure 8 Immunofluorescence experiments were performed 8 hours later using mouse RASA3 and rabbit CEBP / β antibodies. Immunofluorescence images were collected and statistically analyzed after the experiment.

[0265] The results show: Figure 8 A: Immunofluorescence detection of colocalization of RASA3 and CEBP / β; Figure 8 B is the use of ImageJ software Figure 8 Yellow fluorescence quantification statistics of A.

[0266] The results showed that RASA3 and CEBP / β colocalized in the cytoplasm to a large extent, and the colocalization in the cytoplasm increased significantly after CQ treatment, indicating that RASA3 caused CEBP / β protein degradation, thereby reducing the interaction, but by inhibiting protein degradation by CQ, the colocalization of RASA3 and CEBP / β increased.

[0267] Example 8: RASA3 interacts with CEBP / β in primary cells

[0268] BMDM cells from 8-week-old WT mice (C57BL / 6J background) were plated on confocal microplates. PLA experiments were performed after the BMDM matured on the seventh day. The experimental group was incubated with mouse RASA3 and rabbit CEBP / β antibodies, while the control group was incubated with mouse RASA3 antibody. After the experiment, immunofluorescence images were collected and statistically analyzed.

[0269] The results show: Figure 9 A: RASA3 interacts with CEBP / β in BMDM cells; Figure 9 B is for Figure 9 The experimental results of A are quantitatively analyzed.

[0270] The results indicate that RASA3 interacts with CEBP / β in primary BMDM cells.

[0271] Example 9: CEBP / β protein levels significantly increased after RASA3 knockout in primary BMDM cells

[0272] BMDM cells from 8-week-old WT mice (C57BL / 6J background) and Rasa3-cKO mice were obtained. After the BMDM matured on the seventh day, they were infected with VSV (MOI = 0.5). The expression of RASA3 and CEBP / β was detected by Western Blot, with GAPDH as an internal control.

[0273] The results show: Figure 10Western Blot was used to detect the effect of RASA3 myeloid knockout on CEBP / β protein under VSV infection.

[0274] The results showed that RASA3 knockout promoted the protein level of CEBP / β under VSV infection.

[0275] Example 10: RASA3 myeloid knockout exacerbates VSV-induced lung inflammation in mice

[0276] Female WT mice (C57BL / 6J background) and Rasa3-cKO mice were intraperitoneally infected with 5×10 7 PFU of VSV was added, and RT-qPCR was used to detect the expression of Il-6 and Tnfα in lung tissues and the viral load 6 hours later; ELISA was used to detect the levels of IL-6, TNF-α, and IP-10 in serum; HE staining of tissue sections was used to detect lung tissue damage in mice; WT mice (n=11) and Rasa3-cKO mice (n=11) were injected with 1×10 8 PFU of VSV, and the survival of the two groups of mice were monitored at different time points.

[0277] The expression of Il-6 and Tnfα in lung tissue, as well as the viral load Figure 11 As shown in A, the levels of IL-6, TNF-α and IP-10 in serum were Figure 11 As shown in B, the lung tissue damage of mice Figure 11 As shown in C, the survival of mice is as follows Figure 11 As shown in D.

[0278] The results showed that: (1) RASA3 myeloid knockout exacerbated lung inflammation caused by VSV infection. The Il-6 transcript level of Rasa3-cKO mice was significantly higher than that of WT mice, while there was no difference in gene Tnfα and viral load. Serum ELISA test results showed that Rasa3-cKO mice had increased IL-6 and TNF-α levels, but had no effect on IP-10. Similarly, HE staining of lung tissue 6 hours after infection showed that there was a large amount of immune cell infiltration in the lungs of Rasa3-cKO mice, and tissue damage was observed. (2) The survival rate of Rasa3-cKO mice decreased under VSV infection. Mouse survival analysis showed that compared with WT mice, the survival rate of Rasa3-cKO mice under VSV virus infection was significantly reduced, and they were more sensitive to VSV virus.

[0279] The results showed that RASA3 knockout promoted the massive production of inflammatory factors such as IL-6, ultimately leading to inflammatory outbreaks and death in mice.

[0280] Example Eleven, RASA3 myeloid knockout exacerbates lung inflammation induced by PR8 infection in mice

[0281] Female WT mice and Rasa3-cKO mice were infected intranasally with 200 PFU of PR8, and 3 days later, the content of IL-6 and TNF-a in lung tissues was detected by ELISA; TCID 50 The viral titer in lung tissues was detected, and the lung tissue damage of mice was detected by HE staining of tissue sections.

[0282] The content of IL-6 and TNF-a in lung tissues was detected by ELISA, and the viral titer in lung tissues was detected by RT-qPCR. Figure 12 The content of IL-6 and TNF-a in lung tissues was detected by ELISA, and the viral titer in lung tissues was detected by RT-qPCR. Figure 12 The content of IL-6 and TNF-a in lung tissues was detected by ELISA, and the viral titer in lung tissues was detected by RT-qPCR. Figure 12 The content of IL-6 and TNF-a in lung tissues was detected by ELISA, and the viral titer in lung tissues was detected by RT-qPCR.

[0283] The results showed that RASA3 myeloid knockout exacerbated lung inflammation induced by PR8 infection. Compared with WT mice, the levels of IL-6 and TNF-a in the serum of Rasa3-cKO mice were significantly increased, the lung tissue damage was more serious, there were a large number of immune cell infiltration, and the viral load did not change significantly.

[0284] The results showed that RASA3 myeloid knockout exacerbated lung inflammation induced by PR8 infection. Compared with WT mice, the levels of IL-6 and TNF-a in the serum of Rasa3-cKO mice were significantly increased, the lung tissue damage was more serious, there were a large number of immune cell infiltration, and the viral load did not change significantly.

[0285] Example Twelve, RASA3 myeloid knockout exacerbates lung inflammation induced by RSV infection in mice

[0286] Female WT mice (C57BL / 6J background) and Rasa3-cKO mice were infected intranasally with 2x10 8 PFU of VSV, and 5 days later, the expression of Il-6 and Tnf a in lung tissues and the viral load were detected by RT-qPCR, the content of IL-6 and TNF-a in serum was detected by ELISA, and the lung tissue damage of mice was detected by HE staining of tissue sections.

[0287] The expression of Il-6 and Tnf a in lung tissues and the viral load were detected by RT-qPCR, and the content of IL-6 and TNF-a in serum was detected by ELISA. Figure 13 The expression of Il-6 and Tnf a in lung tissues and the viral load were detected by RT-qPCR, and the content of IL-6 and TNF-a in serum was detected by ELISA. Figure 13 The expression of Il-6 and Tnf a in lung tissues and the viral load were detected by RT-qPCR, and the content of IL-6 and TNF-a in serum was detected by ELISA. Figure 13 The expression of Il-6 and Tnf a in lung tissues and the viral load were detected by RT-qPCR, and the content of IL-6 and TNF-a in serum was detected by ELISA.

[0288] The results showed that myeloid knockout of RASA3 exacerbated lung inflammation induced by RSV infection. Il-6 and Tnfa transcript levels were significantly higher in Rasa3-cKO mice than in WT mice. Serum ELISA assays revealed increased IL-6 levels in Rasa3-cKO mice. Similarly, HE staining of lung tissue revealed significant immune cell infiltration and tissue damage in the lungs of Rasa3-cKO mice.

[0289] The results showed that RASA3 knockout promoted the production of inflammatory factors such as IL-6, which ultimately led to an outbreak of inflammation in mice.

[0290] MDKQEAAIPLVRLLLHYGRVVPFISAIASAEVKRTQDPNTIFRGNSLTSKCIDETMKLAGMHYLHVT

[0291] LKPTIEEICQSHKSCEIDPVKLKDGENLENNMESLRQYVDRIFTVITKSGVSCPTVMCDIFFSLREAAA

[0292] KRFQDDLDVRYTAVSSFIFLRFFAPAILSPNLFQLTPHHTDPQTSRTLTLISKTIQTLGSLSKSKSASFKE

[0293] SYMATFYEFFNEQKYADAVKNFLDLISSSGRRDPKSIEQPILLKEGFMIKRAQGRKRFGMKNFKKRW

[0294] FRLTNHEFTYQKSKGDQPLCNIPIENILAVERLEEESFRMKNMFQVIQPERALYIQANNCVEAKDWID

[0295] ILTKVSQCNQKRLTVFHPSAYLNGHWLCCRASSDTAAGCTPCTGGLPANIQLDIDGDRETERIYSLFN

[0296] LYMGKLEKMQEACGSKSVYDGPEQEEYSTFVIDDPQETYKTLKQVIAGVGTLEQEHAQYRRDKFK

[0297] KTRYGSQEHPIGDKSFQNYIRQQSEISTHSI

[0298] SEQ ID NO. 2, GAP domain

[0299] DKQEAAIPLVRLLLHYGRVVPFISAIASAEVKRTQDPNTIFRGNSLTSKCIDETMKLAGMHYLHVTLKPTIEEICQSHKSCEIDPVKLKDGENLENNMESLRQYVDRIFTVITKSGVSCPTVMCDIFFSLREAAAKRFQDDLDVRYTAVSSFIFLRFFAPAILSPNLFQLTPHHTDPQTSRTLTLISKTIQTLGS

[0300] SEQ ID NO.3,PH / Btk结构域

[0301] ILLKEGFMIKRAQGRKRFGMKNFKKRWFRLTNHEFTYQKSKGDQPLCNIPIENILAVERLEEESFRMKNMFQVIQPERALYIQANNCVEAKDWIDILTKVSQCNQKRLTVFHPSAYLNGHWLCCRASSDTAAGCTPCTGG

[0302] SEQ ID NO.4,C2A结构域

[0303] MAVEEEGLRVFQSVRIKIGEAKNLPSYPGPNKMRDCYCTVNLDQEEVFRTKIVEKSLCPFYGEDFYCEIPRSFRHLSFYIFDRDVFRRDSIIGKVAIQKEDLQRYHNRDTWF

[0304] SEQ ID NO.5,C2B结构域

[0305] VQGKVHLELRLSEVITDTGVVCHKLAARIFECQGLPIVNGQCDPYATVTLAGPFRSEAKKTKVKKKTNNPQFDEVFYFEVTRPCSYSKKSHFDFEEEDVDKLEIRVDLWNASNLKFGDEFLGELRLPLKILRHSSSYEAWY

[0306] SEQ ID NO.6,RASA3(Mus musculus)

[0307] MAVEEEGLRVFQSVRIKIGEAKNLPSYPGPNKMRDCYCTVNLDQEEVFRTKIVEKSLCPFYGEDFYC

[0308] EIPRSFRHLSFYIFDRDVFRRDSIIGKVAIQKEDLQRYHNRDTWFQLQHVDADSEVQGKVHLELRLSE

[0309] VITDTGVVCHKLAARIFECQGLPIVNGQCDPYATVTLAGPFRSEAKKTKVKKKTNNPQFDEVFYFEV

[0310] TRPCSYSKKSHFDFEEEDVDKLEIRVDLWNASNLKFGDEFLGELRLPLKILRHSSSYEAWYFLQPRD

[0311] NGNKSLKPDDLGSLRLNVVYTEDHVFSSEYYSPLRDLLLKSADVEPVSASAAHILGEVCRDKQEAAI

[0312] PLVRLLLHYGRVVPFISAIASAEVKRTQDPNTIFRGNSLTSKCIDETMKLAGMHYLHVTLKPTIEEICQ

[0313] SHKSCEIDPVKLKDGENLENNMESLRQYVDRIFTVITKSGVSCPTVMCDIFFSLREAAAKRFQDDLD

[0314] VRYTAVSSFIFLRFFAPAILSPNLFQLTPHHTDPQTSRTLTLISKTIQTLGSLSKSKSASFKESYMATFYEF

[0315] FNEQKYADAVKNFLDLISSSGRRDPKSIEQPILLKEGFMIKRAQGRKRFGMKNFKKRWFRLTNHEFT

[0316] YQKSKGDQPLCNIPIENILAVERLEEESFRMKNMFQVIQPERALYIQANNCVEAKDWIDILTKVSQCN

[0317] QKRLTVFHPSAYLNGHWLCCRASSDTAAGCTPCTGGLPANIQLDIDGDRETERIYSLFNLYMGKLEK

[0318] MQEACGSKSVYDGPEQEEYSTFVIDDPQETYKTLKQVIAGVGTLEQEHAQYRRDKFKKTRYGSQE

[0319] HPIGDKSFQNYIRQQSEISTHSI

[0320] SEQ ID NO. 7, RASA3 (Homo sapiens)

[0321] MAVEDEGLRVFQSVKIKIGEAKNLPSYPGPSKMRDCYCTVNLDQEEVFRTKIVEKSLCPFYGEDFYC

[0322] EIPRSFRHLSFYIFDRDVFRRDSIIGKVAIQKEDLQKYHNRDTWFQLQHVDADSEVQGKVHLELRLSE

[0323] VITDTGVVCHKLATRIVECQGLPIVNGQCDPYATVTLAGPFRSEAKKTKVKRKTNNPQFDEVFYFEV

[0324] TRPCSYSKKSHFDFEEEDVDKLEIRVDLWNASNLKFGDEFLGELRIPLKVLRQSSSYEAWYFLQPRD

[0325] NGSKSLKPDDLGSLRLNVVYTEDHVFSSDYYSPLRDLLLKSADVEPVSASAAHILGEVCREKQEAA

[0326] VPLVRLFLHYGRVVPFISAIASAEVKRTQDPNTIFRGNSLASKCIDETMKLAGMHYLHVTLKPAIEEIC

[0327] QSHKPCEIDPVKLKDGENLENNMENLRQYVDRVFHAITESGVSCPTVMCDIFFSLREAAAKRFQDD

[0328] PDVRYTAVSSFIFLRFFAPAILSPNLFQLTPHHTDPQTSRTLTLISKTVQTLGSLSKSKSASFKESYMATF

[0329] YEFFNEQKYADAVKNFLDLISSSGRRDPKSVEQPIVLKEGFMIKRAQGRKRFGMKNFKKRWFRLTN

[0330] HEFTYHKSKGDQPLYSIPIENILAVEKLEEESFKMKNMFQVIQPERALYIQANNCVEAKDWIDILTKV

[0331] SQCNQKRLTVYHPSAYLSGHWLCCRAPSDSAPGCSPCTGGLPANIQLDIDGDRETERIYSLFNLYMS

[0332] KLEKMQEACGSKSVYDGPEQEEYSTFVIDDPQETYKTLKQVIAGVGALEQEHAQYKRDKFKKTKY

[0333] GSQEHPIGDKSFQNYIRQQSETSTHSI

[0334] SEQ ID NO. 8, pcDNA3.1-RASA3

[0335] GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCAT

[0336] AGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATT

[0337] TAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTT

[0338] TTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATA

[0339] GTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTA

[0340] AATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC

[0341] CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCC

[0342] ACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAAT

[0343] GGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACG

[0344] TATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGT

[0345] TTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAA

[0346] AATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGC

[0347] GTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGG

[0348] CTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGCCACCATGGCGGTGG

[0349] AGGAAGAGGGACTGCGGGTCTTCCAGAGCGTGAGGATCAAGATTGGTGAAGCCAAGAATCTTC

[0350] CTTCCTACCCGGGGCCAAACAAGATGAGGGACTGCTACTGCACTGTGAACCTGGACCAGGGAGG

[0351] AGGTTTTCCGGACCAAAATTGTCGAGAAGTCACTCTGCCCCTTTTACGGAGAGGACTTTTACTGT

[0352] GAAATCCCTCGGAGTTTCCGCCACCTGTCCTTTTACATTTTTGATAGAGATGTTTTCCGAAGGGAC

[0353] TCCATCATAGGCAAGGTGGCCATCCAGAAGGAAGACTTGCAGAGGTACCACAACAGGGACACAT

[0354] GGTTCCAGCTGCAGCACGTGGACGCAGACTCAGAGGTGCAGGGCAAGGTCCACCTGGAGCTGA

[0355] GACTAAGTGAGGTCATTACGGACACTGGTGTCGTCTGCCACAAACTCGCTGCACGCATCTTCGA

[0356] GTGCCAGGGTCTCCCCATTGTGAACGGGCAGTGTGACCCTTACGCCACAGTGACGCTGGCCGGA

[0357] CCCTTCAGGTCTGAAGCAAAGAAAACGAAGGTGAAGAAGAAAACCAACAACCCCCAGTTTGAC

[0358] GAGGTGTTTTATTTTGAGGTGACCAGACCCTGCAGCTACAGCAAAAAGTCCCACTTTGACTTTG

[0359] AGGAAGAGGACGTGGACAAACTTGAAATCCGAGTTGACCTCTGGAACGCCAGCAACCTGAAGT

[0360] TTGGGGATGAGTTTCTCGGGGAACTGAGGCTTCCTCTCAAGATTCTGCGACACTCGAGCTCTTAT

[0361] GAAGCCTGGTACTTCCTCCAGCCCCGAGACAACGGCAATAAGAGCCTGAAGCCAGATGACTTGG

[0362] GGTCTCTGAGGTTAAATGTCGTTTATACAGAAGACCATGTCTTCTCCTCTGAGTACTACAGCCCA

[0363] CTGCGTGACCTGTTACTGAAGTCTGCAGATGTGGAGCCTGTCTCAGCCTCAGCAGCTCACATCCT

[0364] GGGTGAGGTGTGCAGAGACAAGCAAGAGGCAGCCATCCCGCTGGTACGGCTCCTGCTGCACTAT

[0365] GGCAGGGTAGTGCCCTTCATCAGTGCCATCGCTAGCGCAGAGGTGAAGAGGACCCAGGACCCC

[0366] AATACCATCTTCCGAGGAAACTCACTGACATCCAAGTGCATAGATGAGACGATGAAGCTGGCAG

[0367] GCATGCACTATCTCCATGTGACCCTGAAGCCCACCATTGAGGAGATCTGCCAGAGCCACAAGTC

[0368] CTGTGAGATCGACCCTGTGAAACTGAAAGACGGCGAAAACCTCGAGAACAACATGGAGAGCCT

[0369] GAGGCAGTATGTGGATCGCATCTTCACTGTCATCACCAAGTCCGGGGTGAGCTGCCCCACCGTCA

[0370] TGTGTGACATCTTTTTCTCCTTGCGGGAGGCGGCTGCCAAGCGCTTCCAAGATGACTTGGATGTG

[0371] AGGTACACGGCCGTGAGCAGCTTCATCTTCCTCAGGTTCTTCGCCCCTGCCATCCTGTCCCCAAA

[0372] CCTTTTCCAGCTGACACCCCACCACACGGATCCACAGACTTCTAGAACCCTGACACTTATCTCAA

[0373] AGACGATCCAGACCCTCGGCAGCTTGTCCAAGTCCAAGTCTGCCAGTTTTAAGGAGTCGTACAT

[0374] GGCGACATTCTATGAATTCTTCAATGAGCAGAAGTATGCAGATGCTGTAAAAAATTTTCTGGATTT

[0375] GATCTCATCCTCGGGGAGAAGGGACCCCAAGAGCATAGAGCAGCCCATCCTGCTTAAAGAAGGG

[0376] TTCATGATCAAGCGGGCCCAGGGAAGGAAACGGTTTGGAATGAAGAATTTCAAGAAGAGGTGG

[0377] TTTCGCCTGACGAACCACGAGTTCACCTACCAGAAAAGCAAAGGTGATCAGCCACTCTGCAACA

[0378] TCCCCATCGAGAACATCTTGGCTGTGGAGAGGCTAGAGGAGGAGTCCTTCCGAATGAAAAACAT

[0379] GTTCCAGGTCATCCAGCCAGAGCGTGCCCTGTACATCCAGGCCAACAACTGTGTGGAGGCCAAG

[0380] GACTGGATCGACATCCTCACCAAAGTGAGCCAGTGCAACCAGAAGCGGCTCACCGTCTTCCACC

[0381] CGTCGGCCTACCTGAACGGCCACTGGCTCTGCTGCAGGGCCTCCTCAGACACGGCTGCTGGCTG

[0382] CACTCCCTGCACTGGTGGGCTCCCGGCCAACATCCAGCTGGACATTGATGGGGACCGTGAAACA

[0383] GAGCGCATCTACTCTCTCTTTAACCTGTACATGGGCAAGCTGGAAAAGATGCAGGAGGCCTGCG

[0384] GCAGCAAGTCTGTGTATGACGGCCCTGAGCAGGAGGAGTACTCAACGTTCGTCATCGACGACCC

[0385] CCAGGAGACGTACAAGACGCTGAAGCAGGTCATCGCTGGGGTGGGGACCCTTGAGCAGGAGCA

[0386] CGCACAGTACAGGAGAGACAAGTTCAAGAAGACGAGATACGGGAGCCAGGAGCACCCTATTGG

[0387] AGACAAGAGCTTCCAGAACTACATCCGGCAGCAGTCTGAGATCTCCACCCATTCCATTGGAAGC

[0388] TTTCTAGATTACAAGGATGACGACGATAAGTGAGTTTAAACGGTCTCCAGCTTAAGTTTAAACCG

[0389] CTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTC

[0390] CTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATT

[0391] GTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTG

[0392] GGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACC

[0393] AGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGG

[0394] TGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTC

[0395] CCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGG

[0396] GTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTA

[0397] GTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGT

[0398] GGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGG

[0399] ATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAA

[0400] TTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTAT

[0401] GCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGC

[0402] AGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCAT

[0403] CCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTAT

[0404] GCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAG

[0405] GCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACA

[0406] GGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGT

[0407] GGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTC

[0408] CGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATG

[0409] AACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTG

[0410] TGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGG

[0411] ATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGG

[0412] CTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGC

[0413] ACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTC

[0414] GCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTG

[0415] ACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGA

[0416] CTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTG

[0417] AAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCG

[0418] CAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATG

[0419] ACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAA

[0420] GGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATG

[0421] CTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGC

[0422] ATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATC

[0423] AATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGC

[0424] TGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAG

[0425] TGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGC

[0426] TTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGC

[0427] GGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCT

[0428] GCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAAC

[0429] GCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTT

[0430] GCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG

[0431] AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGC

[0432] GCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTG

[0433] GCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGG

[0434] CTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGT

[0435] CCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGC

[0436] GAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGA

[0437] ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTG

[0438] ATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGA

[0439] AAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAA

[0440] ACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATT

[0441] AAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCT

[0442] TAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCG

[0443] TCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGA

[0444] GACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCA

[0445] GAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTA

[0446] AGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACG

[0447] CTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCC

[0448] CATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCG

[0449] CAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGAT

[0450] GCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGT

[0451] TGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCAT

[0452] CATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGA

[0453] TGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGA

[0454] GCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATA

[0455] CTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACAT

[0456] ATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC

[0457] CTGACGTC

[0458] SEQ ID NO.9,pcDNA3.1-△1-329

[0459] GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCAT

[0460] AGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATT

[0461] TAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTT

[0462] TTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATA

[0463] GTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTA

[0464] AATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC

[0465] CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCC

[0466] ACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAAT

[0467] GGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACG

[0468] TATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGT

[0469] TTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAA

[0470] AATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGC

[0471] GTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGG

[0472] CTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGCCACCATGGACAAGC

[0473] AAGAGGCAGCCATCCCGCTGGTACGGCTCCTGCTGCACTATGGCAGGGTAGTGCCCTTCATCAGT

[0474] GCCATCGCTAGCGCAGAGGTGAAGAGGACCCAGGACCCCAATACCATCTTCCGAGGAAACTCAC

[0475] TGACATCCAAGTGCATAGATGAGACGATGAAGCTGGCAGGCATGCACTATCTCCATGTGACCCTG

[0476] AAGCCCACCATTGAGGAGATCTGCCAGAGCCACAAGTCCTGTGAGATCGACCCTGTGAAACTGA

[0477] AAGACGGCGAAAACCTCGAGAACAACATGGAGAGCCTGAGGCAGTATGTGGATCGCATCTTCA

[0478] CTGTCATCACCAAGTCCGGGGTGAGCTGCCCCACCGTCATGTGTGACATCTTTTTCTCCTTGCGG

[0479] GAGGCGGCTGCCAAGCGCTTCCAAGATGACTTGGATGTGAGGTACACGGCCGTGAGCAGCTTCA

[0480] TCTTCCTCAGGTTCTTCGCCCCTGCCATCCTGTCCCCAAACCTTTTCCAGCTGACACCCCACCAC

[0481] ACGGATCCACAGACTTCTAGAACCCTGACACTTATCTCAAAGACGATCCAGACCCTCGGCAGCT

[0482] TGTCCAAGTCCAAGTCTGCCAGTTTTAAGGAGTCGTACATGGCGACATTCTATGAATTCTTCAAT

[0483] GAGCAGAAGTATGCAGATGCTGTAAAAAATTTTCTGGATTTGATCTCATCCTCGGGGAGAAGGGA

[0484] CCCCAAGAGCATAGAGCAGCCCATCCTGCTTAAAGAAGGGTTCATGATCAAGCGGGCCCAGGGA

[0485] AGGAAACGGTTTGGAATGAAGAATTTCAAGAAGAGGTGGTTTCGCCTGACGAACCACGAGTTC

[0486] ACCTACCAGAAAAGCAAAGGTGATCAGCCACTCTGCAACATCCCCATCGAGAACATCTTGGCTG

[0487] TGGAGAGGCTAGAGGAGGAGTCCTTCCGAATGAAAAACATGTTCCAGGTCATCCAGCCAGAGC

[0488] GTGCCCTGTACATCCAGGCCAACAACTGTGTGGAGGCCAAGGACTGGATCGACATCCTCACCAA

[0489] AGTGAGCCAGTGCAACCAGAAGCGGCTCACCGTCTTCCACCCGTCGGCCTACCTGAACGGCCA

[0490] CTGGCTCTGCTGCAGGGCCTCCTCAGACACGGCTGCTGGCTGCACTCCCTGCACTGGTGGGCTC

[0491] CCGGCCAACATCCAGCTGGACATTGATGGGGACCGTGAAACAGAGCGCATCTACTCTCTCTTTAA

[0492] CCTGTACATGGGCAAGCTGGAAAAGATGCAGGAGGCCTGCGGCAGCAAGTCTGTGTATGACGGC

[0493] CCTGAGCAGGAGGAGTACTCAACGTTCGTCATCGACGACCCCCAGGAGACGTACAAGACGCTG

[0494] AAGCAGGTCATCGCTGGGGTGGGGACCCTTGAGCAGGAGCACGCACAGTACAGGAGAGACAA

[0495] GTTCAAGAAGACGAGATACGGGAGCCAGGAGCACCCTATTGGAGACAAGAGCTTCCAGAACTA

[0496] CATCCGGCAGCAGTCTGAGATCTCCACCCATTCCATTGGAAGCTTTCTAGATTACAAGGATGACG

[0497] ACGATAAGTGAGTTTAAACGGTCTCCAGCTTAAGTTTAAACCGCTGATCAGCCTCGACTGTGCCT

[0498] TCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCAC

[0499] TCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT

[0500] TCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATG

[0501] CTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTA

[0502] TCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACC

[0503] GCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTC

[0504] GCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACG

[0505] GCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAG

[0506] ACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGG

[0507] AACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTAT

[0508] TGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAG

[0509] TTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATT

[0510] AGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGC

[0511] ATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCC

[0512] AGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCC

[0513] TCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAA

[0514] GCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCAT

[0515] GATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATG

[0516] ACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCG

[0517] CCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCG

[0518] CGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAG

[0519] CGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGC

[0520] TCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTA

[0521] CCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGG

[0522] TCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCC

[0523] AGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGC

[0524] CGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCG

[0525] GACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGC

[0526] TGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCT

[0527] TCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACC

[0528] TGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTC

[0529] CGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCA

[0530] ACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAG

[0531] CATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATA

[0532] CCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTAT

[0533] CCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAAT

[0534] GAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCG

[0535] TGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTT

[0536] CCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCAC

[0537] TCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAA

[0538] AAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCC

[0539] GCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGAC

[0540] TTCGGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGG

[0541] TTCGGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGG

[0542] TTCGGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGG

[0543] TTCGGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGG

[0544] TTCGGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGG

[0545] TTCGGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGG

[0546] TTCGGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGG

[0547] TTCGGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGG

[0548] TTCGGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGG

[0549] TTCGGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGG

[0550] TTCGGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGTTTTCGGG

[0551] GATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGA

[0552] GGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATT

[0553] TATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGC

[0554] CTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCG

[0555] CAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAG

[0556] CTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCT

[0557] CCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCA

[0558] GCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCA

[0559] ACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGA

[0560] TAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAA

[0561] AACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGA

[0562] TCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCG

[0563] CAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATT

[0564] GAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAAC

[0565] AAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCSEQ ID NO.10,pcDNA3.1-PH / Btk

[0566] GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCAT

[0567] AGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATT

[0568] TAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTT

[0569] TTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATA

[0570] GTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTA

[0571] AATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC

[0572] CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCC

[0573] ACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTTGACGTCAATGACGGTAAAT

[0574] GGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTGGCAGTACATCTACG

[0575] TATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGT

[0576] TTGACTCACGGGGATTTCCAAGTCTCCACCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAA

[0577] AATCAACGGGACTTTCCAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGC

[0578] GTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGG

[0579] CTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGCCACCATGATCCTGC

[0580] TTAAAGAAGGGTTCATGATCAAGCGGGCCCAGGGAAGGAAACGGTTTGGAATGAAGAATTTCA

[0581] AGAAGAGGTGGTTTCGCCTGACGAACCACGAGTTCACCTACCAGAAAAGCAAAGGTGATCAGG

[0582] CACTCTGCAACATCCCCATCGAGAACATCTTGGCTGTGGAGAGGCTAGAGGAGGAGTCCTTCCG

[0583] AATGAAAAACATGTTCCAGGTCATCCAGCCAGAGCGTGCCCTGTACATCCAGGCCAACAACTGT

[0584] GTGGAGGCCAAGGACTGGATCGACATCCTCACCAAAGTGAGCCAGTGCAACCAGAAGCGGCTC

[0585] ACCGTCTTCCACCCGTCGGCCTACCTGAACGGCCACTGGCTCTGCTGCAGGGCCTCCTCAGACA

[0586] CGGCTGCTGGCTGCACTCCCTGCACTGGTGGGGGAAGCTTTCTAGATTACAAGGATGACGACGA

[0587] TAAGTGAGTTTAAACGGTCTCCAGCTTAAGTTTAAACCGCTGATCAGCCTCGACTGTGCCTTCTA

[0588] GTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCC

[0589] ACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTG

[0590] GGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGG

[0591] GGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCC

[0592] CACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTA

[0593] CACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCG

[0594] GCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCAC

[0595] CTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGT

[0596] TTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAA

[0597] CACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTT

[0598] AAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGG

[0599] GTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCA

[0600] GCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTC

[0601] AATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTC

[0602] CGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGC

[0603] CTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTC

[0604] CCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATT

[0605] GAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACT

[0606] GGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCC

[0607] GGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGG

[0608] CTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGG

[0609] GAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCC

[0610] TGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCT

[0611] GCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCT

[0612] TGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGG

[0613] CTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGA

[0614] ATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGAC

[0615] CGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGA

[0616] CCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCT

[0617] TGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGC

[0618] CATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGG

[0619] GACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTT

[0620] GTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATT

[0621] TTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCG

[0622] TCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCG

[0623] CTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAG

[0624] TGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGC

[0625] CAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCG

[0626] CTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCA

[0627] AAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAA

[0628] GGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCC

[0629] CCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTAT

[0630] AAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTT

[0631] ACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAG

[0632] GTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGC

[0633] CCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCG

[0634] CCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGT

[0635] TCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTG

[0636] AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTA

[0637] GCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTT

[0638] GATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGA

[0639] GATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAA

[0640] GTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGA

[0641] TCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGG

[0642] GCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTA

[0643] TCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCT

[0644] CCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCA

[0645] ACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCT

[0646] CCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCC

[0647] TTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGC

[0648] ACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAAC

[0649] CAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATA

[0650] ATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAA

[0651] ACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGAT

[0652] CTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGC

[0653] AAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTG

[0654] AAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACA

[0655] AATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC

[0656] SEQ ID NO.11,pFGEH载体

[0657]

[0658]

Claims

1. Use of recombinant RASA3 in the preparation of a product for preventing and / or treating diseases and / or symptoms associated with viral infection, characterized in that: The recombinant RASA3 comprises a Δ1-329 truncation that interacts with the CEBP / β protein; The Δ1-329 truncation promotes the degradation of CEBP / β protein by interacting with the CEBP / β protein; preferably, the degradation of the CEBP / β protein includes degradation through the protein autophagy-lysosome pathway; The Δ1-329 truncation comprises the PH / Btk domain of RASA3 and, optionally, the GAP domain of RASA3; Optionally, the GAP domain comprises the sequence shown in SEQ ID NO. 2, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity to the sequence shown in SEQ ID NO. 2; Optionally, the PH / Btk domain comprises the sequence shown in SEQ ID NO. 3, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity to the sequence shown in SEQ ID NO. 3; Preferably, the Δ1-329 truncation comprises the sequence shown in SEQ ID NO. 1, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity to the sequence shown in SEQ ID NO. 1; The virus is an RNA virus.

2. The use according to claim 1, characterized in that The recombinant RASA3 further comprises at least one of a C2A domain and a C2B domain; Optionally, the C2A domain comprises the sequence shown in SEQ ID NO. 4, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity to the sequence shown in SEQ ID NO. 4; Optionally, the C2B domain comprises the sequence shown in SEQ ID NO.5, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the sequence shown in SEQ ID NO.

5.

3. The use according to claim 1 or 2, characterized in that The virus includes at least one of influenza virus, coronavirus, novel coronavirus, adenovirus, respiratory syncytial virus and vesicular stomatitis virus; Preferably, the virus is vesicular stomatitis virus, influenza virus and / or respiratory syncytial virus; More preferably, the influenza virus is influenza A virus A / PR / 8 / 34 strain.

4. The use according to any one of claims 1 to 3, characterized in that The symptoms of the viral infection-related disease and / or symptoms include at least one of the following (i) to (iv): (i) Increased expression of inflammatory factors in the body; (ii) increased immune cell infiltration; (iii) inflammatory damage caused by infection; (iv) inflammatory diseases caused by viral infection; Optionally, the inflammatory factors include IL-6, IL-1β, IP-10, and TNF-α; Optionally, the inflammatory damage includes inflammatory damage to the respiratory system, especially inflammatory damage to the lungs; Optionally, the inflammatory disease comprises pneumonia, preferably acute inflammatory pneumonia; Optionally, the organism is an individual who is likely to suffer from a disease and / or symptom associated with viral infection; the individual includes mice, rats, guinea pigs, cows, sheep, cats, dogs, horses, rabbits, pigs, monkeys and humans.

5. The use according to any one of claims 1 to 4, characterized in that The product comprises a recombinant RASA3 protein, a polynucleotide encoding the recombinant RASA3, a vector carrying the recombinant RASA3, and a vector carrying the polynucleotide encoding the recombinant RASA3; The vector includes any one or more of plasmid, adeno-associated virus, retrovirus, liposome, lipid nanoparticle, N-acetylgalactosamine and extracellular vesicle.

6. The use according to any one of claims 1 to 5, characterized in that The product also includes a drug that promotes autophagic degradation of CEBP / β protein; Optionally, the drug that promotes autophagic degradation of CEBP / β protein includes an mTOR inhibitor, an AMPK activator and / or a natural compound; The natural compounds include curcumin and / or resveratrol.

7. The use according to any one of claims 1 to 6, characterized in that The products for preventing and / or treating diseases and / or symptoms associated with viral infection include drugs and pharmaceutical compositions; Optionally, the pharmaceutical composition further comprises one or more drugs for clinically treating diseases and / or symptoms associated with viral infection and / or one or more pharmaceutically acceptable carriers; Optionally, the pharmaceutically acceptable carrier includes one or a combination of two or more of solvents, solubilizers, cosolvents, emulsifiers, flavoring agents, olfactory agents, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, surfactants and preservatives.

8. Use of recombinant RASA3 in the preparation of CEBP / β inhibitors, characterized in that: The recombinant RASA3 comprises a Δ1-329 truncation that interacts with the CEBP / β protein; The Δ1-329 truncation promotes the degradation of CEBP / β protein through the interaction of CEBP / β protein; preferably, the degradation of CEBP / β protein includes degradation through the protein autophagy-lysosome pathway; The Δ1-329 truncation comprises the PH / Btk domain of RASA3 and, optionally, the GAP domain of RASA3; Optionally, the GAP domain comprises the sequence shown in SEQ ID NO. 2, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity to the sequence shown in SEQ ID NO. 2; Optionally, the PH / Btk domain comprises the sequence shown in SEQ ID NO. 3, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity to the sequence shown in SEQ ID NO. 3; Preferably, the Δ1-329 truncation comprises the sequence shown in SEQ ID NO.1, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the sequence shown in SEQ ID NO.

1.

9. The use according to claim 8, characterized in that The recombinant RASA3 further comprises at least one of a C2A domain and a C2B domain; Optionally, the C2A domain comprises the sequence shown in SEQ ID NO. 4, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity to the sequence shown in SEQ ID NO. 4; Optionally, the C2B domain comprises the sequence shown in SEQ ID NO.5, or a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the sequence shown in SEQ ID NO.

5.

10. The use according to claim 8 or 9, characterized in that The CEBP / β inhibitor further comprises a polynucleotide encoding recombinant RASA3, a vector carrying recombinant RASA3, and / or a vector carrying a polynucleotide encoding recombinant RASA3; The vector includes any one or more of plasmid, adeno-associated virus, retrovirus, liposome, lipid nanoparticle, N-acetylgalactosamine and extracellular vesicle.

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