CN111588840A - Application of histone deubiquitinating enzyme in preparation of medicine for treating systemic lupus erythematosus - Google Patents

Abstract

Application of histone deubiquitinating enzyme in preparing medicine for treating systemic lupus erythematosus is disclosed. The invention belongs to the technical field of biotechnology and biological medicine, and discloses application of histone deubiquitinating enzyme in preparing a medicament for treating systemic lupus erythematosus, wherein the histone deubiquitinating enzyme has a nucleotide sequence of SEQ ID NO: 1. the protein amino acid sequence is SEQ ID NO: 2. the invention selects lentivirus with human MYSM1 as a gene therapy tool, and carries out gene therapy by taking peripheral blood lymphocytes separated from the blood of a systemic lupus erythematosus patient as a target. The result shows that the lentivirus overexpression MYSM1 can obviously inhibit interferon and inflammatory factors produced by peripheral blood lymphocytes of systemic lupus erythematosus patients. The invention effectively applies the anti-inflammatory effect of the MYSM1 protein to the treatment of autoimmune diseases, and has obvious anti-inflammatory effect, so that MYSM1 has potential application prospect in the gene treatment of systemic lupus erythematosus and even in the gene treatment of autoimmune diseases.

Description

Application of histone deubiquitinating enzyme in preparation of medicine for treating systemic lupus erythematosus

Technical Field

The invention belongs to the technical field of biotechnology and biomedicine, and particularly relates to application of histone deubiquitinating enzyme in preparation of a medicine for treating systemic lupus erythematosus.

Background

Currently, Systemic Lupus Erythematosus (SLE) is a very serious autoimmune disease, and has various clinical manifestations, and various Systemic and visceral injuries, such as skin, joints, serosa, heart, kidney, central nervous system, blood system, and the like, can occur. Due to cellular and humoral immune dysfunction, many autoantibodies are produced, severely harming the health and even life threatening diseases. Is widely prevalent throughout the world, with 40 million new patients each year. Comprises discoid lupus erythematosus, subacute cutaneous lupus erythematosus, systemic lupus erythematosus and other types, wherein the systemic lupus erythematosus is the most serious type of lupus erythematosus. Systemic lupus erythematosus is an autoimmune inflammatory connective tissue disease which is frequently generated in young women and involves multiple organs, most patients have multiple system damage manifestations when in onset, and a few patients develop other types of lupus erythematosus. Some patients also have other connective tissue diseases, such as scleroderma, dermatomyositis, sjogren's syndrome, etc., which form various overlapping syndromes. Systemic lupus erythematosus is characterized by various clinical manifestations, complicated and complicated clinical manifestations, and more serious manifestations, which can endanger the life of patients due to lupus nephritis, lupus encephalopathy and the side effects of long-term use of a large amount of drugs.

Many studies have shown that abnormal activation of the immune system and abnormal expression of inflammatory factors are one of the main features of systemic lupus erythematosus and also one of the targets for clinical treatment.

Research finds that high expression of type I interferon and proinflammatory cytokines is widely existed in systemic lupus erythematosus patients, and suggests that abnormal activation of the innate immune system is a potential cause of systemic lupus erythematosus. Studies have found that systemic lupus erythematosus produces abnormal self-DNA, and inappropriate activation of the immune system by abnormal self-DNA results in high levels of type I interferon and proinflammatory cytokines. Thus, targeting inflammation-related pathways is a potential therapeutic approach.

So far, the main method for clinically treating lupus erythematosus patients is drug therapy, which mainly comprises the following steps:

first, glucocorticoid drugs, glucocorticoids, are often the first choice in the treatment of immune diseases, especially lupus erythematosus, especially in the early stages of the disease process.

Second, immunosuppressive agents, which are also important for achieving therapeutic effects, especially in the maintenance phase, can alleviate the condition and maintain the therapy.

Thirdly, lupus is now available as a biological agent or as a targeted drug, such as belimumab and interleukin-2, which have been relatively new therapeutic approaches in recent years.

The medicine treatment needs to be taken for life, and most patients can generate symptoms such as drug resistance, adverse reaction and the like, including osteoporosis, susceptibility to infection, obesity and the like, so that the development of a new treatment means is very critical to the treatment of the lupus erythematosus disease.

Gene therapy (gene therapy) is a new therapeutic approach that has been developed in recent years, and refers to introducing exogenous normal genes into target cells to correct or compensate diseases caused by defective and abnormal genes, so as to achieve the purpose of treatment. The method also includes the technical application of transgenosis and the like, namely, the exogenous gene is inserted into a proper receptor cell of a patient through a gene transfer technology, so that a product made of the exogenous gene can treat a certain disease. To date, gene therapy has been widely used for the treatment of various diseases. By 7 months 2005, the worldwide approved clinical protocol for gene therapy reached 1076, of which 66% are cancer-directed therapies. Over a decade of development, gene therapy research has advanced a lot. Therefore, the development of a new gene therapy target for treating lupus erythematosus has important clinical value.

Previous studies showed that histone H2A deubiquitinase (MYSM1), also known as Myb-like SWIRM and MPN domain-containing protein 1(Myb-like, SWIRM and MPN domain-containing protein 1) can inhibit inflammatory activation and interferon response caused by self-DNA, and MYSM1 was shown to protect mice from organ damage caused by inflammatory storm in a mouse model.

Through the above analysis, the problems and defects of the prior art are as follows: (1) the medicine treatment needs to be taken for life, and most patients can generate symptoms such as drug resistance, adverse reaction and the like, including osteoporosis, susceptibility to infection, obesity and the like, so that the development of a new treatment means is very critical to the treatment of the lupus erythematosus disease.

(2) In the prior art, no means related to combined gene therapy exists, and MYSM1 protein is not further developed to be used as a gene therapy technology, so that the drug for treating systemic lupus erythematosus has poor treatment effect. And can not provide theoretical basis for further gene therapy application of the MYSM1 protein.

The difficulty in solving the above problems and defects is: the main problem of gene therapy is target selection, a proper target site gene is needed, the therapeutic efficiency is ensured, other side effects are prevented from being caused, lupus erythematosus is a systemic autoimmune disease, the pathogenesis is complex, the participation passage is wide, and therefore, the selection of the proper target gene is particularly critical.

The significance of solving the problems and the defects is as follows: the invention is based on the early research foundation, combines with the related means of gene therapy, and further develops the MYSM1 protein as the gene therapy technology for treating systemic lupus erythematosus and even treating other autoimmune diseases.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides application of histone deubiquitinating enzyme in preparing a medicine for treating systemic lupus erythematosus. The invention provides a new treatment technology for treating systemic lupus erythematosus clinically. The MYSM1 overexpression can obviously reduce inflammatory factors and type I interferon produced by lymphocytes of lupus erythematosus patients in vitro cell experiments. Therefore, the MYSM1 combined gene therapy means is further developed to be used as a new technology and a method for preventing/treating systemic lupus erythematosus and other autoimmune diseases, and has wide application prospect.

The invention is realized in such a way that the application of histone deubiquitinating enzyme (slow virus processing cell, mol is 0.1) in preparing the medicine for preventing and treating systemic lupus erythematosus is provided, the histone deubiquitinating enzyme has a nucleotide sequence of SEQID NO: 1.

another object of the present invention is to provide a protein encoded by the histone deubiquitinating enzyme gene in the above application, wherein the amino acid sequence of the protein is SEQ ID NO: 2.

the invention also aims to provide an application of the protein in preparing a medicament for preventing/treating autoimmune diseases.

Another purpose of the invention is to provide a lentivirus expression vector constructed by using the protein.

Another objective of the invention is to provide a metalloprotease obtained by monoubiquitination modification at the K119 site of said protein.

By combining all the technical schemes, the invention has the advantages and positive effects that: the invention discloses application of histone H2A deubiquitinase (MYSM1), also called Myb-like SWIRM and MPN structural domain protein 1(Myb-like SWIRM and MPNdomain-relating protein 1) in treating Systemic Lupus Erythematosus (SLE). The invention selects lentivirus with human MYSM1 as a gene therapy tool, and carries out gene therapy by taking peripheral blood lymphocytes separated from the blood of a systemic lupus erythematosus patient as a target. The result shows that the lentivirus overexpression MYSM1 can obviously inhibit interferon and inflammatory factors produced by peripheral blood lymphocytes of systemic lupus erythematosus patients. The invention effectively applies the anti-inflammatory effect of the MYSM1 protein to the treatment of autoimmune diseases, and has obvious anti-inflammatory effect, so that MYSM1 has potential application prospect in the gene treatment of systemic lupus erythematosus and even in the gene treatment of autoimmune diseases.

Compared with the prior art, the invention has the advantages that:

MYSM1 is closely related to systemic lupus erythematosus, MYSM1 has anti-inflammatory effect and potential for treating systemic lupus erythematosus, has solid basis and theoretical foundation, and is strong in innovation and good in application prospect.

Compared with the traditional anti-inflammatory related therapeutic drugs, the gene therapy is a new treatment means at present, and has small side effect and strong targeting property.

Currently, no related gene therapy technology is applied to lupus erythematosus.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.

FIG. 1 is a chart that illustrates the correlation of MYSM1 with systemic lupus erythematosus.

In the figure: a and B: expression of MYSM1mRNA and MYSM1 protein in Peripheral Blood Mononuclear Cells (PBMC) of systemic lupus erythematosus patients and healthy people. C-H: expression of IFN- β mRNA (C), ISG56 mRNA (D), ISG15mRNA (E), TNF- α mRNA (F), IL-6 mRNA (G) and CXCL10 mRNA (H) in Peripheral Blood Mononuclear Cells (PBMC) of systemic lupus erythematosus humans and healthy humans.

FIG. 2 is a study of the effect of Lenti-Mysm1 on inhibiting systemic lupus erythematosus, provided by an embodiment of the present invention. A and B: systemic lupus erythematosus patients infected with MYSM1 expressing lentivirus (Lenti-Mysm1) or its control lentivirus (Lenti-Ctrl) in Peripheral Blood Mononuclear Cells (PBMC) for 48 hours, and after treatment, the mRNA (A) and protein (B) expression of MYSM1 were detected. C-H: peripheral Blood Mononuclear Cells (PBMC) of a systemic lupus erythematosus patient are infected with a lentivirus (Lenti-Mysm1) expressing MYSM1 or a control lentivirus (Lenti-Ctrl) thereof for 48 hours, and the expression levels of IFN-beta mRNA (C), ISG56 mRNA (D), ISG15mRNA (E), TNF-alpha mRNA (F), IL-6 mRNA (G) and CXCL10 mRNA (H) in the Peripheral Blood Mononuclear Cells (PBMC) are detected after treatment.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In the prior art, no means related to combined gene therapy exists, and MYSM1 protein is not further developed to be used as a gene therapy technology, so that the drug for treating systemic lupus erythematosus has poor treatment effect. And can not provide theoretical basis for further gene therapy application of the MYSM1 protein.

Aiming at the problems in the prior art, the invention provides the application of histone deubiquitinating enzyme in preparing the medicine for treating systemic lupus erythematosus, and the invention is described in detail by combining the attached drawings.

The invention selects histone H2A deubiquitinase (MYSM1), also called Myb-like SWIRM and MPN domain-containing protein 1(Myb-like SWIRM and MPN domain-containing protein 1), a metalloprotease, which can be used for single-ubiquitination modification on K119 site of deubiquitinase histone H2A. Meanwhile, MYSM1 has important functions in hematopoietic stem cells, lymphocytes and mature blood cells. MYSM1 has been previously reported to regulate mouse hematopoietic and tissue development by interacting with p53 and to be involved in regulating mouse skin development and hereditary bone marrow failure syndrome (1).

M,Belle JI,Petrov JC,RyderEJ,Clare S,Nijnik A.Deubiquitinase MYSM1 Is Essential for Normal Fetal LiverHematopoiesis and for the Maintenance of Hematopoietic Stem Cells in AdultBone Marrow.Stem Cells Dev.(2015):24(16):1865-77；

2. Gatzka M, Tasdogan A, Hainzl A, Allies G, Maity P, Wilms C, Wlaschek M, Schaffetter-Kochanek K. Interlay of H2A deubiquitinase 2A-DUB/Mysm1and the P19(ARF)/P53 axis in hematology, early T-cell definition and tissue differentiation cell definition Differ (2015):22(9): 1451-62.). The invention combines MYSM1 with a lentivirus vector (the used vector is a lentivirus PLENTI vector purchased from ADDGENE), packages the lentivirus with stable expression, infects a receptor cell, and realizes the high-efficiency ectopic expression of related protein.

The invention provides an application of histone deubiquitinating enzyme (H2A) in preparing a medicine for preventing and treating systemic lupus erythematosus, wherein the nucleotide sequence of the histone deubiquitinating enzyme is SEQ ID NO: 1.

in the prepared medicine for preventing and treating systemic lupus erythematosus, the amount of the histone deubiquitinating enzyme is 0.1 mol.

The invention provides a protein encoded by a histone deubiquitinating enzyme gene in the application, wherein the amino acid sequence of the protein is SEQ ID NO: 2.

protein amino acid sequence:

MAAEEADVDIEGDVVAAAGAQPGSGENTASVLQKDHYLDSSWRTENGLIPWTLDNTISEENRAVIEKMLLEEEYYLSKKSQPEKVWLDQKEDDKKYMKSLQKTAKIMVHSPTKPASYSVKWTIEEKELFEQGLAKFGRRWTKISKLIGSRTVLQVKSYARQYFKNKVKCGLDKETPNQKTGHNLQVKNEDKGTKAWTPSCLRGRADPNLNAVKIEKLSDDEEVDITDEVDELSSQTPQKNSSSDLLLDFPNSKMHETNQGEFITSDSQEALFSKSSRGCLQNEKQDETLSSSEITLWTEKQSNGDKKSIELNDQKFNELIKNCNKHDGRGIIVDARQLPSPEPCEIQKNLNDNEMLFHSCQMVEESHEEEELKPPEQEIEIDRNIIQEEEKQAIPEFFEGRQAKTPERYLKIRNYILDQWEICKPKYLNKTSVRPGLKNCGDVNCIGRIHTYLELIGAINFGCEQAVYNRPQTVDKVRIRDRKDAVEAYQLAQRLQSMRTRRRRVRDPWGNWCDAKDLEGQTFEHLSAEELAKRREEEKGRPVKSLKVPRPTKSSFDPFQLIPCNFFSEEKQEPFQVKVASEALLIMDLHAHVSMAEVIGLLGGRYSEVDKVVEVCAAEPCNSLSTGLQCEMDPVSQTQASETLAVRGFSVIGWYHSHPAFDPNPSLRDIDTQAKYQSYFSRGGAKFIGMIVSPYNRNNPLPYSQITCLVISEEISPDGSYRLPYKFEVQQMLEEPQWGLVFEKTRWIIEKYRLSHSSVPMDKIFRRDSDLTCLQKLLECMRKTLSKVTNCFMAEEFLTEIENLFLSNYKSNQENGVTEENCTKELLM

the genotype sequences were as follows: SEQ ID NO: 1

atggcgg ctgaagaggc ggatgtggat atcgaagggg acgtggtagc ggcggcgggggcacagccag gaagtggtga aaatacagca tcagttttac aaaaagatca ctatcttgat tcatcttggagaacagagaa tggccttatt ccttggacct tggataacac catcagtgaa gagaacagag ctgttattgagaaaatgttg ttggaagaag aatattattt atctaaaaaa tcacaaccgg aaaaagtctg gcttgatcaaaaggaagatg ataaaaaata catgaagagt ctgcagaaaa cagcaaaaat catggtacac tctcctacaaaaccagccag ttactcagta aagtggacga tagaagaaaa agagctgttt gaacaagggc tggctaaatttggccgaaga tggaccaaaa tttcaaagct aattggaagc cgcactgttt tacaagtgaa gagttatgcaagacagtatt ttaaaaataa ggtcaaatgc ggtctggata aagaaacacc aaatcagaag accggccataatcttcaagt taaaaatgaa gataaaggga caaaggcatg gacaccatca tgtttaaggg gacgtgctgatcccaacttg aatgctgtaa aaattgaaaa gttatctgat gatgaagaag tagacatcac agatgaggtggacgagttgt cttctcaaac accccagaag aattctagca gtgatctctt gttagacttt cctaatagtaaaatgcatga aaccaatcaa ggagaattca ttacttctga cagccaggaa gctctctttt ctaagtcttccaggggctgt cttcaaaatg aaaagcaaga tgaaacactt tcaagctcag aaattacact gtggactgagaaacagagca atggtgacaa aaaatcaatt gaattaaatg accagaaatt taatgaattg attaaaaactgcaacaagca tgatggaaggggaataatag ttgatgccag gcagttgcct tctccagagc cttgtgaaattcagaaaaat ttgaatgata atgaaatgct ttttcattct tgccaaatgg tagaggaaag ccatgaggaagaagagctta agccaccaga acaggaaata gaaatagata gaaatatcat tcaagaagaa gaaaaacaagcaattcctga gttttttgag gggcgccaag ctaaaacacc agaacgctat ttgaaaatta gaaattatattttggatcaa tgggagatat gcaaaccaaa atacttaaat aagacctcag tacgtcctgg cctgaagaactgtggagatg ttaattgtat tggacggatt catacatacc tcgaattgat aggagcaatc aattttggatgtgaacaggc tgtgtataat aggccacaaa cagttgacaa agtacgaatc agagacagaa aagatgcagtagaagcatac caacttgccc agcgtctgca gtctatgcgt acaaggagac gtagggtccg agacccatggggaaactggt gtgatgcaaa ggacttagaa ggacaaacgt ttgagcatct ctctgctgag gagttggcaaaaagaagaga agaggaaaaa ggcagacctg ttaaatcttt aaaagtgcca agaccaacaa aaagctcgtttgatcccttc caactgatac cttgtaattt ttttagtgaa gaaaagcagg agccatttca ggtgaaagtggcttcagaag cacttttaat aatggatttg catgctcatg tttctatggc agaagtgatt ggtctgttaggaggaagata ctcagaagtt gataaagtag ttgaagtctg tgcagcagaa ccatgtaaca gtctgagtacaggactacag tgtgagatgg atcctgtatc acaaacacag gcctcagaaa ccttggctgt tagaggcttcagtgttattg gatggtatca ttctcatcct gcttttgatc ctaatccttc cttacgagat attgacacacaagctaaata ccagagttac ttctccagag gaggtgcaaa gttcattggg atgattgtta gtccctataatcgaaataat cccttaccat attctcagat tacctgcctg gttataagtg aggaaattag cccagatggctcttatcgct taccttacaa atttgaagta cagcagatgt tagaagaacc tcagtgggga ttagtatttgaaaagacaag atggataata gaaaaataca ggctctccca tagcagcgtc cccatggata aaatctttcgccgggattct gacctgactt gtttgcagaa acttttggag tgtatgagga agactctgag caaagtgaccaattgcttta tggctgaaga attcttgact gaaatagaaa atttgttcct ttccaattat aaaagcaaccaagagaatgg agtaaccgaa gagaactgta caaaggaatt gttaatgtga。

The invention provides an application of the protein in preparing a medicament for preventing/treating autoimmune diseases.

The invention is further described below with reference to specific examples and related experiments.

Example 1

The invention provides an effect of MYSM1 in systemic lupus erythematosus treatment, which comprises the following steps:

A. confirmation of the relevance of MYSM1 to systemic lupus erythematosus

Peripheral blood lymphocytes (PBMCs) were isolated from healthy human blood and from systemic lupus erythematosus patients. The method comprises the following steps:

1. a suitable amount of lymphocyte separation medium was added to a 15ml centrifuge tube.

2. Heparin anticoagulated venous blood is taken and fully mixed with Hanks buffer solution or RPMI1640 with the same amount, and the mixture is slowly overlapped on a layered liquid surface along the tube wall by using a dropper, and a clear interface is kept. Centrifuge horizontally at 2000rpm for 20 minutes.

3. After centrifugation, the tube is divided into three layers, the upper layer is plasma and Hanks buffer solution, and the lower layer is mainly red blood cells and granulocytes. The middle layer is lymphocyte separation liquid, a white cloud layer narrow band mainly comprising mononuclear cells is arranged at the interface of the upper layer and the middle layer, and the Peripheral Blood Mononuclear Cells (PBMCs) comprise lymphocytes and monocytes.

4. And sucking the lymphocyte layer. Put into another short tube, add Hanks buffer or RPMI1640 in a volume of 5 times more, centrifuge horizontally at 1500rpm for 10 minutes, and wash the cells twice.

5. After centrifugation, the supernatant was discarded, RPMI1640 containing 10% calf serum was added, the cells were resuspended and counted, and cultured at 37 ℃.

Blood samples were drawn at random from healthy populations (15 cases) and from Systemic Lupus Erythematosus (SLE) patients (14 cases). Peripheral Blood Mononuclear Cells (PBMCs) were isolated from the blood sample. The expression of MYSM1mRNA and MYSM1 protein in PBMCs was detected by RT-PCR and Western blot methods, respectively, and beta-Interferon (Interferon-beta, IFN-beta) mRNA, Interferon stimulated gene 56(IFN-stimulated gene 56, ISG56) mRNA, Interferon stimulated gene15 (IFN-stimulated gene15, ISG15) mRNA, Tumor necrosis factor-alpha (Tumor necrosis factor-alpha, TNF-alpha) mRNA, Interleukin-6 (Interleukin-6, IL-6) mRNA and chemokine (C-X-C motif ligand 10, CXCL10) mRNA were detected by RT-PCR. The results show that expression of MYSM1mRNA and MYSM1 protein were significantly reduced in Peripheral Blood Mononuclear Cells (PBMCs) of systemic lupus erythematosus patients. In contrast, interferon and proinflammatory cytokine mRNA levels, including IFN- β, ISG56, ISG15, IL-6, TNF- α and CXCL10 were significantly increased in Peripheral Blood Mononuclear Cells (PBMCs) of systemic lupus erythematosus patients. These results suggest that MYSM1 expression is closely associated with systemic lupus erythematosus.

MYSM1 treatment of lupus erythematosus

Blood samples were drawn randomly from healthy populations in Wuhan's Hospital (5 cases) and from patients with Systemic Lupus Erythematosus (SLE) (5 cases). Peripheral Blood Mononuclear Cells (PBMCs) were isolated from the blood sample. The MYSM 1-expressing lentivirus (Lenti-Mysm1) or its control lentivirus (Lenti-Ctrl) was then infected for 48 hours. MYSM1mRNA and MYSM1 protein levels were detected by RT-PCR and Western blot methods. In the assay, RT-PCR was used to detect IFN-. beta.mRNA, ISG56 mRNA, ISG15mRNA, TNF-. alpha.mRNA, IL-6 mRNA and CXCL10 mRNA. Experiments show that the lentivirus system can stably express MYSM1 in PBMC, the system is proved to be feasible, the level of inflammatory factors after treatment is further analyzed, the MYSM1 overexpression is shown to remarkably inhibit the activation of the inflammatory factors in systemic lupus erythematosus patients, and the MYSM1 is proved to have a treatment effect and potential application value.

Example 2 confirmation of the relevance of MYSM1 to systemic lupus erythematosus

Confirming the relevance of MYSM1and systemic lupus erythematosus, which comprises the following steps:

1. experimental materials:

1.1 cells

Peripheral Blood Mononuclear Cells (PBMCs) isolated from blood of a healthy person and blood of a patient with systemic lupus erythematosus.

1.2 reagents

1640 medium and Fetal Bovine Serum (FBS) from GIBCO; RNA extraction kits were purchased from members of the Zhuang Union.

1.3 Experimental instruments:

LC480 is a product of Roche; the cell culture box is a product of Thermofisiher company.

2. Experimental methods and results:

2.1 Add appropriate amount of lymphocyte isolate into 15ml centrifuge tube.

2.2 heparin anticoagulant venous blood is taken and fully mixed with Hanks liquid or RPMI1640 with the same amount, and the mixture is slowly overlapped on a layered liquid level along the tube wall by a dropper, and a clear interface is kept. Centrifuge horizontally at 2000rpm for 20 minutes.

2.3 after centrifugation, the tube is divided into three layers, the upper layer is plasma and Hanks liquid, and the lower layer is mainly red blood cells and granulocytes. The middle layer is lymphocyte separation liquid, and a white cloud layer narrow band mainly comprising mononuclear cells including lymphocytes and monocytes is arranged at the interface between the upper layer and the middle layer.

2.4 suction of lymphocyte layer. Put into another short middle tube, add Hanks' solution or RPMI1640 with volume more than 5 times, centrifuge horizontally at 1500rpm for 10 minutes, and wash the cells twice.

2.5 centrifugation, discard the supernatant, add RPMI1640 containing 10% calf serum, resuspend and count the cells, incubate at 37 ℃.

2.6 blood samples were drawn at random from healthy populations (15 cases) and from Systemic Lupus Erythematosus (SLE) patients (14 cases). PBMCs were isolated from blood samples. The RT-PCR and Western blot methods are respectively used for detecting MYSM1mRNA and MYSM1 protein expressed in PBMCs, and the RT-PCR is used for detecting IFN-beta mRNA, ISG56 mRNA, ISG15mRNA, TNF-alpha mRNA, IL-6 mRNA and CXCL10 mRNA. Results mean and standard deviation were calculated by GraphPad Prism 6 software.

2.7 Using the results of the calculations in step (6) to draw a relevant conclusion, as shown in FIG. 1(A, B: expression of MYSM1mRNA and MYSM1 protein in Peripheral Blood Mononuclear Cells (PBMC) of systemic lupus erythematosus patients and healthy persons C-H: expression of IFN-. beta.mRNA (C), ISG56 mRNA (D), ISG15mRNA (E), TNF-. alpha.mRNA (F), IL-6 mRNA (G) and CXCL10 mRNA (H)) in Peripheral Blood Mononuclear Cells (PBMC) of systemic lupus erythematosus patients and healthy persons.

Example 3 treatment of systemic lupus erythematosus with MYSM1

1. Experimental Material

1.1 cells

Blood from healthy persons and blood from patients with systemic lupus erythematosus are obtained, and Peripheral Blood Mononuclear Cells (PBMCs) are isolated.

1.2 reagent:

1640 medium and Fetal Bovine Serum (FBS) from GIBCO; RNA extraction kits were purchased from members of the Zhuang Union.

1.3 Experimental instruments:

LC480 is a product of Roche; the cell culture box is a product of Thermofisiher company.

2. Experimental methods and results:

2.1 Add appropriate amount of lymphocyte isolate into 15ml centrifuge tube.

2.2 heparin anticoagulant venous blood is taken and fully mixed with Hanks liquid or RPMI1640 with the same amount, and the mixture is slowly overlapped on a layered liquid level along the tube wall by a dropper, and a clear interface is kept. Centrifuge horizontally at 2000rpm for 20 minutes.

2.3 after centrifugation, the tube is divided into three layers, the upper layer is dish pulp and Hanks liquid, and the lower layer is mainly red blood cells and granulocytes. The middle layer is lymphocyte separation liquid, and a white cloud layer narrow band mainly comprising mononuclear cells including lymphocytes and monocytes is arranged at the interface between the upper layer and the middle layer.

2.4 suction of lymphocyte layer. Put into another short middle tube, add Hanks' solution or RPMI1640 with volume more than 5 times, centrifuge horizontally at 1500rpm for 10 minutes, and wash the cells twice.

2.5 centrifugation, discard the supernatant, add RPMI1640 containing 10% calf serum, resuspend and count the cells, incubate at 37 ℃.

2.6. Blood samples were drawn at random from healthy populations (5 cases) and from Systemic Lupus Erythematosus (SLE) patients (5 cases). Peripheral Blood Mononuclear Cells (PBMCs) were isolated from the blood sample. The MYSM 1-expressing lentivirus (Lenti-Mysm1) or its control lentivirus (Lenti-Ctrl) was then infected for 48 hours. MYSM1mRNA and MYSM1 protein levels were detected by RT-PCR and Western blot methods. The mean and standard deviation were calculated by GraphPad Prism 6 software after RT-PCR for IFN-. beta.mRNA, ISG56 mRNA, ISG15mRNA, TNF-. alpha.mRNA, IL-6 mRNA and CXCL10 mRNA.

2.7 Using the results of the calculation in step 2.6, relevant conclusions are drawn, as shown in FIG. 2(A, B: SLE patients Peripheral Blood Mononuclear Cells (PBMC) infected with MYSM1 expressing lentivirus (Lenti-Mysm1) or its control lentivirus (Lenti-Ctrl) for 48 hours, C-H: SLE patients Peripheral Blood Mononuclear Cells (PBMC) infected with MYSM1 expressing lentivirus (Lenti-Mysm1) or its control lentivirus (Lenti-Ctrl) for 48 hours, and IFN-. beta.mRNA (C), ISG56 mRNA (D), ISG15mRNA (E), TNF-. alpha.mRNA F), IL-6 mRNA (G) and CXCL10 (mRNA H) expression levels after treatment.

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Guangdong Dragon sail Biotechnology Ltd

Application of <120> histone deubiquitinating enzyme in preparation of medicine for treating systemic lupus erythematosus

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>2487

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

atggcggctg aagaggcgga tgtggatatc gaaggggacg tggtagcggc ggcgggggca 60

cagccaggaa gtggtgaaaa tacagcatca gttttacaaa aagatcacta tcttgattca 120

tcttggagaa cagagaatgg ccttattcct tggaccttgg ataacaccat cagtgaagag 180

aacagagctg ttattgagaa aatgttgttg gaagaagaat attatttatc taaaaaatca 240

caaccggaaa aagtctggct tgatcaaaag gaagatgata aaaaatacat gaagagtctg 300

cagaaaacag caaaaatcat ggtacactct cctacaaaac cagccagtta ctcagtaaag 360

tggacgatag aagaaaaaga gctgtttgaa caagggctgg ctaaatttgg ccgaagatgg 420

accaaaattt caaagctaat tggaagccgc actgttttac aagtgaagag ttatgcaaga 480

cagtatttta aaaataaggt caaatgcggt ctggataaag aaacaccaaa tcagaagacc 540

ggccataatc ttcaagttaa aaatgaagat aaagggacaa aggcatggac accatcatgt 600

ttaaggggac gtgctgatcc caacttgaat gctgtaaaaa ttgaaaagtt atctgatgat 660

gaagaagtag acatcacaga tgaggtggac gagttgtctt ctcaaacacc ccagaagaat 720

tctagcagtg atctcttgtt agactttcct aatagtaaaa tgcatgaaac caatcaagga 780

gaattcatta cttctgacag ccaggaagct ctcttttcta agtcttccag gggctgtctt 840

caaaatgaaa agcaagatga aacactttca agctcagaaa ttacactgtg gactgagaaa 900

cagagcaatg gtgacaaaaa atcaattgaa ttaaatgacc agaaatttaa tgaattgatt 960

aaaaactgca acaagcatga tggaagggga ataatagttg atgccaggca gttgccttct 1020

ccagagcctt gtgaaattca gaaaaatttg aatgataatg aaatgctttt tcattcttgc 1080

caaatggtag aggaaagcca tgaggaagaa gagcttaagc caccagaaca ggaaatagaa 1140

atagatagaa atatcattca agaagaagaa aaacaagcaa ttcctgagtt ttttgagggg 1200

cgccaagcta aaacaccaga acgctatttg aaaattagaa attatatttt ggatcaatgg 1260

gagatatgca aaccaaaata cttaaataag acctcagtac gtcctggcct gaagaactgt 1320

ggagatgtta attgtattgg acggattcat acatacctcg aattgatagg agcaatcaat 1380

tttggatgtg aacaggctgt gtataatagg ccacaaacag ttgacaaagt acgaatcaga 1440

gacagaaaag atgcagtaga agcataccaa cttgcccagc gtctgcagtc tatgcgtaca 1500

aggagacgta gggtccgaga cccatgggga aactggtgtg atgcaaagga cttagaagga 1560

caaacgtttg agcatctctc tgctgaggag ttggcaaaaa gaagagaaga ggaaaaaggc 1620

agacctgtta aatctttaaa agtgccaaga ccaacaaaaa gctcgtttga tcccttccaa 1680

ctgatacctt gtaatttttt tagtgaagaa aagcaggagc catttcaggt gaaagtggct 1740

tcagaagcac ttttaataat ggatttgcat gctcatgttt ctatggcaga agtgattggt 1800

ctgttaggag gaagatactc agaagttgat aaagtagttg aagtctgtgc agcagaacca 1860

tgtaacagtc tgagtacagg actacagtgt gagatggatc ctgtatcaca aacacaggcc 1920

tcagaaacct tggctgttag aggcttcagt gttattggat ggtatcattc tcatcctgct 1980

tttgatccta atccttcctt acgagatatt gacacacaag ctaaatacca gagttacttc 2040

tccagaggag gtgcaaagtt cattgggatg attgttagtc cctataatcg aaataatccc 2100

ttaccatatt ctcagattac ctgcctggtt ataagtgagg aaattagccc agatggctct 2160

tatcgcttac cttacaaatt tgaagtacag cagatgttag aagaacctca gtggggatta 2220

gtatttgaaa agacaagatg gataatagaa aaatacaggc tctcccatag cagcgtcccc 2280

atggataaaa tctttcgccg ggattctgac ctgacttgtt tgcagaaact tttggagtgt 2340

atgaggaaga ctctgagcaa agtgaccaat tgctttatgg ctgaagaatt cttgactgaa 2400

atagaaaatt tgttcctttc caattataaa agcaaccaag agaatggagt aaccgaagag 2460

aactgtacaa aggaattgtt aatgtga 2487

<210>2

<211>828

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Met Ala Ala Glu Glu Ala Asp Val Asp Ile Glu Gly Asp Val Val Ala

1 5 10 15

Ala Ala Gly Ala Gln Pro Gly Ser Gly Glu Asn Thr Ala Ser Val Leu

20 25 30

Gln Lys Asp His Tyr Leu Asp Ser Ser Trp Arg Thr Glu Asn Gly Leu

35 40 45

Ile Pro Trp Thr Leu Asp Asn Thr Ile Ser Glu Glu Asn Arg Ala Val

50 55 60

Ile Glu Lys Met Leu Leu Glu Glu Glu Tyr Tyr Leu Ser Lys Lys Ser

65 70 75 80

Gln Pro Glu Lys Val Trp Leu Asp Gln Lys Glu Asp Asp Lys Lys Tyr

85 90 95

Met Lys Ser Leu Gln Lys Thr Ala Lys Ile Met Val His Ser Pro Thr

100 105 110

Lys Pro Ala Ser Tyr Ser Val Lys Trp Thr Ile Glu Glu Lys Glu Leu

115 120 125

Phe Glu Gln Gly Leu Ala Lys Phe Gly Arg Arg Trp Thr Lys Ile Ser

130 135 140

Lys Leu Ile Gly Ser Arg Thr Val Leu Gln Val Lys Ser Tyr Ala Arg

145 150 155 160

Gln Tyr Phe Lys Asn Lys Val Lys Cys Gly Leu Asp Lys Glu Thr Pro

165 170 175

Asn Gln Lys Thr Gly His Asn Leu Gln Val Lys Asn Glu Asp Lys Gly

180 185 190

Thr Lys Ala Trp Thr Pro Ser Cys Leu Arg Gly Arg Ala Asp Pro Asn

195 200 205

Leu Asn Ala Val Lys Ile Glu Lys Leu Ser Asp Asp Glu Glu Val Asp

210 215 220

Ile Thr Asp Glu Val Asp Glu Leu Ser Ser Gln Thr Pro Gln Lys Asn

225 230 235 240

Ser Ser Ser Asp Leu Leu Leu Asp Phe Pro Asn Ser Lys Met His Glu

245 250 255

Thr Asn Gln Gly Glu Phe Ile Thr Ser Asp Ser Gln Glu Ala Leu Phe

260 265 270

Ser Lys Ser Ser Arg Gly Cys Leu Gln Asn Glu Lys Gln Asp Glu Thr

275 280 285

Leu Ser Ser Ser Glu Ile Thr Leu Trp Thr Glu Lys Gln Ser Asn Gly

290 295 300

Asp Lys Lys Ser Ile Glu Leu Asn Asp Gln Lys Phe Asn Glu Leu Ile

305 310 315 320

Lys Asn Cys Asn Lys His Asp Gly Arg Gly Ile Ile Val Asp Ala Arg

325 330 335

Gln Leu Pro Ser Pro Glu Pro Cys Glu Ile Gln Lys Asn Leu Asn Asp

340 345 350

Asn Glu Met Leu Phe His Ser Cys Gln Met Val Glu Glu Ser His Glu

355 360 365

Glu Glu Glu Leu Lys Pro Pro Glu Gln Glu Ile Glu Ile Asp Arg Asn

370 375 380

Ile Ile Gln Glu Glu Glu Lys Gln Ala Ile Pro Glu Phe Phe Glu Gly

385 390 395 400

Arg Gln Ala Lys Thr Pro Glu Arg Tyr Leu Lys Ile Arg Asn Tyr Ile

405 410 415

Leu Asp Gln Trp Glu Ile Cys Lys Pro Lys Tyr Leu Asn Lys Thr Ser

420 425 430

Val Arg Pro Gly Leu Lys Asn Cys Gly Asp Val Asn Cys Ile Gly Arg

435 440 445

Ile His Thr Tyr Leu Glu Leu Ile Gly Ala Ile Asn Phe Gly Cys Glu

450 455 460

Gln Ala Val Tyr Asn Arg Pro Gln Thr Val Asp Lys Val Arg Ile Arg

465 470 475 480

Asp Arg Lys Asp Ala Val Glu Ala Tyr Gln Leu Ala Gln Arg Leu Gln

485 490 495

Ser Met Arg Thr Arg Arg Arg Arg Val Arg Asp Pro Trp Gly Asn Trp

500 505 510

Cys Asp Ala Lys Asp Leu Glu Gly Gln Thr Phe Glu His Leu Ser Ala

515 520 525

Glu Glu Leu Ala Lys Arg Arg Glu Glu Glu Lys Gly Arg Pro Val Lys

530 535 540

Ser Leu Lys Val Pro Arg Pro Thr Lys Ser Ser Phe Asp Pro Phe Gln

545 550 555 560

Leu Ile Pro Cys Asn Phe Phe Ser Glu Glu Lys Gln Glu Pro Phe Gln

565 570 575

Val Lys Val Ala Ser Glu Ala Leu Leu Ile Met Asp Leu His Ala His

580 585 590

Val Ser Met Ala Glu Val Ile Gly Leu Leu Gly Gly Arg Tyr Ser Glu

595 600 605

Val Asp Lys Val Val Glu Val Cys Ala Ala Glu Pro Cys Asn Ser Leu

610 615 620

Ser Thr Gly Leu Gln Cys Glu Met Asp Pro Val Ser Gln Thr Gln Ala

625 630 635 640

Ser Glu Thr Leu Ala Val Arg Gly Phe Ser Val Ile Gly Trp Tyr His

645 650 655

Ser His Pro Ala Phe Asp Pro Asn Pro Ser Leu Arg Asp Ile Asp Thr

660 665 670

Gln Ala Lys Tyr Gln Ser Tyr Phe Ser Arg Gly Gly Ala Lys Phe Ile

675 680 685

Gly Met Ile Val Ser Pro Tyr Asn Arg Asn Asn Pro Leu Pro Tyr Ser

690 695 700

Gln Ile Thr Cys Leu Val Ile Ser Glu Glu Ile Ser Pro Asp Gly Ser

705710 715 720

Tyr Arg Leu Pro Tyr Lys Phe Glu Val Gln Gln Met Leu Glu Glu Pro

725 730 735

Gln Trp Gly Leu Val Phe Glu Lys Thr Arg Trp Ile Ile Glu Lys Tyr

740 745 750

Arg Leu Ser His Ser Ser Val Pro Met Asp Lys Ile Phe Arg Arg Asp

755 760 765

Ser Asp Leu Thr Cys Leu Gln Lys Leu Leu Glu Cys Met Arg Lys Thr

770 775 780

Leu Ser Lys Val Thr Asn Cys Phe Met Ala Glu Glu Phe Leu Thr Glu

785 790 795 800

Ile Glu Asn Leu Phe Leu Ser Asn Tyr Lys Ser Asn Gln Glu Asn Gly

805 810 815

Val Thr Glu Glu Asn Cys Thr Lys Glu Leu Leu Met

820 825

Claims (6)

1. The application of histone deubiquitinating enzyme in preparing the medicine for preventing and treating systemic lupus erythematosus is characterized in that the nucleotide sequence of the histone deubiquitinating enzyme is SEQ ID NO: 1.

2. the use according to claim 1, wherein the histone deubiquitinating enzyme dose in the preparation of the medicament for preventing and treating systemic lupus erythematosus is 0.1 mol.

3. A protein encoded by the histone deubiquitinating enzyme gene in the use of claim 1, wherein the amino acid sequence of the protein is SEQ ID NO: 2.

4. use of the protein of claim 3 for the preparation of a medicament for the prevention/treatment of autoimmune diseases.

5. A lentiviral expression vector constructed using the protein of claim 3.

6. A metalloprotease obtained by monoubiquitination modification at the K119 site of the protein according to claim 3.

Images