Target TRIM55 for preventing and / or treating autoimmune diseases and medicine and application thereof

Abstract

The invention provides a target TRIM55 for preventing and / or treating autoimmune diseases as well as a medicine and application thereof, and discloses the target TRIM55 for the autoimmune diseases and application of an inhibitor for the target TRIM55 in preparation of the medicine for preventing and / or treating the autoimmune diseases. The invention also provides a pharmaceutical composition for preventing and / or treating autoimmune diseases, and a method for screening out a drug for treating autoimmune diseases based on a target TRIM55, the drug targets TRIM55, its structural domain or TRIM55 protein truncation, and inhibits or weakens the function of TRIM55; the TRIIM55 regulates and controls activation of a non-classical NF-kappa B signal channel through targeted combination, ubiquitination modification of p100 and promotion of p100 processing, and the TRIIM55 can be used as an effective target for treatment of the autoimmune diseases, thereby participating in regulation and control of development of the autoimmune diseases, finding a new way for prevention and treatment of the autoimmune diseases, and having a broad application prospect. The compound has an extremely important application value in preparation of drugs for treating autoimmune diseases.

Description

Technical Field

[0001] The present invention belongs to the new application field of human host factor TRIM family protein TRIM55, and relates to a target TRIM55 for preventing and / or treating autoimmune diseases, and a medicine and application thereof. Background Art

[0002] With the development of basic medical research and the deepening of disease understanding, precision medicine has been valued in many countries, including China and the United States. The biological functions of the non-classical NF-κB signaling pathway correspond to its unique regulatory mechanism. The non-classical NF-κB pathway is not involved in the rapid response of the host to pathogens or infections, but the pathway plays an important role in the development of lymphoid organs, B cell immune responses, autoimmune diseases and chronic inflammation. The non-classical NF-κB signaling pathway is also involved in B cell-mediated autoimmune diseases such as systemic lupus erythematosus (SLE). SLE is an autoimmune and chronic inflammatory disease that affects multiple tissues and organs. The etiology of SLE is multifactorial, including genetic, environmental, hormonal and immune factors. SLE is a heterogeneous autoimmune disease with multiple complications, often accompanied by B cell dysfunction. As the source of autoantibodies, B cells play a central role in the pathogenesis of systemic lupus erythematosus and have been developed as a major target for the treatment of SLE. Belimumab, as a BAFF (B cell activating factor, BAFF) neutralizing antibody, has been approved for the treatment of SLE by inhibiting non-classical NF-κB activation and B cell function. In this regard, the non-classical NF-κB pathway is a potential drug target for the treatment of SLE, and further understanding of the molecular regulatory events of this pathway is needed. At the same time, how the non-classical NF-κB pathway is activated, the source cells of the activator, and the effector cells are still subject to further research and identification.

[0003] In the prior art, research on TRIM55 mainly focuses on its functions in muscle development, autophagy and tumorigenesis-related fields, but has not yet involved research on the role of TRIM55 in other diseases. Summary of the invention

[0004] The purpose of the present invention is to provide a target TRIM55 for preventing and / or treating autoimmune diseases, and a drug and application thereof. The present invention discloses a target TRIM55 for autoimmune diseases, and an application of an inhibitor targeting the target TRIM55 in preparing a drug for preventing and / or treating autoimmune diseases. A pharmaceutical composition for preventing and / or treating autoimmune diseases, and a method for screening a drug for treating autoimmune diseases based on the target TRIM55 are also provided. The drug targets TRIM55, its domain or a TRIM55 protein truncate, and inhibits or weakens the function of TRIM55. TRIIM55 regulates the activation of a non-classical NF-κB signaling pathway by targeting and ubiquitin-modifying p100 and promoting p100 processing. TRIM55 can serve as an effective target for the treatment of autoimmune diseases, thereby participating in regulating the development of autoimmune diseases, and finding a new approach for the prevention and treatment of autoimmune diseases.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] A target TRIM55 for preventing and / or treating autoimmune diseases, wherein the autoimmune diseases are autoimmune diseases associated with a non-classical NF-κB signal transduction pathway.

[0007] Furthermore, the autoimmune disease is systemic lupus and related complications, and rheumatoid arthritis.

[0008] Use of the target TRIM55 in the preparation of a drug for preventing and / or treating an autoimmune disease, wherein the autoimmune disease is an autoimmune disease associated with a non-classical NF-κB signal transduction pathway.

[0009] Furthermore, the autoimmune disease is systemic lupus and related complications, and rheumatoid arthritis.

[0010] Furthermore, the drug targets TRIM55, its domain or TRIM55 protein truncation, and inhibits or weakens the function of TRIM55, that is, the drug is an agent that reduces the expression level of TRIM55 and / or antagonizes the effect of TRIM55.

[0011] Furthermore, the target TRIM55 regulates p100 processing and non-classical NF-kB activation, the target TRIM55 binds to p100, and the binding ability of p100-TRIM55 is enhanced with the activation of the non-classical NF-kB pathway; the mutual binding of p100-TRIM55 depends on the phosphorylation modification of p100 S866 / 870; that is, the target TRIM55 regulates the activation of the non-classical NF-κB signaling pathway by targeting and ubiquitinating p100 and promoting p100 processing, thereby participating in the regulation of the development of autoimmune diseases.

[0012] Use of TRIM55 in the preparation of a drug for treating an autoimmune disease, wherein the autoimmune disease is an autoimmune disease associated with a non-classical NF-κB signal transduction pathway, wherein the drug targets TRIM55 and affects the function of TRIM55. The autoimmune disease is SLE and related complications, and rheumatoid arthritis.

[0013] The non-canonical NF-κB signaling pathway is involved in B cell-mediated autoimmune diseases such as SLE. TRIM55 participates in the pathogenesis of SLE by binding to p100 and mediating the K48 ubiquitination modification of p100, thereby promoting p100 processing and activation of the non-canonical NF-κB signaling pathway.

[0014] SLE is an autoimmune and chronic inflammatory disease that affects multiple tissues and organs. The etiology of SLE is multifactorial, including genetic, environmental, hormonal, and immune factors. SLE is a heterogeneous autoimmune disease with multiple complications, often accompanied by B cell dysfunction. As a source of autoantibodies, B cells play a central role in the pathogenesis of systemic lupus erythematosus and have been developed as a major target for SLE treatment. Belimumab, a BAFF neutralizing antibody, has been approved for the treatment of SLE by inhibiting non-classical NF-κB activation and B cell function.

[0015] The immune system has many negative regulatory mechanisms to ensure immune homeostasis in the host. If the immune homeostasis is broken, autoimmune diseases will occur. After studying the role of TRIM55 in autoimmune diseases, the present invention provides the application of TRIM55 in the treatment of autoimmune diseases. Specifically, it relates to autoimmune diseases related to non-classical NF-κB signal transduction pathways, including but not limited to: SLE, lupus-related complications, rheumatoid arthritis, etc.

[0016] The present invention provides a use of TRIM55 in preparing a drug for treating an autoimmune disease. The drug can further affect the function of TRIM55 by affecting the function of a key domain of TRIM55, thereby playing a role in treating the autoimmune disease.

[0017] After immunization with NP-KLH and NP-Ficoll, the serum concentrations of IgA, IgM, and IgG antibodies in B cell-specific knockout TRIM55 mice were significantly reduced. After immunization with NP-KLH, the development of germinal center B cells and plasma cells in B cell-specific knockout TRIM55 mice was impaired. These results indicate that TRIM55 knockout inhibits the humoral immune response of mice.

[0018] In the mouse lupus disease model constructed, the proportion of germinal center B cells and plasma cells in the spleen of TRIM55 knockout mice was significantly reduced; the concentration of autoantibodies such as ANA (Anti-nuclear antibody, ANA) and anti-dsDNA (autoantibody against double-stranded DNA, anti-dsDNA) in the serum of TRIM55 knockout mice was significantly reduced; and the IgG antibody deposition in the kidneys of TRIM55 knockout mice was significantly reduced. These results indicate that the knockout of TRIM55 alleviates the symptoms of lupus disease in mice.

[0019] A method for screening drugs for preventing and / or treating autoimmune diseases based on the target TRIM55, wherein the method uses TRIM55 as a target and screens out candidate drugs or drug combinations by detecting the effects of the drugs on the activity of TRIM55, that is, detecting the inhibitory and / or antagonistic properties of specific drugs on the activity of TRIM55 to screen out candidate drugs or drug combinations.

[0020] Furthermore, the method uses TRIM55 as a target, and further screens the target drug or drug combination by detecting the effect of the candidate drug or drug combination on the activation of the non-classical NF-κB signaling pathway of B cells, that is, detecting the inhibitory and / or antagonistic performance of the candidate drug or drug combination on the activation of the non-classical NF-κB signaling pathway of B cells to screen out the target drug or drug combination.

[0021] A drug for preventing and / or treating an autoimmune disease, wherein the autoimmune disease is an autoimmune disease associated with a non-classical NF-κB signal transduction pathway, and the drug is an agent or a combination of agents that reduces the expression level of TRIM55 and / or antagonizes the effect of TRIM55.

[0022] Furthermore, the drug is an agent or a combination of agents that inhibits and / or antagonizes the activation of the non-classical NF-κB signaling pathway of B cells.

[0023] Beneficial effects:

[0024] 1) TRIM55 regulates p100 processing and non-canonical NF-kB activation.

[0025] 2) TRIM55 can bind to p100, and the binding ability of p100-TRIM55 is enhanced with the activation of the non-classical NF-kB pathway. The binding of p100-TRIM55 depends on the phosphorylation modification of p100 S866 / 870.

[0026] 3) TRIM55 knockout affects germinal center formation and humoral immune response in mice.

[0027] 4) TRIM55 knockout alleviated lupus disease symptoms in mice.

[0028] In summary, the present invention clearly reveals a new regulatory mechanism of the p100 processing-dependent signaling pathway, and establishes that TRIIM55 regulates the activation of the non-classical NF-κB signaling pathway by targeting and ubiquitinating p100 and promoting p100 processing, thereby participating in regulating the development of autoimmune diseases. Therefore, a new intervention strategy for controlling autoimmune diseases is proposed, and a new approach is found for the prevention and treatment of autoimmune diseases. The use of TRIM55 in the preparation of drugs for treating autoimmune diseases is disclosed, which provides useful suggestions for related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the technical route of the present invention;

[0030] Figure 2 A schematic diagram illustrating that TRIM55 specifically binds to p100 and mediates K48 ubiquitination modification of p100, and regulates p100 processing, thereby affecting the activation of the non-classical NF-κB signaling pathway, thereby regulating B cell-mediated humoral immune responses and autoimmune diseases;

[0031] Figure 3 Schematic diagram to illustrate that silencing TRIM55 expression in MEF (mouse embryonic fibroblasts, MEF) cells and M12 B cells can weaken p100 processing and p52 / RelB nuclear entry, thereby inhibiting the activation of the non-classical NF-κB signaling pathway.

[0032] Figure 4 Schematic diagram to illustrate that in TRIM55 knockout MEF cells and B cells, p100 processing and p52 / RelB nuclear import were significantly weakened, and the transcriptional activity of non-classical NF-κB was significantly reduced;

[0033] Figure 5 To illustrate the results of co-immunoprecipitation of TRIM55 and p100;

[0034] Figure 6 The results are shown in Figure 2 to illustrate that the concentrations of IgA, IgM and IgG antibodies in the serum of B cell-specific knockout TRIM55 mice were lower than those of wild-type mice after NP-KLH and NP-Ficoll immunization;

[0035] Figure 7 The results are shown to illustrate that germinal center formation in the spleen of B cell-specific TRIM55 knockout mice is impaired after NP-KLH immunization, wherein: Figure 7 a is the flow cytometry analysis of the development of GCB (Germinal centre B cell, GCB) in mouse spleen; Figure 7 b is flow cytometry analysis of mouse spleen plasma cell development;

[0036] Figure 8 A graph showing that germinal center formation in the spleen of TRIM55 knockout mice is impaired in a mouse lupus disease model, wherein: Figure 8 a and Figure 8 b Flow cytometry analysis of GCB development in mouse spleen; Figure 8 c and Figure 8 d is flow cytometry analysis of mouse spleen plasma cell development;

[0037] Fig. 9 Figure 1 is a graph showing the impairment of autoantibody production in the serum of TRIM55 knockout mice in a mouse lupus disease model, wherein: Fig. 9 a is the result graph of antinuclear antibodies; Fig. 9 b is the result graph of double-stranded DNA antibody;

[0038] Fig.10 A graph illustrating the reduced IgG antibody deposition in the kidneys of TRIM55 knockout mice in a mouse lupus disease model;

[0039] Fig.11 This is a comparison of NF-κB transcriptional activity in mouse MEF cells after TRIM55 knockdown;

[0040] Fig.12 Diagram of the strategy for constructing Trim55 knockout mice;

[0041] Fig.13 The genotype is Ella-Cre + Trim55 wt / wt Wild-type mice and Ella-Cre + Trim55 flox / floxComparison of mRNA expression in skeletal muscle, myocardium, MEF cells and B cells of Trim55 knockout mice. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0043] The present invention identifies the key function of TRIM55 in the pathogenesis of autoimmune diseases, which is achieved by binding to p100 and promoting p100 processing. Specifically, TRIM55 affects the activation of the non-classical NF-κB signaling pathway by regulating p100 processing. The results in mice showed that TRIM55 regulates B cell-mediated humoral immune responses and autoimmune diseases by regulating the activation of the non-classical NF-κB signaling pathway.

[0044] The non-classical NF-κB signaling pathway is mainly activated by some members of the TNF receptor superfamily (TNFRSFs), such as LTβR (lymphotoxin-βreceptor, LTβR), CD40, B cell activating factor receptor (B cell activating factor receptor, BAFFR) and NF-κB receptor activator ligand (Receptor activator for NF-κB ligand, RANKL), and the biological functions regulated by this pathway are relatively specific. Under steady-state conditions, the key kinase NIK (NF-κB-inducing kinase, NIK) of the non-classical NF-κB signaling pathway is degraded through a ubiquitination-proteasome-dependent mechanism mediated by TRAF2 (TNFR associated factor 2, TRAF2)-TRAF3-cIAP (cellular inhibitor of apoptosis, cIAP), resulting in the protein level of NIK being maintained at a very low level. Under stress conditions, TRAF2-TRAF3-cIAP is recruited and binds to the intracellular receptor, and cIAP mediates TRAF2-TRAF3 ubiquitination and leads to TRAF2-TRAF3 ubiquitination-dependent degradation, which slowly increases the protein level of NIK in the cytoplasm. The accumulation of NIK leads to the activation of downstream IKKα (IκB kinaseα, IKKα). NIK and activated IKKα induce the phosphorylation of p100, so that the E3 ubiquitin ligase β-TrCP binds to the phosphorylated p100 and ubiquitinates p100, leading to the processing of p100 and the transfer of the p52 / RelB transcription complex to the nucleus, thereby initiating the transcription of the target gene. As a key event in the activation of the non-classical NF-kB pathway, p100 processing is related to many human diseases. p100 mutations, resulting in inability to process or constitutively process p100, are characteristic of many human diseases such as B-cell immunodeficiency and B-cell chronic lymphocytic leukemia, suggesting potential therapeutic targets for p100 processing. In vivo results highlight the regulatory role of TRIM55 in humoral immune responses and autoimmune diseases by regulating p100 processing and activation of the non-canonical NF-kB pathway.

[0045] The present invention further provides the use of TRIM55 in immune diseases, especially autoimmune diseases. In order to verify the function of TRIM55, the present invention uses MEF and B cells as research objects, uses anti-LTβR, anti-CD40 and BAFF as stimuli, and conducts a comprehensive study on the interaction between TRIM55 and p100 and the signal pathway mediated by p100 processing. The results show that under stimulation conditions, the ubiquitination of p100 in Trim55-deficient cells is significantly reduced, accompanied by weakened p100 processing and p52 / RelB nuclear translocation, thereby leading to weakened activation of the non-classical NF-κB signaling pathway. The experimental results showed that compared with wild-type mice, mice with B cell-specific deletion of Trim55 had reduced induction of GCB cells and plasma cells in the spleen of mice when immunized with T cell-dependent antigens (NP-KLH); when mice with B cell-specific deletion of Trim55 were immunized with two antigens, T cell-dependent antigens (NP-KLH) and T cell-independent antigens (NP-Ficoll), the production of antigen-specific antibodies in the mouse serum was reduced, indicating that Trim55 plays a key role in the activation of B cells and the humoral immune response mediated by them. In the BM12 CD4+T cell-induced lupus disease model, TRIM55-deficient mice showed weakened B cell-mediated autoimmunity, as shown by reduced GCB and plasma cells in the spleen of TRIM55-deficient mice, reduced production of ANA and anti-dsDNA autoantibodies in their serum, and reduced IgG antibody deposition in the kidneys. That is, TRIM55 deficiency inhibited the onset symptoms of lupus disease in mice.

[0046] Through Figure 1 The technical route schematic diagram shown in the figure shows that the present invention reveals a new regulatory mechanism of the p100 processing-dependent signaling pathway, and establishes that TRIIM55 regulates the activation of the non-classical NF-κB signaling pathway by targeting and ubiquitinating p100 and promoting p100 processing, thereby participating in the regulation of the development of autoimmune diseases. Therefore, a new intervention strategy for controlling autoimmune diseases is proposed, and a new approach is found for the prevention and treatment of autoimmune diseases, such as Figure 2 shown.

[0047] The following is a description of the role of TRIM55 in cells with reference to specific examples, thereby verifying its effectiveness as a target for the treatment of autoimmune diseases.

[0048] Embodiment 1:

[0049] Wild-type, TRIM55 knockdown, and TRIM55 knockout MEF cells or B cells were stimulated with anti-LTβR, anti-CD40, and BAFF to explore the effect of TRIM55 knockdown or knockout on the activation of the non-classical NF-κB signaling pathway.

[0050] (1) The expression of TRIM55 was knocked down in MEF cells and M12 B cells. Wild-type and TRIM55 knockdown MEF cells or M12 B cells were stimulated with anti-LTβR, anti-CD40, and BAFF at 0 h, 4 h, and 8 h. Western blotting was used to detect the processing of p100 and the nuclear entry of p52 / RelB to explore the effect of TRIM55 on the activation of the non-classical NF-kB signaling pathway. Results are shown in Figure 3 ;Depend on Figure 3 As can be seen, immunoblot analysis found that compared with the control group, MEF cells with knockdown of Trim55 ( Figure 3 a) and M12 B cells ( Figure 3 b, 3c) Under the corresponding stimulation conditions, p100 accumulates at a high level in the cytoplasm, and the protein level of p52 / RelB in the nucleus is low. Since p100 is processed in the cytoplasm and then enters the nucleus to play a role, after knocking down Trim55, p100 accumulates in the cytoplasm. The above experimental results show that knocking down the expression of TRIM55 in MEF cells and M12 B cells will lead to weakened p100 processing, and then reduce the nuclear entry of p52 / RelB, thereby affecting the activation of the non-classical NF-κB signaling pathway.

[0051] (2) Wild-type, TRIM55 knockout MEF cells or B cells were stimulated with anti-LTβR, anti-CD40, and BAFF. Immunoblotting was used to detect p100 processing and p52 / RelB nuclear entry. Electrophoretic mobility-shift assay (EMSA) was used to detect NF-κB transcriptional activity to explore the effect of TRIM55 on the activation of the non-classical NF-κB signaling pathway. Results are shown in Figure 4 ;

[0052] Figure 4 a is the expression of p100 in the cytoplasm, p52 and RelB in the nucleus after anti-LTβR stimulation of wild-type and TRIM55 knockout MEF cells at 0h, 2h, 4h and 8h, and the NF-κB transcriptional activity was detected by EMSA (Electrophoretic mobility-shift assay, EMSA). The experimental results showed that compared with the wild-type, the expression level of p100 in the cytoplasm was higher, the expression levels of p52 and RelB in the nucleus were lower, and the NF-κB transcriptional activity was reduced.

[0053] Figure 4b is the expression of p100 in the cytoplasm, p52 and RelB in the nucleus after anti-CD40 stimulation of wild-type and TRIM55 knockout B cells at 0h, 2h, 4h and 8h, and the EMSA experiment was used to detect the NF-κB transcriptional activity. The experimental results showed that compared with the wild-type, the expression level of p100 in the cytoplasm was higher, the expression levels of p52 and RelB in the nucleus were lower, and the NF-κB transcriptional activity was reduced.

[0054] Figure 4 c is the expression of p100 in the cytoplasm, p52 and RelB in the nucleus after BAFF stimulation of wild-type and TRIM55 knockout B cells at 0h, 2h, 4h and 8h, and the EMSA experiment was used to detect the NF-κB transcriptional activity. The experimental results showed that compared with the wild-type, the expression level of p100 in the cytoplasm was higher, the expression levels of p52 and RelB in the nucleus were lower, and the NF-κB transcriptional activity was reduced.

[0055] like Figure 4 As shown, further experiments found that the p100 processing of TRIM55 knockout MEF cells and B cells was weakened, the p52 / RelB nuclear entry was reduced, and the transcriptional activity of the non-classical NF-κB pathway was reduced. These experimental results indicate that TRIM55 can regulate the activation of the non-classical NF-κB signaling pathway.

[0056] Example 2

[0057] Interaction between TRIM55 and p100:

[0058] (1) p100-HA and TRIM55-Flag overexpression plasmids were prepared and expressed in HEK293T cells by calcium phosphate transfection. Cell samples were collected and proteins were extracted. Immunoprecipitation (IP)-immunoblotting (IB) analysis was then performed using an anti-Flag tag antibody. Figure 5 The results are shown in Figure 5 a, p100-HA and TRIM55-Flag were expressed separately, and p100-HA and TRIM55-Flag were co-expressed. In the IB experiment, anti-HA antibody, anti-Flag antibody, and anti-β-Actin antibody were used to incubate, and the expression of the corresponding proteins could be detected, confirming that the experimental process was correct. In the IP experiment, after magnetic bead sorting, anti-Flag and anti-HA antibodies were used to incubate, and TRIM55 and p100 could be detected in the co-expression system. The experimental results show that TRIM55 can bind to p100.

[0059] (2) Overexpression of empty plasmid and TRIM55-HA in M12 B cells, stimulate M12 B cells overexpressing empty plasmid and TRIM55-HA with anti-CD40, collect cell samples and extract proteins, and perform immunoprecipitation to detect whether TRIM55 and p100 interact. Figure 5 b, The binding ability of p100 and TRIM55 was detected after 0h and 3h of stimulation. The IP experimental results showed that the expression of TRIM55 and the binding of p100 were not detected in the cells expressing the empty plasmid; the expression of TRIM55 and the binding of p100 could be detected in the cells expressing TRIM55-HA, and the binding amount of TRIM55 and p100 increased after stimulation. The experimental results showed that the binding ability of p100-TRIM55 would be enhanced with the activation of the non-classical NF-κB pathway.

[0060] (3) Overexpression plasmids of TRIM55, p100 and p100 point mutation were prepared, and TRIM55, p100 and p100 point mutation overexpression plasmids were transfected into HEK 293 cells by calcium phosphate method to make them co-overexpressed. Cell samples were collected and proteins were extracted, and immunoprecipitation was performed to detect whether there was any interaction between the two. The results are shown in Figure 5 c, TRIM55-Flag and p100WT-HA were expressed respectively, TRIM55-Flag and p100 WT-HA were co-expressed, TRIM55-Flag and p100S707A / S711A were co-expressed, and TRIM55-Flag and p100 S866 / 870 were co-expressed. In the IB experiment, anti-Flag antibody and anti-p100 antibody were used to incubate respectively, and the expression of the corresponding proteins could be detected, confirming that the experimental process was correct. In the IP experiment, after magnetic bead sorting, the cells were incubated with anti-Flag antibody and anti-p100 antibody, respectively. The binding of TRIM55 and p100 was detected in the co-expression of TRIM55-Flag and p100WT-HA, TRIM55-Flag and p100 S866 / 870, while the binding of TRIM55 and p100 was not detected in the co-expression of TRIM55-Flag and p100S707A / S711A, indicating that the binding of p100-TRIM55 depends on the phosphorylation modification of p100S866 / 870.

[0061] Example 3

[0062] Exploring the effect of TRIM55 on humoral immune response

[0063] (1) Wild-type mice and B-cell-specific TRIM55-deficient mice were immunized with NP-KLH (T cell-dependent immunogen) and NP-Ficoll (T cell-B-independent immunogen), and mouse sera were collected on days 0, 7, and 14 of immunization. The concentrations of IgA, IgM, and IgG antibodies were detected by ELISA. The results are shown in Figure 6 , Figure 6 a is the concentration of IgA, IgM and IgG antibodies detected by ELISA after 0, 7 and 14 days of NP-KLH immunization. Compared with the wild type, the antibody levels of mice with B cell-specific deletion of TRIM55 were reduced. Figure 6 b is the concentration of IgA, IgM and IgG antibodies detected by ELISA after NP-Ficoll immunization 0, 7, and 14 days. Compared with the wild type, the antibody levels of mice with B cell-specific deletion of TRIM55 were also reduced. From the experimental results, it can be concluded that after NP-KLH and NP-Ficoll immunization, the concentrations of IgM, IgA and IgG antibodies in the serum of TRIM55 knockout mice were significantly reduced.

[0064] (2) Wild-type mice and B cell-specific TRIM55-deficient mice were immunized with NP-KLH, and GCB and plasma cell differentiation in the spleen of mice was detected by flow cytometry before and on day 14 after immunization. Figure 7 , Figure 7 a is the flow cytometry analysis of the development of GCB cells in the spleen of mice after NP-KLH immunization, and its statistical results. Compared with the wild type, the proportion of GCB cells in mice with B cell-specific deletion of TRIM55 was significantly reduced. Figure 7 b is the flow cytometry analysis of the development of plasma cells in the spleen of mice after NP-KLH immunization, and its statistical results, the proportion of plasma cells in mice with B cell-specific deletion of TRIM55 was significantly reduced. From the experimental results, it can be concluded that TRIM55 knockout affects the formation of germinal centers and humoral immune responses in mice.

[0065] Example 4

[0066] Exploring the effect of TRIM55 on lupus-like disease in mice

[0067] (1) Wild-type and TRIM55 knockout mice were intraperitoneally injected with 7.5x10 6 CD4 of BM12 mice + T cells were used to construct a mouse lupus-like disease model, and flow cytometry was used to detect the GCB and plasma cell differentiation in the spleen of mice on the 14th day of modeling. Figure 8 Flow cytometric analysis of GCB in lupus disease models of wild-type and Trim55 knockout mice Figure 8 a) and plasma cells ( Figure 8 c) development, and GCB ( Figure 8 b) and plasma cells ( Figure 8 d) Statistical results. The experimental results showed that in the induced systemic lupus disease model, the number of GCB cells and plasma cells in Trim55 knockout mice was significantly reduced compared with that in wild-type mice.

[0068] (2) The mouse serum was collected on days 0, 7, and 14 of the establishment of the mouse lupus-like disease model, and the concentrations of ANA and anti-dsDNA autoantibodies were detected by ELISA. Fig. 9 Compared with wild-type mice, the serum ANA ( Fig. 9 a) and anti-dsDNA( Fig. 9 b) The autoantibody concentrations were significantly reduced.

[0069] (3) The mouse kidneys were collected on the 14th day after the establishment of the mouse lupus-like disease model. The results are shown in Fig.10 , immunofluorescence analysis of IgG antibody accumulation in the kidneys of wild-type and Trim55 knockout mice ( Fig.10 a), and its statistical results ( Fig.10 b). Fig.10 The IgG fluorescence intensity in the kidneys of TRIM55 knockout mice was lower ( Fig.10 a), IgG antibody deposition was significantly reduced. These results indicate that TRIM55 knockout alleviated lupus disease symptoms in mice.

[0070] Special Notes:

[0071] 1. Description of TRIM55 knockdown of the present invention:

[0072] The TRIM family members that regulate the activation of the non-classical NF-κB signaling pathway were screened by shRNA library combined with dual luciferase reporter assay. The control group MEFs or MEF cells with shRNA knockdown of TRIM family members were stimulated with anti-LTβR for 12 hours, and the transcriptional activity of NF-κB was detected by dual luciferase reporter assay. Fig.11 As shown. It was found that the NF-κB transcriptional activity of MEF cells with Trim55 knockdown was significantly lower than that of the control group ( Fig.11 A), indicating that TRIM55 may affect the activation of the non-classical NF-κB signaling pathway. To verify the knockdown efficiency of TRIM55 shRNA plasmid, we overexpressed TRIM55, vector shRNA, and TRIM55 shRNA plasmids in HEK293T cells and found that the two TRIM55 shRNA plasmids could indeed knock down the expression of TRIM55 in the overexpression system ( Fig.11B). In addition, TRIM55 shRNA plasmid was used to knock down the expression of Trim55 in MEF and M12 B cells. RT-PCR experiments showed that the mRNA level of Trim55 gene in MEF and M12 B cells with knockdown of Trim55 was significantly reduced ( Fig.11 C, Fig.11 D), indicating that TRIM55shRNA plasmid can knock down the expression of Trim55 in MEF and M12 B cells.

[0073] Dual luciferase reporter assay:

[0074] 1) One day before, MEF cells were cultured at 1×10 6 Evenly spread into a 10 cm cell culture dish.

[0075] 2) On the next day, MEF cells were transfected with corresponding amounts of plasmids: NF-κB-Luc (3 μg) + RL-TK (12 ng), pLKO.1puro Trim55 shRNA (2 μg) or pLKO.1puro luciferase (2 μg) by electroporation. Fresh complete medium was carefully replaced 8 hours after transfection and culture was continued for 24 hours.

[0076] 3) 24 hours after transfection, MEF cells were plated into 12-well plates at 1×105 per well, and anti-LTβR antibody was added for stimulation for 12 hours 12 hours after plating.

[0077] 4) Collect cells and detect relative luciferase activity of cells according to the instructions of the Dual-Luciferase Reporter Gene Assay Kit (Promega).

[0078] TRIM55 knockout

[0079] Wild-type and Trim55 knockout MEF cells were cultured using wild-type and Trim55 knockout mice.

[0080] 3. Construction and identification of Trim55 knockout mice

[0081] To further study how TRIM55 regulates the non-canonical NF-κB signaling pathway and the biological function of TRIM55, we constructed the Trim55 flox / flox Mice, whose construction strategy is as follows Fig.12 .

[0082] Using Trim55 flox / flox The mice were crossed with Ella-Cre, which expresses Cre enzyme systemically, and the genotype was Ella-Cre +

[0083] Trim55wt / wt Wild-type mice and Ella-Cre + Trim55 flox / flox Trim55 knockout mice (eg Fig.13 , Fig.13 A). The skeletal muscle, myocardium, MEF cells and B cells of wild-type mice and Trim55 knockout mice were taken, and mRNA was extracted to detect the mRNA expression of Trim55. It was found that the mRNA expression of Trim55 in the four tissues or cells (including skeletal muscle, myocardium, MEF cells and B cells) of Trim55 knockout mice was significantly lower than that of wild-type mice (such as Fig.13 , Fig.13 B), indicating that the knockout of Trim55 in knockout mice is effective.

[0084] In summary, TRIM55 plays a key role in the immune signaling pathway and can be used as a research target for drugs for treating autoimmune diseases. The present invention discloses a target TRIM55 for preventing and / or treating autoimmune diseases, and a drug and application thereof. The present invention discloses a target TRIM55 for autoimmune diseases, and an inhibitor targeting the target TRIM55 in the preparation of a drug for preventing and / or treating autoimmune diseases. A pharmaceutical composition for preventing and / or treating autoimmune diseases, and a method for screening drugs for treating autoimmune diseases based on the target TRIM55 are also provided. The drug targets TRIM55, its domain or TRIM55 protein truncation, and inhibits or weakens the function of TRIM55.

[0085] TRIIM55 regulates the activation of the non-classical NF-κB signaling pathway by targeting and ubiquitinating p100 and promoting p100 processing. TRIM55 can serve as an effective target for the treatment of autoimmune diseases, thereby participating in regulating the development of autoimmune diseases and finding new ways to prevent and treat autoimmune diseases. It has extremely important application value in the preparation of drugs for the treatment of autoimmune diseases.

[0086] The TRIM55 KO mouse sequence is:

[0087] tgaagcaaaccaaaccaaaccgaaccaaaaaaaaaaaaaaaaaaaaaaaggtccgatttaggctccctcgttaaaagacatactacacacgtacacacatatact

[0088] tcagtcaagggcagaattattagccttttgacgattataataagtcataattcctttgtacattttagagtcctgggcaacctgtgtgcttttgtttccagtgtatacaatctgct

[0089] gcaatttctagctgtcttccactttctatctttccatttttaatcctattttgttattttcaataaagcttttgattaaatctttgtcacttaaatatatccccctacataaaattatgtattt

[0090] ttcttattttacaaaggttccaagtaactcctcggggcctgcaggtgtggcatgttcaccaggcctcttctcaaaggcttcaggagctgggcttgagttgcctgtgcctccc

[0091] aagtaccgtgtgtgtgcgtgtgcgtgcgcgtgtgcgtgtgtgtgtgtatgcgtgtgccagagcagggtgtgcaggcctctagctcttcactgtcttcctccgctttcccag

[0092] aactggggttattggtgttcaccacagtgctttgccacttaaatcttatttgtgaacaataacaattcctgtaagtgtatgtggtaaaaagagaaaggaatgtcaagtcagat

[0093] agtagggatggtatctgcaccagaatccagacacttcctgaggtcagcaatccagacagggaagtgcagaggttcagatgtgaaaatcaggaggctgggtcacaga

[0094] gaggttagtcacacaggaagtgagagccagtggcttgtgttgactctgtaggacattggacagggtgctcagcatctgggttatttccctttcatcttccataaagcagg

[0095] atggccctgtggcacttcctctcacttaagatagaatttcactctcttagcagaggtcctggaacacagtaagaatcaataagaagggggatgcggatggaggaaaag

[0096] gaaacgggaggggaggagagtgagaggcataaatgcagcctggtattctgctggcacactgcagagcctgggacagacttcagtaccaaggggcctggtgaaca

[0097] tgctacacccgcaggccccgaggagttacgtggaatctttgcatatgtgttgtctaaacacatatgtgctgtctaaacacataagacagtgcagaaaaggggtgctggg

[0098] ctttcccaagccaaggaagtagaccaagactgaaaatacagaatgtggttaaacacattatgggatggccatgtaatagcgtatgtgtaaaagtaaccactggcatttta

[0099] ctaagagcttgtaagaacatggcggcaggggtgtgtagttccaatggtgctaaggaggactagagcctggtgaaggcgtcagataggaccacttgctgggcatctca

[0100] ctgggtatttctagcacagaaattgtgccaaaaagctaagaagctgagtggtggtggcgcacaccttcaatcccaggaattgggtggcagaggcaggcagatttctga

[0101] gttcgaggccagtctggtctacagagtgagttccaggacagccagggctacacagagaaaccctgtctcggaaaaactaaaaccatgcaggggctgaggatggttt

[0102] cttagaaccatgtggacttctgagcaccgccctgctgaagcaacacaggtttccaacctcttgaacttgtcacaaactcttcagaggacagaaaatattgctgtaagcc

[0103] aatgaggttgtttgtcttgatcatcaacataactgataacttgtttggtttgtccttaatgggacaagtatttacaatcccatgtatggccatggtctctgttgaacatttgcacg

[0104] gtttgggttatttcaaatttttggcaggccttgacggtggagacttaatttctattgtttgaagcttgtatttctcttgtcctttgtgggttttgtctgtttctttaaacttttattggaca

[0105] tatgaaaacgtcttgttgacttgcaatgcacattttgtagttaaagtctgtaatcagcaataaaaaacccaaaatgtgcaaaacaaaaaaaacaaaaaaaaaaaacaaaaaaa

[0106] cccaaaaaacaaaaaaacaaaaaaagaaaaaactaaaaccaacacaaaacaaaaaccaacaacaacaacaacaaaaattgtgccaagaaatgttcagaagaaagta

[0107] tggtatgctaatttttactcattttctcaaagatcttcgtgatgaagtgaaatctagaaagaacaactttgaaggtggaacaattatcaagataaaacacagcttccagtgag

[0108] cagtttgcaatcagcttaatacatgaaatgccagcgtgtaaagcacttgaaattagtcccttccttgtgatttttatctttatgaaatgtcatttctcattcaaaatttaaagtcat

[0109] atgttcaaactccttgttgccaggtagcaagaaaaactgtaacagagccaatgagcagaggcaacatggcaagtgaccagtggagtttctgatcaggtactgggctctt

[0110] ctgcgtaggtaataactgggatctcactacacagatgaaggctagggctggtttatttctcagtaacttttaagatcgatgcataaaagcagaaattctcaggatgagggt

[0111] cttgcctctgttgtcactgtaaggggtggtggttttgtgtattctagactggccttcaatagggtgcggccttggctctacctcccacacactgggaaaacaggaatgcgc

[0112] aagtagctcacattttttaggaagtggggttttttgtttgtttgtttgtttgtttgtttttatccacagctttggctataggaaaccaaaggaagagccattgtatctgggtgatcta

[0113] ccaagccttatggagagaaggtgaagccccagctgacagtatattgttaatatggaatctgtgtcaaggaccccagtgtagcagacccaaggagggaaaaagcctac

[0114] cccaggtgaggtccacagggatgacaaagctttcctctgttcccagaactagtgaggagtgactggcctttagttaaaaccagtgtatgcagaaactgtcaaccagag

[0115] cttcctgcctctgtatgtgtgcatgctgagggtgctgcttctcagaggcctccctctggaggagttagccccttccctgtgtgatatacacaggaactttctggggggattt

[0116] attgtatcatgggtgaccccacacagcttttctagctgtcatttcttcattcttgtgtcccttgagtcatgtttccagggccaagttgtgcagaacacagcagagcctggtact

[0117] tggaacaaacccagttcatctaaatttcaaataaagtgtagccttctttatggaatgtgtagtgtattttgcttataatagttgtggggaggaggacggatggatgtgtattgc

[0118] agaaaactactaacatttttagcccaggagttggctcaagtacccacataaaaacacttgcaaccctagtgctgggcgatgaagacagaagagtcccaggggctcact

[0119] aaccagtcagcctgtgcaattgtcctgtcttcagaagattcagagcatgagagctcatggcagctgatgtcagtgtctggctgtcatatgcacgtacacgcagagctag

[0120] cacatctgtgcccacacaccaagaacacttggtttacccaagattaattatttgctgttttgttttgttctgggattaaagtaatttggaggagaattaatcaatgctgcaagc

[0121] catgtcctgcgtgtgtgtgtccactcttcctaagccccctccttcagggctcaccagtgcctccagcattcacatcatctggtgttcttaccttgttttgtttgtatttttctctgtt

[0122] ttaatgatggaatcagtcataacttcctcgattttactgggtataagatattaagctagaagcaagactggccttgcaattcctggttttctttcagtctatgttgatcttgattg

[0123] ccaaaggactgattttagaatcttttaggaagcctttgggcatgtctgtgaaggcacttcaagatggatttaactaaatggaagaccaaccctggatatgtggagctctgtt

[0124] ccccattccctggggtggagtcccacaccctgtaaaagtgtaagcaccagcatttagattcccccctgctccctgagcaccagcatttagattcccccctgctccctgag

[0125] caccagcatttagattcccccctgctccctgagcaccagcatttagatcccccccccctcctcaccatgattcagtgaccagctgtcctaggttcccaataccttgccaga

[0126] gctgctccggcttccacaccttctctgtcatgatagattatcctctcagattgtcaatacagtaaaccctttcctccttaaactgttctcatcaggtattttatcccagcaataag

[0127] caaagtagctaatacagctgtatctaatacttcctgttggtttcacctcgtctctgaccataaatcctattttgtatggtgaacatatatttgttgaagccaagggcctgtactg

[0128] atgagacagaaggggccaggaataaataaaagccccctaggtactctggagtgctgagaaatgttaccacttttgactaatgcctcaaattcctcctattccccgaatttt

[0129] ctgacctattctggaagttggggtaaatttctgattatttttctaaccatctggagccaatgggcatttacatttgagctttaagcacagtctcccaatggtgctcgccaagct

[0130] cctgttggagaatccttgatatatactttagtttccccaagatttttttttttaattttacattcaagggctgttcgtccaataagaaaccactcaccacatgtggtcatttaaacat

[0131] aaacttaaattaaatgatgcttaaaattcaatttaagactttttagtaaaatagaactggagagtctcatattaaatgagaccacaatggttcagaagacaaatgacacatgt

[0132] gtcgcacacacgttacacatgagtgtatctgtggggatgagtgtgggtataagctatgaaactaagaaccaggcccacagaggagagaccagggacttaggaaggg

[0133] gcaggcgtgatcaagcacatgtagcatgaaagtggagagggatgctcaggaaaacgagcagacagggagatgaagaaacaggagtttggggaccaaccaaagt

[0134] aaacatgggtgcacatgtcctggtgaactggttaccttgtgctctgaccgaaaggaagaaagaggcagctcccctatgccaaggccagaagagtttcagcctagacc

[0135] tcacaggcttctggattcttcataggaccttgtcactcactgtcaccaaggatacaaacatcaagtccagcttgtgtctcctgatgcttcagcatctgctgtctccctagaag

[0136] gagagcctgataaagcacatgcagcatggagggagagctgtctccagccctttctggcacatgttcaagtccatctttattaatgacagtgctttaagtttttgcacagtca

[0137] tctaacaagaaatgcaccgggttgttcttagataatcggaagtgttttaagggcgtgaaacacccaggcattaaacacatcagtgaacaaaatggcttcctgtcacaaaa

[0138] atgtaagtttatttaatatatttaaaacattaaactccagtctagatctatacacattctcatcaggcagtagaaaggttggacagtgcagatagaacatcttcatcaccatag

[0139] aaagtcctgctggaactttcatagaagaagatcctgacaactatacccctggacaactgttttcagccacagctggccacagtgataataacagttattatgagatgcatt

[0140] atacagactgactggaactaggtgtgtacttatttattgtgctgaaacaggagtaaaaataattttagagtctccgttccccagccctgaagatcatactgaggttttagtga

[0141] atgatagagaatttggcactggtcccaccttctctaaattcttagaatttcccgaatgatgtcattgataatttatgctaataagatgtctcaggacagactgaacaggatgg

[0142] ctagagtgaccctatgattagaatgttggagttctgaggcacggagtatcagccaggcctctgtgaagcaaggagggctacagctagtcacatgaccagcaatcaaa

[0143] cccagcatgcctctgtgataaatttgtactcagaagcacgggtgagcctccctggataacatttctctgtctggtcttacatctatgtgcctggaggatggtgtatcttcatg

[0144] ccacttagagaggatatgcaagttcacatctggagactaccattcaagtccttcctggtgtgtcttgtcttctggctggtcttaagttttatctttctccttttactctcttaaaaa

[0145] aaatatataaggataatgtctattatatatacatacacacatatatatgtgtaaatgtatatataaatacacacatatatgtatgtatgtatgtatgtatgtatgtatacatatatatg

[0146] gcttgcattagctacttttctcatcgctgagacaaaattcttgccaaaagaaagcttgctgttctacaaggcaccagttgttgtggagggaaagacatggcagaagcatgt

[0147] agctggcactccttgtctctggaagcagagagcaaacaggaagagaggcaggcctacaaccagtgaccttctctgtccagtgaggctccgtctcctagacttaaagat

[0148] ttcacaactttccaaaggctcaccaccaggcaagatccaaggggtcaaacacatgggcaaggagtggaaggggtgaggaagtggggagtgggtagttggggtag

[0149] ggggacattgtatatttagcttgtattgctggaacgtaattacaatgcttttctgaattatgagtcatcattagcaatttttcaaactgaagatgtagtgagaatccttgagtatg

[0150] tagccactggatcagaagtttaggtgaccttggaacccactttgctgacagagctagaatgaggccctgtgtccttcatgtgtcaagtgtggctcagttgcatgtggtca

[0151] gaattgccttgccctggcccctgaagccttcttccatttgatctggtggctagaggcaggagaatgtcataaacctttctgctgtcatctgtcatagaccatcccagttggg

[0152] catgggggtccctgggacgagagtccttgaggctagatgggtaaggatttggtggtgacaataaaaaacaaatacactggcagtttgaatccgagtatattttgtagctc

[0153] tcaaacaagggtttttatgtataaagaaacaagtattacaactctgaaaagttgtgtttagtgaagcagtgaaatagtgatattggaataatcatttggcacagtaaaatata

[0154] aaaatcatctaagactagtggcaaaattttaagaacaagttaataaaattttttgatcagtttctatttaacatctagtacctggtattttgaaaatctttaaagcataaatttaaa

[0155] ctgttttaattttttatttaaggctttttaaaacaatatattaaagcctacttcttataacacatgtgcagccatataaaattttatgattgagaaagtttttatttatcaacactatctt

[0156] atacatgatttataaagtgaagcactggttttcacacagataaggtcatggttagcaattttatcttgagggattttttttttacccattattttcacttaagatcttagcctaggct

[0157] atcagaaacatcttgtaaataggaaaaggaataaaaagataaaatggataagttgtcaagagaactatggctcaataatttttctctctccaagaattttacaatcagttcca

[0158] ggaggactctttcttttgaagtctattgccttagtctgtggaacttgtcacacagatcttactggggttggtggcagtcatcaattaataccaaatacactttggagactgtca

[0159] tcattgccccagactcacttcagaaattagaaatttcagtttctcaagatttctaaatatgattccaccactttgatggactcaggcatgaagacttaatgaggccttctacca

[0160] ccccagtgtcccatgtaattgagggatttcaggctctgtcgggtgtgtgtggtaagcctgcacggagaaggggaggggccagggaccctccaggccactgtgttgtg

[0161] ttcacaactccatgaagaccttaaatatgtagtcctggatggtttctgtctcctgtacttcaaagaatttggaagtagactaaatattctggctcttctctagagcacaccctgt

[0162] gtttcacttccaacacacaaaccagccacatagatggctgatgtttgaaagaaggcagagtcaagagatggaaaaactaggatgagggcaaagtgaagattattcagt

[0163] gcttgcatatattcctatggtgtttagctgcttcccatgtggtctacagggacaaaagatgaaagatagcccaatgaagcagaagatacaaaggaagaaggctgggag

[0164] ttgagtatatatccaataatggaagcgggatattcaaatgaaaagtaatattgtttctgagatatttcctccatacctgtagcctcctggcctggagcaggttgagaggcaa

[0165] cacagattagcactctagtgtggctttaaaagattgacagtctcccgaagttctagctctctaactatactgaaaatgtaatttaggactttaaaatgtcataaaaacctttcttg

[0166] ctctttctgagtgtaaaaaagctactggcttagacagttgacacctgtagcctaaactcagaggataataatgtggcctttgttctatctcagctgcatggttctaactttca

[0167] ccctgctagtagaaggtctcaggaagaaaaaggacactctgaaaaggtgattgtagtatttattactaagttgtaaaatgtttcctattaaaagttctcttaacaaaggggggac

[0168] aaaggaatgaatgaaaaaagagggataagggaaattctttcctctaatttgtctaagaagtgttctgttcataaaagttcttttagctcagttctgactcttctctttgtcctca

[0169] gcacttatacatctttcagaatacatgatcacatggtcaaagttcatcacaagttcacgtataaaattcaaatcataaatcgaataagaagtttacaacagtgaatgtttacat

[0170] gcatatccattacgagtaattatctggctaaacattcgtcacctgtcacagttccacaggttcatggagagttaaaaaaccatgactaagttatagtaaatttctgtatggata

[0171] aattcactcaagattttatcttctgtcccagcacctgtaatctgtcctagcacctataataatcgttagttcccttttaatgacctttggttaatggctttacaacctcttggaat

[0172] gtgctctgggtagtagaaagtctggttcctatctagaagcaattaactggagacacaatgggagactggcagtgttctcattgcagttttgactatcaggaaaggaccta

[0173] atagcagtcccactataaaagagcttaataatcactgatatatttaaggaattcttataggatcatcataaataattgtcatcataaataattaagaaggtatctatttgactata

[0174] tagcaccactacaagacagtacatcttcatagatctactcaaaagagtggactaatacatagtgatcatcacatatgtttaattatatcaggaaaagcataacaatagcag

[0175] goesctttcttaaaatgattctcctgggccttgcatactgtagattttaatctaaggtggttatctcagaaagatcacctgctagttattagcttgtcctatgatggctcctgaca

[0176] aaatactgctaagttatttatgaaccctaatcagttgtagatggcacaatctaaatccagttaggtgaaggtactttcctgatggtacaaggtaaggcagctgcagaaccc

[0177] agagagtctgcctgctgactcactgtagcatcccctgcagcaacagcactggtggaccatgctaggcatctgagtcatctccattgagtttactatcataaccccagagt

[0178] cccttcctactgtcctttccactgctactgccctgtgcaagggcttgctgacctcttatttgaactattggaagttgccaatcttgtctttagaaagtcccctgcattgttgtct

[0179] gtccccatcacagcaggaaggtcattccaacaacacagacatgagttcatggaaacattctcaagagaaatttttaatgactttctagtgcctggttttagtaagttttaaag

[0180] aaacaacccaatgcatcagtagggcagtcttcactgtaggccaaactactaagagctctgatgctccctgtacgcctgtgcctccaggtctctgcaaatgtgtcttttccc

[0181] agttgatttattccaaagctatcaaaccctgaagctttcctcattccttgcactcctcattagtgccttctcaccatgtggttcctacatgtgaatgtgttatctctatcatattga

[0182] gctccttgcaggcaacagcctaatctctataatctttcaattcccagagcattgttcaataggaacatatggtaagtactcagtgaattttttttttttttagaaggaatgggtta

[0183] tttccacgaaaggccctgcctcagtgtacaactcatgtgccgcacaactttcagccattgatggagctttggtatatatactgatagccccatgagattacatcacttggtg

[0184] acttctctggttataacgacactgatagtgcattatggacatgtttgtagtgatgctcctagagaggagtctagattgatgttgcatacttatgtcttaattttcagtgaaagga

[0185] ataaaagaaacacaaaagaggcttatataatgaaaaatatgcagaaagtagtttacagttgtacagctgtgtttaataaatgtttattacagctgtacagctgtatttaataaa

[0186] tgtttattacagctgtacagctgtgtttaacaaatgtttatcaagcaaaagtacaaaattaaacatttacaaggtaaaaagtaatactaacagagtcggagagatgatcaac

[0187] agttaagagtactggctgttcttccagaggatccaggttggattcccagcaaccatatggtggcaaacaaccatatgaactccagttccgggggatctgacacctcctgt

[0188] tagcctctgagagtactgcatgtacaggatacaaagcaaagtaaacctcaaaaaaaaaaataaaataaaataaaattacagtaagctaatgtcaaataattttgaagagt

[0189] aacttttatttattcattcactcatttataaattgagcagccctgagcacaggtccagcacagtctacagtagtctgccttcctgtcccaggcttcatagcaactcacaatctc

[0190] acctagagcagcttccaatcttttcatcctttatatccatttctattactgtacttcaggtgtgcaatgcttttaccactaagttataattgcctgaagcattcagcaccacacca

[0191] tgctgttccagcttgtagtctaggagcagtaaaacataccaaagagcccaagtgcccagttacctctgcctgctaggcttgagtaactctgacctgatggtcacacactg

[0192] atagacttgtccagggatgaagttctcaggatacagccccactgacaaatgagtcatgagtacactattagtttaaagaccatagtggttgatcttggtcattaacttgatta

[0193] aagactcctagaatatcagtcaactacctctctgggtgtgctgtgaagctgtctctagagccaaatctcatgtcagtcttcaaactgataggcaagacatactcacaattg

[0194] agggaagttggagcctaggaacaaagagaggaagaggagggcatccaaacttgttagcaccctgggatcttttggggtggtttggtgtttgctgctgctctcattctca

[0195] gacactggacttcaggatttttagcctttgacttgtattaatgactcttcagggccttcctgaccttctgccttgggctgagactacatcattggtctctcttgtcctgaacttca

[0196] agcttctgcattaagaacctattggtttctgcagacctctagtcttcagaagagcattatgcatctatacagcctttgatgatgtgatctgatcaggccaacccttcttatgat

[0197] actacacatacacacactcatgagcgtctaatgcttctactactgattctactcccctggagaaccctgactgataggcgcttttctttagatttcaagggcttttacttctga

[0198] aatagaaactcccctggaaattaatctcatatatagagctgagagccccatagccagctataaacagaatctaactggtgaagtgaggtgacagggtgtaattcatcag

[0199] gaaagaggcatgaattcccatcttcactgcaattgctacataaatattgaaagtttttgagagactcctccaaaatttccccctgatccttatgtattcatataacttcatattag

[0200] ataacagggcttacaaataacactgcccatgcatatagaacaagactgcacagccagtcacaatgacctacgttttattgtactaactgtgtgtatgtgttttgctttggattt

[0201] ttgaaatcatagagaaataagataaaaaaaattcacataaaaactggcataagaattagaacaacatctcctgtagctcagccttgactcaggctgtgtagcttgattttct

[0202] gtcctaagctcacaaaatgatggaatgcgatgtgatagaaccatccaaacgaaccaacaaagcagtgaacaaactaaccaaccatccaactaatcaaaccaactaaa

[0203] taactaaccaattgactaactaatcaagtaaccaaccaaaacccatccaactaacctatcaaccaaccaaccaaccaaccaagggaagaaacttcattgtctgagatgc

[0204] ttttggtcagaggatttaatcccagcaccagagtagcaagttttaacaaagaattggcctgcagaactgattctgatattggacagaacctgggaataatcttttttttttttttt

[0205] ttgagaaatgtgggaatataagaacttgggatggcaaaaggcatagaaaattgcagagggctcaattggctcttcttgtaggacctggaaggtcggtgtgtcgggagtt

[0206] gtatagaccaagggactgcctggtcataagatttcagtggaacatagtttccttgagaaattcaactagatatcactgtgatattttggcccccaatctttcttattctgcttat

[0207] gccgtgtggaattaagttaggcagaactagaagacaatgggctgctgtcttagactctcccagatgcccctggctctggctatgctctcagttttgtttataataaggattc

[0208] tcttgcacattatttggaaaaaaaactggcataaaatttctcttgcatttttatttgattcctggttttacatacaacacatattgattaacatttttacaaattcctggcaaatgtca

[0209] gaatttgtgttcatgattcaagagcagaccaagaatatgagaaattaccagggttattatcattttaaaacatgaccttgaatgaaggctaaaatgcccaaacatttaggag

[0210] cagattgggaagttatataggacaccttattcactgcagctatcaaaggcactggtatcttcctccaacaggtggagaagtgcaattatagtgtgtaatgcacatgccaac

[0211] tgttatcccattgagtcttactgtcacagagcaagcttaattttaaaaagactcttacagattgtctctatgttttagggtgtatgtggggtgggtagaggatgaacgagggc

[0212] tgtgtgtggagagccacatactcaacattttggttcttaatttttttaaaaaagaggtctggggacatggctctgtggataaatcccttgatatacaagtgtgatgactccagt

[0213] tcagatccccaggactcacgtaaatgcctgccagttgtggctgcccacctgtaatttcagggaaaccagaggaatcctctaagcaagacactagccagacttgtcctct

[0214] tggctaatgttggcttcacttgagatatcctgacttcatgaataatatagagtgatggaggaagattcttgatagcagccacatgtcccccacatgcatgtgcacaacatgt

[0215] tcacccacacacatgtaaacatgcacacacacacacacacatacatgtgtaagtgtctaagtacactcttttatggtgaaagcattcttaaattggaattttcctatactgta

[0216] atattggcaaacatgaaatttttaatatcatggatggctttttacactattgccctggtcagatcaggatggagtttttacagaacaagctttgcttacttgcacctgagaagtt

[0217] gctaccatgttcctttggctggcttgtttcctgctgtctgaagaaacaatgcttcttatacagttacactgtagcgctgcattctacagaagcctgtcactaagttcttcccaa

[0218] gtgctcacgagggcatggctcagatactaagcatgcacatttgcttttgttgttaacaactgtgggcttcagattgccctccttcataacccctgctaaatgggacttgtaat

[0219] ccccttcccacccactctcttactccccagggcaagaatgttgactcctgttctctgtcacctgtcagaatccatctgcctgtcactggcttatatggacaagtcagtccag

[0220] ggtcatgtgaccttgggttgctaaaaacagctccgtcacccatctgagccaggcctggaacaatgtcctccacagagagagagacataaaggacacttgatcataca

[0221] gtccttggaattatttccaggaaccacttacagaagccgttcgggagcacctttcccttggcagcacactcagggacagggacggcaaggaaatgagcacttctctga

[0222] attacaagtctttctccaaagagcagcagaccatggataacttggaaaagcaactgatctgtcccatctgcctagagatgttcacgaagcctgtggtcattctcccttgcc

[0223] agcacaacctgtgcaggaaatgtgccagtgacatcttccaggtaggtttgtttggaatctggaagctgtagagtgtccctgtctcctgcagtcttgagatcaaagccca

[0224] gttcccttagagagttttctgagactgttcgtgtatttgaattgtttccccaggtttgattgtcaacctgctcctcacaagtcctgctgtgtttttaattcttagaagcagatttgttt

[0225] gtagtaaaacaaaactgatccactgaatctgtttaagaattgaaaatgttaccgctcatgtctgagcattgggtaccggcgcgcctagtggatccccgtagtttaaacaca

[0226] gataacttcgtatagatcatacattatacgaagttataagctgtcgacagatctagataatgctggcctgaaggagtgtcatttacagaaatgggaaggggacacatttc

[0227] aggctggttccctgctatacaatactctttgcaggcacttggtgattgtctggtatgtcacctgaatccattcactaggacatttaaaagggcttcttatttatcacaccacat

[0228] aagctttacattcctagagacaaaactacagagtttattcctaaaaggcatgaaggtgtgaagaccctgatctccattctgcacaggtcatttgagtctaaagccttggtag

[0229] ccatatggaaattgtgtagaaatgggcccgttgtgatccttattacaagaggcctgtgcttatgaaacatctgcaggactataaacgccttgcactctttgcaggaaagtc

[0230] aatcaattaatataacgtgtaattattgtgattattatggccatttgtttctgctcaagattcagagctaaattagatagcaccaggctatagatggaactgaggaagatctgc

[0231] agatgacgaaccgcagccacctgttttcttgcttcctaatctcttgagaaggaaagtgttaccatgttccagatcagattgcattgtattttgttgtgttcaacagagttatccc

[0232] aatggatacatctataaaccatccccacaccaaaggcttaggaaacattgcagaagaagcggtggaaagactctaagagccagtagatcagggagtttgctgtgagg

[0233] ctatgtttcctattagtattagaagctacacccgttactgcccctacagcacgactgcttaaacatgaggtgaacaaggaaaacaacagtagacatgcaaaagtggatg

[0234] gggaaagacttcaacttgcaaagaatcacaggcagctaagaagtgctgagagtggggaatgtcttccccagggaagagcacacccacagggttatccaacaccata

[0235] atggtcatacctgaaaacataataagagtaaaattgtatagactgagcagatcttatttagtaatgtacacatgtatagatatatgcatgcaataacaattttaaaagagtcta

[0236] tgagtttgaaagatcaagagggctagatgggagggtttggagggagaaaagtgaaaggaaagatggtgtctacttaagagataatcctggcaactagtaagcaacca

[0237] gttattaaaagacatttctgtgacagcagtttcagtatcagatccagtgtttttccacagagcaggggctccacaatgttagttctgtaagcagcacaggcaagcatagta

[0238] cagtgttctgtgttttatttattataatgctttttctgtctctctactcacctggtaggaacttcagatatgaacataatagcatttggtatttggtatgaaatcttgaggacagaaa

[0239] atgttaaaacgtgtgttagtaaaactgcctaaccaatggagtcagcctgatgtcccttcaagggctggggaccaaactgtagagaaccagtgagtcaaatccctaccac

[0240] taaagcaaaaaccaagtgaaacagggatatttttgtgattataaaattcttacattgtttattaaactcaagttgcttttggagctggctaaattgtgcttagtcacataaaatta

[0241] gatgttcctgaaattgggaagtcataacagacctgaactccaagccttgagatatattagatgaccttagaataactctgggtgcacagaataaactaaccccaggtata

[0242] gactttagtactgtcaacagaaaggggaaatactgccacaatgggtttttgaagtatctttcaattctggcctcatacagaacagctggactcttgaaaatgctggccttat

[0243] ctagcattttgtatgatgatgcactatccacagttaatttggtttatattattattgaacatagtgattaatttgttaatgtaattaacaaaagggctctgtgttgtgcgttacactct

[0244] tttcagaagctttacctcggattcaggtgggattctttgattttcccaaaaacttttgaggtgggttctattatcaacctaccaaattgtggtgtgtgtgtgtgtgtgtgtgtgtgt

[0245] gtgtgtgtgtgtgtgtgtgatgcacgtctgcatgttcatgtacaggcaggtctttagacacatgtgtgtagaggccagaggtggatcagatgtcttgctctgttactcccca

[0246] ctttatcttttaagacagagtctgtcactgaaacaggagcacacctagctagtgcgcctgagcctcgccagaattaggaccataggcacacaccactatactgtttcctgt

[0247] cctttttaaaggttgcttagaatcaatttcaggtcctaacgcttgcatggcaagtactttactgactcagacatttccccagcccttttaaatgcttcagtgggaatgaaattta

[0248] taaatattttaattttaaatgagtcttaaacaagactacaaggcctttagtgagaagggttgttccaatctttatctctcacactcctcaaaaattttatttatttatatttttgcttatt

[0249] cactttacatctcactcactgccccccgcccagtcatcccctcccacaatccttcccccatccttccctccccttctctctgagcagtctctgtgaggttcaatgcaacctcc

[0250] ccctctgaggcagacaagacagtccagctagaagaacagactccacatataggcaacagcttttaggatagcccccgctccagttgttctggacccacgggaagac

[0251] caagctgcatatctgttacatatgtgtggggtctctcacgctctcaactctcatttcccaaatgctagaactcaattttccaagcgacatcctgctgtacatcaccaatgata

[0252] actgtgttactggtttgtccttttgtaacaaaacaaaaaaattttagacttattttattttattttattttattttattttattttatatgtatgaatgttttgcctggatgcatgtatgtgtgc

[0253] tatgtacgtgcctaatgcccgtaaaggccaaaagagagtgtcaggtcctctagaactggagttatgcatggtcatgagcttaccacatgggtgctgggaaccaaactct

[0254] ggtcctctgcaagagcaatatgtgcttttaaccactgggacaactctcccaccccatgtgtatcttactgtgatgttcctattccctggtttttgtttttcgtgttgcgcacaga

[0255] ctatcagctttcttctgcagtttgagccttttcagtgctgggccctactctctgcttgggaattgctcttatttactcccacttcaacccatgtcttcctctcttggcttacagtgtt

[0256] gcttttatggagcacatttccatatgcaatctccatatgcaatctccttatcttcctcctcattaaatgtcctgcccttacacttgcaccttcacgatatcccataagttctgctga

[0257] agattctggctgcagttcttgaaatttcctaggactcccagcttcttgtaggcatctgtatagtgggcaaccagcagaaattgtctagctgtttccagttgttggcatttggta

[0258] gacatctattcctgtcatcttgccctttgtgtttattgctttatgtagttttatttccttggtctttttagggagacttccacaggaaacacaaacaactgccgctgctcattctgcc

[0259] acatttagttagaagacccttagtcttttttctagatgagagaaactgatgtcaaaggtttaatcctgcgtttaaggccacacatcaacacatgcagaaattgagctggaat

[0260] caaactttcagctcactctaactacagcagcatgaactgaactttctcaccaggacttcctgctcactcttcagtaaaggctctgctaaccccagagacctggagaccac

[0261] agcgaacaacctataggtctccacagtattgaacatcgagaggcacttccaacattcatccctgagcctggagccacggctgtgggcccagccactctaacagacag

[0262] cgcttacaccctcagttctctacgccagacttgtttcagatctgttatttggtgtaagctgtatgcaggatttttcatatgtatttatttagaagagattggtgactttagagccat

[0263] gttgcccctaagcaaagcaaaaacaaaaagccaaaccaaaccaaaccaatgaaataaaggaagcaattctcaatgtattatgaaaaaaagagtaaagtgaatcatga

[0264] acaaaaataaaatatatcaccatggctgtttccatatgttcagtgtgtgtgtgtgtgctttctgcagtcccactccgtaagttcatatctgctgatatcataagagcgcaagg

[0265] gcattagcaatatgcgctcatgtgggcctgcccagggaagataaagggcgttggcatgcctgtgctgtgatatgctggcaaatatgctccagacttcagcaaagccca

[0266] agttaacataatgtacacatagaaggctttgaccaggcccgcttgtgaatcctactctccagatgaccacaaagtctcaatcaattctggtctcctctcaatatttcagcag

[0267] ggctcctctctctgaggctctgctccaggcacaggatattcagaaaaaagagaaaagaaaagcaaagccttgaattctggggcagacctgggaaggggaacaatctt

[0268] cccattcatagccgtgcttccttgcttgagtcccagattcgtgggtgaatctggaggccatagatttcattaacatatacagctatgggagcagacaaaaattcctcttgca

[0269] acatcttgggcctagaacctcatgtagcttccttagaactgtaagattcttctataagatgaaataatagatatttcccagcatttattggggccaatgttaaatactgcctagt

[0270] acagtagagatatattgtctgtcctctgccttcggggtttggaactgcagctacataatcccacacaccaatactatggtgttcaaacacttggatttatgacccatagtgg

[0271] acagaatgttttacattgcaacctatcttttctttctttttttttttctttttttattagctattttctttatttacatttcaaatgctatccggaaagttcgttgtaacccgccccccaaga

[0272] cacacacacacacacacacacacacacacacacacacacaccgccctgctccgatacccacccactctcacttcttggtcctggtgttcccctgtactggggcatata

[0273] aagtttgcaagaccaagcggcctctcttcccagtgatggccgactaggccatcttctgctacatatgcagctagagacatgagctccgggggtactggttagttcatattt

[0274] ttgttccacctatagggttgcagaccccttcagctccttgggtactttctctagctcctccattgggggccctgtgttccatctaatagatgactgtgaccatccacttctgtat

[0275] ttaccaggcactgccatagcttcacaagagacagctatatcagggtcctttcagcgaaatcttgctggcatatgcaatagtgtctgcatttggtggctgattatgggatgg

[0276] atcccctaatggggtagtctctggatattccatcctttcgtcatacctccaaactttgtctctgtaactcctttcatgggtattttgttccctattctaaggaggaatgaagtatg

[0277] cacatgttggtcttcctcttcttgattttcttctgttttgcaaattgtatcttgggtattctaagtttggggctaatcagtgagtgcatatcaagtgacttttttgtgattgggtta

[0278] cctcactcaggatgatatcctccaaatacatccatttgcccaagaatttcattaattcattgtttttaatatagctgaatagtactcattgtgttaatgtaacaccgttttgtatcc

[0279] attcctctgttgagggacatctgggttcttcccaacttctggatgttataaataaggctgcaatgaacatagtggagcatgtgtccgttttaccagttggagcatcttctggg

[0280] tatatactcaagagaggtattgctggatcttccggtagtactatgtccaatttctgaggaaccaccagactgacttccagagtggttgtaccagcttgcaatcccaccag

[0281] caatggaggagtgttcctctttctccacatcctcaccagcatgtgctgtcacctgagttttttatcttagccattctgactggtgtgagatggaatctcagggttgttttaatttg

[0282] catttccctgatgattaaggatgttgaacattttttcaggtgcttctcagccattcggtattcctcagttgagaattctttgtttagatctgtacaccatttttaatggggttatttga

[0283] atttccggagtccagcttcttaagctctttgtatatatgggatattagtcccctatcagatttaagatggtaaaatcctttctcaatctgttggtggtcttttggtcttattgaca

[0284] gtgtcttttgccttacagaagctttgcaattttatgaggtcccatttgtcaattcttgatcttacagcataagccattgctgttctgtttaggaattttttcccctgtgcccatatctt

[0285] tgaggcttttccccattttctcctctataagtttcactgtctctggttttatgtggagttccttgatccacttagatttgaccttagtacaaggagataggaatggatcaattcgc

[0286] attcttctacatgataaccgccagttgtgccagcaccatttgttgaaaatgctgtctttttcccactggatggttttagctcccttggcaaagatcaagtgatcataggtgtgt

[0287] ggattcatttctgggtcttcaattctattccattgatctacctgtctgtctctataccagtaccatgcagtttttatcacaattgctctgtagtaaagctttagatcaggcatggtg

[0288] attccaccagaggttcttttattgttgaaaatagtttttgctatcctaggtttttttgttattccagatgaatttgcaaattgccctttcctgctcattgaagcattgagttggaatttt

[0289] gatggggactgcattgcatctgtagattgctttcggcaggataaccattttgactatattaattctgccaatccatgagcatgggaggtctttccatcttctgagatcttctttg

[0290] atttctttcttcagagacttgaagttcttatcatacagatctttcacttccttagttagagtcacaccaaggtattttatattatttgtgactattgagaagggtgttgtttccctaatt

[0291] tctttctcagcctgtttatcctttgggtagaagaaaggccattgacttgtttgagttaattttatatccagctatggcactgaagctatttcttaggtttaagagttttctggtggaat

[0292] ttttagggtcacttatatatatactatcatataatctacaaataatgatattttgacttcttcctttccaatttgaattcccttgatctccttttgttgttgaattgctctggctaggactt

[0293] caagtactatattcaatagttaggaagaaagtaggcaggcttgtctagtccctgattttagtgggattgcttctagtttctctccatttagtttgatgttggccactggtttgctg

[0294] tacattgcttttattatgtttagatataggccttgaattcttgatcgttccaagagttttatcatgaaggggtattagattttgtcatatgctttctcagcatctaataagatgatcat

[0295] gtggtttttgtctttgagtttgtttatatagtggcttacgttgatggatttccgtatattaaaccatccttacatccctgggatgaagcctacttggtcatggtggatgatggtttg

[0296] atgtgttcttggatttggtttgcaaggattttattgagtattttttcatctatattcaagggaaattggtttgtctgtttctattttgtggaatagtttgaggagagttggaattagg

[0297] tcttctttgaaggtctgatagaactctgcactaaacccatgtggttctgggctttttttggttaggagactattgatgactgcttctatttctttaggggaaatgggacagtttag

[0298] atcgataatatgatcatgatttaactgtggtacctggtatctgtctaggaagttgtccatttcatccaggttttctagttttgttaagtatggccttttgtagtaggatctgatgat

[0299] gttttggatttcctcaggttctgttgttatgtctcctttttcatttttgattttgttaattaggatactttccttatgccctctagtgagtctggctaagggtttatctatcttgttgattttc

[0300] tcaaagaaccagctcctggtttggttgattctttgaatagttctttttgtttccacttggttgattttagcccagagtttgattatttcctgccgtctactccccttgggtgaattttc

[0301] ttcctttagttctagagcttctaggggtgctgtcaggctgctagtgtatgctctctctagtttctttttggaggcactcagggctatgagttttcctcttaggactgccttcattgt

[0302] attccataagtttgggcatattatggcttcattttcattaaactctaaaaagtctttaatttctttctttagttcatccttgtccaaggaatcattgagtaaagtgttgttcagtttcca

[0303] cctgaatgttggctttctattatttatgttgttattgaagatcagccttagtcccgtggtgaccagataagatgcatgggataatttcaataattttgtatctgtggaggcctgttt

[0304] tgtgatcagttatatggtcaattttggaggaggtgccatgaggtgctgagaagaaggtatatccttttgttttaggataaaatgttctgtatttatctattaaatccatttgtttcc

[0305] taacttctgttagtgtccatgtgtctctgtttaatttctgtttccaggatctgtccattggtgagagtagggtgttgaagtctcccactcttttttttaatttttaatattttttattacgt

[0306] atttttctcaattatacttccaatggtatcccaaatgtccctcatactctccccgccacttccctacccacccaatcccattttttttttttttggccctggcgttcccctgtacaca

[0307] tatgaagtttatgtgtccaatgggcctctctttccagtgatgtccgactaggccatcttttgatacatatgcagctagagtcaagatctctggggtactggttagttcataatg

[0308] ttgttccacctatagggttgcagatccctttagctccttgggtactttctctagctcctccattgggagccctgtgatccataagtctcccactcttattgtgtgaggtacaatg

[0309] tgtgcttaggctttatataagtttctttaatgaatgtgggtgcccttgtatttgtagcatagatatttagaattgagagttcattttggtagattttacttttggtgggtatgaagtgc

[0310] ccctccttgtctttttttgataactttgagttggaagtcaattttattcgatattagaatggctactccagcttgtttcttgggaccatttgcttggaaaattgttttccagtctttcac

[0311] tctgaggtagtgtctgtcttttttccctgaggtgggtttcctgtatacaacaaaatgttgggtcctgtttgtgtagccagtctattagtctatgtctttttattggggaattgagtc

[0312] cattgatgttaagagaaattaaagaaaaggaattgttgcttcctgttatttttcgttaaagctgggattctgtgcttggggctctcttcttttaggtttattgaaggtttactttctt

[0313] gctctttctatggtaagttttcatccttgtgttggtgttttccctttattatcgtttgaagggctggatttgtggaaagatattctgttaatttgtgtttgtcatggaatactttggtttc

[0314] tccatctatggtaattgagagttttgctgggtatagtagcctgggttggcatttgtgttcccttagggtctgtataatgtctgtgcaggatcttctgcctttcatagtctctggtg

[0315] agaagcctggtgtaattctaataggcctacctttatatgttacttgacctttttcccttactgcttttaatattctttatttagtgcatttgttgttctgattattatgtgtcgggagga

[0316] atttcttttctggttcagtctatctggagttctctaggcttcttgtatgttcatgggcatctctttctttaggtttgggaagttttcttctatattttgttgaagatatttgctggtccttt

[0317] aaaaattgaaaatcttcattctcatctactcctgttatcagtaggtttgttcttctcattgtgtcctggatttcctggatgatttgagttaggatctttttgcattttctttgattgttgtc

[0318] catgttccctatggaattttctgcacctgagattctctcttccatctcttgtattctgttgctgatgcgcgcatctatgtttcctgacttctttcctagggtttctatctccagagttg

[0319] cctcactttgggttttctttattgtttctacttccatttttaggtcttggatggttttgttcaattccattgcctgtttggttgtattttcccttaattctttaagggatttttgtgtttcctctt

[0320] taaggacttctatctgtttagcagtgttctcctgtatttctttctttttttttctcaatttttttaattgggtatttatttcatttacatttccaatgctatcccaaaagtcccccacatgct

[0321] ccccctacccactcccctctcctgtatttctttaagtgagttattaatgcccttcttaaagtcctctaccagcatcatgagatgattttaaatctgaatcttgcttttcaggtgtgtt

[0322] ggggtatccaggactggctgaggtgggagtgctgggttctgatgatggtgagaggtcttggtttctgttagtaagattcttatgtttgcctttctccatctgataatatctggc

[0323] tttagttataggtgtctctggctggagcttgttcctcctgtgattctgttagcctctgtcagtagtcctgggagtccagctctctcctgagtctcactggtcagagtactctctg

[0324] taggcaagctctcctcttgcggggaaggtgcacagaggtctggcattcagaccagcctactggctgaagatgaaggcctgaaacagagcctttcacataagatgtgtt

[0325] tcctctgcagtctgagcgctcacctgcacagactagtctcagagggacctgggacacaagatggctccctcacctgctctggaggtcagagccctcccaggtggcca

[0326] cctctcctctggcggggaaggtaccgggatatctggagctgaaacagcatctgtcccagaagctgtatcgcttctgccagtcccagaagctgtgtcacttttgcagtcc

[0327] actcactcactgttctcagtttgtgagctgacctgcgcagactcacaatctatctttacacatctgaatgatatatatatatattggtttcacatagatacatttacattatacagt

[0328] gtacatgtacaatccacacacatacttgaaacaaaaccttcatgtgttaatactttgaattctccctacaagtaactctcagttcggtgttctattccagattttagcaatcctgt

[0329] ccattaattgataaattgatttcctatccatataatcctgtgctttttaagaaacgtagttcaatggcaatggggaaaatgtagtggttgtagtgagatgcttcaaaatagctat

[0330] atttgggaatatgaggaaacgtttgaatttgggtttattttcaatttctaacaatctgagccgtaactttaaaatgtttacatgtactcatttgtagtgtgtgtgtgtgtgtgtgtgt

[0331] gtgtgtgtgtgtgtgtgtttgtgtgtgtaatagttattcatagaggacaacttacagcactcagttctctccttctaccatgtaggtcctgggagtgaactcagaccatcagg

[0332] cttggtagcaagtgtcctccttacccattaaagccctctggcctggctgtctttatatgggatccatcagatattctgggttttatgtaaggaggctgcacccacacctatct

[0333] agttttgtttcttcttggcccctcaattctcagacttgtaggtcggaaaggtttccggggctataggctggaggagcagcaccaagcgattccactaacataaaatggag

[0334] atgcattttatcagtggtttcctcttcttcacccataggaagatcgcgtgaggaacctgtaagctcatctgtgggcacagagctggagtgagttccaaatggctggccag

[0335] agactagtgttgaacttttaggaattccacagggcagttttataattttgaactgatgcgaacacactatcaaattagataaatgctaattgaagaatgtcatacatcagtgtc

[0336] tttgttcccaagtgctcaagcagatggagatttgtcagcacttctcaccggacaaatagaagggctctgggagtcttcaggatacggttgggaggttagggaccaatag

[0337] ggtttacaaagtttgtcacccctctgctttgccctggtggaggttgatagctatttatcgtcttatctcctcccaggccagaaaaaaaattggaccagcccatgtgtgaaga

[0338] gcatgaagaggaacgcatcaacatctattgtctgaactgtgaagtgcccacctgttccttgtgcaaggtttttggcgcccataaggactgccaggtggctcccctgactc

[0339] atgtgttccagaggcagaaggtagggagttcaattcctttcttcagcccactttaagcactagtgagttagctccatgtgaccaaagaggggagcagcctcatggaagg

[0340] ccccacaggaagtggaaggcctcacaggaaggggaaggcctcagaggaaggggatggtctcacaggaaggggaaggccccacaggaaggggaaggcccca

[0341] caggaagtggaaatccccacaggaaatggaaggcctcatggaaggccccacaggaagtggaatgcctcacaggaagtggaaggcctcacaggagatggaagac

[0342] cccacagaaagtggaaggcctcacaggaaggggaaggtctcacaggaagtggaaggccccacagaaaggggaaggccccacaggaagtggaagtcccccag

[0343] gaaatggaaggcctcatggaaggccccacaagaagtggaaggcctcacgggaaggggaaggccccacaagaaatggaaggcctcacaggaaatggaaggcc

[0344] ccacagaaagtggaaggcctcacaggaagtagtctcatagtttgtctcaaattccatactcttcaaggttatggagttgcccagagaggtgtgcagagaaaaatgagag

[0345] aaaagagcagaagggctgggtcactctctgtgacatttggaggtggctaaagatatgtagtgttagggggctgatcaaatgccagctgataaagatgactcagagttc

[0346] aaactgcagataagagtgagatgctcttcactctccctttttattgtttatcaataaatgttctcttgccttcttcaaagccttttctcaacaatttcttgcctatgagactcataat

[0347] catgacacacatctacaaaacaactttaaatctgtgcctcttgctggccttcaaagcattctttgaatgtagtacatgtccttcacccctggaactagatctgggtctcctca

[0348] ctctaactcaatacagtctttatagtatctcatcagctctgcaccatgtagcagctgcctaaaatttatgcttctatgcaggttaaggtgtgcatgtgtcagtaggcccatcat

[0349] gcctcagtttcttccacttagaatcattgatttcaattttatatgagagaccagagttgatatcattttggaagtcaaactctgtgacacctgttagaaccagtaaccatgattt

[0350] gtattcgttggtgttatttattttcaacttactttttattttctttaccctgaccccttagccccaaggcccagcatggtagaattgtatggtatctgcttttgactacagtcatcac

[0351] aggtctggaaatccatgggattggcagaggggcaggaagttggacttccagccttttcttctgtggtcagagtagcttcttgtttaataataggcttccaagctaccttcta

[0352] catgtagagaagactgtaggtttcaaatgccttacacccagatttattaaacatctcctttcaaatgtgggaaaattcaggtttatattggtatgtctttctcaaaaatctggag

[0353] aagtttaggtcaaagggagaggagagccagatttccctagtccccattctgcacttcttgtggctccaagtcctgtttaggaggatggtaaataggaacagaaaaatact

[0354] tttggggtcaaaagaacacagggattttagagttgcattgtctctgctaaagccagccatatgttacagttttaagtttgtgcgacagtctagtgagcatttgcatgtatctac

[0355] actgcattagcatagtaggattgaaatatacacaccggataggtgaatacttactgcatttattgctcaagtaaaactagtactgaactgcactagacaaacaattcattt

[0356] tgagttccaaatgtcacatatttgtgtctatctcattgttccacttacgcaatgtttagaagcctccaacttcaatatactattatagatagcttataattaggttttctttgtgttttt

[0357] aacagaatctagttctccatcaataatgttaaaattatggagcctcatccaacacaaatccaagcatatatcaaagttttcttgcaatatcaagaagctcacagatttctcatg

[0358] actggctccagcctcagtccttagatgggattaattatattttctgggttttggataaagagtaatatcccacacatacctcttacaggtcctcaattagcaccttaattaataa

[0359] tgacagaaaattctggctttccttggccaccttcatcctcagacttcctttctgggacctgaggctggtagcactcagcaatccctccagcctaaactggagatgcagctc

[0360] atgagcagcttcctcttcttcatccatggtaagaatatgtcttaggagaaatccgtgagcttggagcttgtttactccttaggaataacacccctttgaggacaatggtgtga

[0361] gcaggccattgtcatctatgattaggaaaaccctaatctcttgagtcttcttttctcttcattagtcagagctcagtgatggtattgctgtacttgtgggaagcaacgatagag

[0362] tccagggtgtgatcagccagctggaggacacctgtaaaactattgaggtaagtcagctggctcaaagggaatcgaaatcaacccggatactccgaggggatagtga

[0363] gtgtctcagtgacacactggaaagcccctcccaacgcgataataatctctgaaacacttcctggtgctttagagaacttaccacacagcttgatgaggtagatttttacca

[0364] cagttgctcagacagttcttgtgtgtggctcttcctcagtgaactccttttgaaagattaccaacaagacaagtcagtagccagcaatggaaatgagtactgttgctattatt

[0365] taacaagatttcttgctgaaaacctaaacagtcttcagatatgctgattggtgtttcaggtgtgagctacatacctgtgctctcctggcagtcatcataaggaccctgagga

[0366] ggagaaaaatctccccaaatagaggctaagaggctgaggtactaaccactcagcttttgggacaccctcctcattgttgtgttcaggcctctcttattacctcttatttcttttt

[0367] gtccctatttcccaaagataacagttctttgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtctcttataaaatacagattgttttatatatgtgcgtcttagttacttttctgttg

[0368] cgaggaaaagacatcatgaccaaggcaacttataaaagtaagcatttaactgagggtttgcttagagttttagaggttcagtccatcacaatgatcgtgaggagcatgg

[0369] caacaggcagggatggcactgtagcagtaattgagagagactgagcctggtcctggcttttgaaacttcacagctgacccctatgacactcctcctccaataaggcca

[0370] ggcctcctaatccttccagagaaagttccaccaagtagggaccaagcattcaagtatatgagcctatggggaccattctcgttcaatccatcatactatgtatgccttttaa

[0371] ttttacaaatgctattgcatgatagtttctttgtgttttgaatctagaattctgtttataagatccacacatattactatcagcgtgtccagtgacttggtttccttttctgtgaaaag

[0372] ggggcaatcacacttattctgtagagattacaggctcaatgaagatgctcagcacaatatctggcatgaagtgtgaggtcttactacttttctattatcaaggcaaaatctat

[0373] cataagtctaaggccattttctagaggatggcttcagggcttcaattagttgtaattatcccatcttagatttacacatcaccaaaataaatttcctttcttctctacactcatatt

[0374] gctaaaaatatcttgggtacttggagatttactttctgaagaaatattttctttggcaaagagccgaagagattcagaaatgaacaatataaactgtttcctcttatttacaatg

[0375] aagatcagtcagtcactggccatgaatcagcagtccactgtgtgctaggttgagtcttgtgtgttataaatagcctgttacttgtgttgagcgaggcaatctctagccacct

[0376] cctgagactctcaatggagagtctggcttcctgtctgggataaagacatcaaaagatgtgtggaaagtcctgtggtccactgaagtcttgagtgagggaaacaagttga

[0377] ttctggctgtgttaggaagggcaacaggcgctttcacagaggccagccagggtcttagaaggaagcagctgtctctagctacacttgagagcaagccaagaaagtac

[0378] aggattaaagctaaatcagcagaaggtgtttagttagggacaatatgaagagcagccaaggtcagaattatcaccctttcttactggtaatgactatccaacagctacag

[0379] atttctaacaaaggtatattaccactacttccaattcattaagatgttggtggtctttaaacttctcactttcataccaggtttggtgacacacacttctaatctcagctactcag

[0380] gaaggtgtgtcaggaggattagaagttctagagtagcctgtataaaaataaattaaaaataaaagctggctgaagataaagaacaattttctggtacacaaagtcaacta

[0381] gcccagtctccagtgctttgaagaaaagtaaactctctccctgttcatgtgtttatttcagttgttggacttcttaaagaaaataagataaaaaaaaaaaacatcgtcagtatag

[0382] ggtcatgcatattgtgtaggatatactggtctaaacatgactattttgaatgtctacattcatgtcttataccagtactttcatgggaagaatagtcaggaggaaaatactaaa

[0383] gggccaggacttttaatagcaggactgggcttggaggttttgtttgttgtttatttgtggcagtttatcagctatatcccaggttggccttgaactcatgtcctttctcagctttc

[0384] tgaatactgggaatagagacaagccatcatgcctggcaattctcaatgccttacttatatatttctgtgtttcccactattgccgagtgagtctgaaatttttaatagctcgtaa

[0385] caactagttgtaaatgagcagatgtgtggctatgcatgtgtttcatgtggagatgatgtaggggtcattaactgtgattcctatgctctgtttcaaaaggattgagaccagg

[0386] caagcatctacctttcactcctataacccccatagg

[0387] The above description is not intended to impose any form of limitation on the present invention. Although the present invention has been disclosed through the above embodiments, it is not intended to limit the present invention. Any technician familiar with the profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. A target TRIM55 for preventing and / or treating autoimmune diseases, characterized in that: The autoimmune disease is an autoimmune disease associated with a non-classical NF-κB signal transduction pathway.

2. A target TRIM55 for preventing and / or treating autoimmune diseases as claimed in claim 1, characterized in that: The autoimmune diseases are systemic lupus and related complications, and rheumatoid arthritis.

3. Use of the target TRIM55 in the preparation of a drug for preventing and / or treating autoimmune diseases, characterized in that: The autoimmune disease is an autoimmune disease associated with a non-classical NF-κB signal transduction pathway.

4. Use of the target TRIM55 as claimed in claim 3 in the preparation of a drug for preventing and / or treating autoimmune diseases, characterized in that: The autoimmune diseases are systemic lupus and related complications, and rheumatoid arthritis.

5. Use of the target TRIM55 as claimed in claim 3 in the preparation of a drug for preventing and / or treating autoimmune diseases, characterized in that: The drug targets TRIM55, its domain or TRIM55 protein truncation, and inhibits or weakens the function of TRIM55, that is, the drug is an agent that reduces the expression level of TRIM55 and / or antagonizes the effect of TRIM55.

6. Use of the target TRIM55 as claimed in claim 3 in the preparation of a drug for preventing and / or treating autoimmune diseases, characterized in that: The target TRIM55 regulates p100 processing and non-classical NF-kB activation, the target TRIM55 binds to p100, and the p100-TRIM55 binding ability is enhanced with the activation of the non-classical NF-kB pathway; The mutual binding of p100-TRIM55 depends on the phosphorylation modification of p100 S866 / 870; that is, the target TRIM55 regulates the activation of the non-classical NF-κB signaling pathway by targeting and ubiquitinating p100 and promoting p100 processing, thereby participating in the regulation of the development of autoimmune diseases.

7. A method for screening drugs for preventing and / or treating autoimmune diseases based on the target TRIM55, characterized in that: The method uses TRIM55 as a target and screens out candidate drugs or drug combinations by detecting the effects of drugs on the activity of TRIM55, that is, detecting the inhibitory and / or antagonistic properties of specific drugs on the activity of TRIM55 to screen out candidate drugs or drug combinations.

8. A method for screening drugs for preventing and / or treating autoimmune diseases based on the target TRIM55 as claimed in claim 7, characterized in that: The method uses TRIM55 as a target, and further screens the target drug or drug combination by detecting the effect of the candidate drug or drug combination on the activation of the non-classical NF-κB signaling pathway of B cells, that is, detecting the inhibitory and / or antagonistic performance of the candidate drug or drug combination on the activation of the non-classical NF-κB signaling pathway of B cells to screen the target drug or drug combination.

9. A drug for preventing and / or treating autoimmune diseases, characterized in that: The autoimmune disease is an autoimmune disease associated with a non-classical NF-κB signal transduction pathway, and the drug is an agent or a combination of agents that reduces the expression level of TRIM55 and / or antagonizes the effect of TRIM55.

10. A drug for preventing and / or treating autoimmune diseases as claimed in claim 9, characterized in that: The drug is an agent or a combination of agents that inhibits and / or antagonizes the activation of the non-classical NF-κB signaling pathway of B cells.

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