CN109985230B

CN109985230B - Application of protein in preparation of medicine for preventing and treating kidney diseases

Info

Publication number: CN109985230B
Application number: CN201810002808.1A
Authority: CN
Inventors: 张英豪; 胡楠; 付晶鹏; 闫桂蕊; 宛佳; 李小灵; 孙荣勋; 王耀
Original assignee: Shanghai Puyou Biomedical Co ltd
Current assignee: Shanghai Puyou Biomedical Co ltd
Priority date: 2018-01-02
Filing date: 2018-01-02
Publication date: 2023-10-17
Anticipated expiration: 2038-01-02
Also published as: CN109985230A; WO2019134482A1

Abstract

The invention relates to application of a protein in preparing a medicament for preventing and treating kidney diseases, which is a medicament for preparing a medicament for preventing and treating kidney diseases by sDSS1 protein. The invention further relates to the use in a kidney disease improving care device. sDSS1 can effectively combine advanced oxidation protein products and reduce cytotoxicity caused by the advanced oxidation protein products, relieve disease symptoms of chronic kidney disease model animals, and has great clinical application value.

Description

Application of protein in preparation of medicine for preventing and treating kidney diseases

Technical Field

The invention relates to application of sDSS1 protein in preparing a medicament for preventing and treating kidney diseases.

Background

Chronic kidney disease (Chronic kidney disease, CKD) is a disease of long-term progressive renal structure and altered renal function, where lesions of the renal structure generally include cysts, carcinomas, malformations, atrophy, and the like; while kidney function abnormality is mainly manifested as edema, hypertension, abnormal urine composition and the like [1,2]. More and more studies have found that creatinine, urea nitrogen and advanced oxidized protein products (Advanced oxidation protein products, AOPPs) are abnormally elevated in the blood of chronic kidney disease patients [3]. AOPPs are considered to be one of the important factors leading to chronic kidney disease [4,5].

AOPPs are a class of oxidized protein products with dual tyrosine structural features, and are one of the recognized biomarkers reflecting oxidative stress levels in vivo [6]. AOPPs in blood comes mainly from the oxidation of albumin. The levels of AOPPs in blood or tissue in renal failure, renal dialysis, diabetes, atherosclerosis and some patients with inflammatory diseases are significantly elevated [7,8]. The increase in blood AOPPs levels was proportional to the injury to glomerular filtration rate [9]. AOPPs can increase the production of reactive oxygen species and inflammatory factors by acting on receptor-activated downstream signaling pathways, thereby impairing the viability and function of vascular endothelial cells, tubular epithelial cells, podocytes, and the like. AOPPs may also activate the renin angiotensin system [10]. Intravenous administration of AOPPs to rats resulted in kidney damage, manifested by increased urine protein and kidney inflammation [11]. In cell experiments, AOPPs antibodies can block cytotoxicity caused by AOPPs in culture solution [12]; injection of the NADPH oxidase inhibitor Apocynin significantly alleviates apoptosis of mouse beta cells resulting from increased AOPPs [13]. AOPPs are closely related to the occurrence and development of chronic kidney disease, and can be a potential approach for preventing or treating chronic kidney disease by eliminating or interfering with AOPPs and downstream signals thereof.

The Shfm1 (split hand/split foot malformation type 1) gene is one of the key genes in human crab claw disease, is highly conserved in evolution, and the coded protein DSS1 is involved in the processes of stable genome, homologous gene recombination, DNA damage repair, protein degradation, cell proliferation and the like [14-18]. The results of the present inventors' studies show that DSS1 protein as a tag can be added to oxidized protein by an energy-consuming enzymatic reaction, helping cells to clear oxidized protein [19]. These results show an important role for DSS1 protein in biological activity.

The citations for the above are as follows:

1.Kidney Disease:Improving Glabal Outcomes(KDIGO)CKD Work Group(2013)KDIGO 2012clinical practice guideline for the evaluation and management of chronic kidney disease.Kidney Int.Suppl.3,1-150.

2.Zaccali C.et al.(2017)The systemic nature of CKD.Nat Rev Nephrol.13(6):344-358.

3.Tucker PS,Dalbo VJ,Han T,Kingsley MI(2013)Clinical and research markers of oxidative stress in chronic kidney disease.Biomarkers 18:103–115.

4.Li HY,Hou FF,Zhang X,Chen PY,Liu SX,Feng JX,Liu ZQ,Shan YX,Wang GB,Zhou ZM,Tian JW,Xie D(2007)Advanced oxidation protein products accelerate renal fibrosis in a remnant kidney model.J Am Soc Nephrol.18(2):528-38.

5.Cao W,Hou FF,Nie J(2011)AOPPs and the progression of kidney disease.Kidney Int Suppl.4(1):102-106.

6.Kuchta A,Pacanis A,Kortas-Stempak B,Cwiklińska A,M,Renke M,Rutkowski B(2011)Estimation of oxidative stress markers in chronic kidney disease.Kidney Blood Press Res.34(1):12-9.

7.Witko-Sarsat V,Friedlander M,Nguyen Khoa T,Capeillère-Blandin C,Nguyen AT,Canteloup S,Dayer JM,Jungers P,DrüekeT,Descamps-Latscha B(1996)Advanced oxidation protein productsas a novel marker of oxidative stress in uremia.Kidney International49(5):1304-13.

8.Witko-Sarsat V1,Friedlander M,Nguyen Khoa T,Capeillère-Blandin C,Nguyen AT,Canteloup S,Dayer JM,Jungers P,DrüekeT,Descamps-Latscha B(1998)Advanced oxidation protein productsas novel mediators of inflammation and monocyte activation inchronic renal failure.The Journal of Immunology 161(5):2524-32.9.K,KlenovicsováK,FerenczováJ,Hedvig J,PodrackáLu,Heidland A(2012)Advanced oxidation protein products andadvanced glycation end products in children and adolescents withchronic renal insufficiency.Journal of Renal Nutrition 22(1):143-8.10.Cao W,Xu J,Zhou ZM,Wang GB,Hou FF,Nie J(2013)Advanced oxidation protein products activate intrarenal renin–angiotensin system via a CD36-mediated,redox-dependentpathway.Antioxidants&redox signaling 18(1):19-35.

11.Li HY,Hou FF,Zhang X,Chen PY,Liu SX,Feng JX,Liu ZQ,Shan YX,Wang GB,Zhou ZM,Tian JW,Xie D(2007)Advancedoxidation protein products accelerate renal fibrosis in a remnantkidney model.J Am Soc Nephrol.18(2):528-38.

12.Liu B,Hou X,Zhou Q,Tian J,Zhu P,Xu J,Hou F,FuN(2011)Detection of advanced oxidation protein products in patientswith chronic kidney disease by a novel monoclonal antibody.FreeRadic Res,45(6):662-71.

13.Liang M,Li A,Lou A,Zhang X,Chen Y,Yang L,Li Y,Yang S,Hou FF(2017)Advanced oxidation protein products promoteNADPH oxidase-dependentβ-cell destruction and dysfunctionthrough the Bcl-2/Bax apoptotic pathway.Lab Invest 97(7):792-805.14.Van Silfhout AT,van den Akker PC,Dijkhuizen T,Verheij JB,Olderode-Berends MJ,Kok K,Sikkema-Raddatz B,van Ravenswaaij-Arts CM(2009)Split hand/foot malformation due to chromosome 7q aberrations(SHFM1):additional support for functional haploinsufficiency as the causative mechanism.Eur JHum Genet 17(11):1432-8.

15.Li J,Zou C,Bai Y,Wazer DE,Band V,Gao Q(2006)DSS1 is required for the stability of BRCA2.Oncogene 25:1186–1194.

16.Liu J,Doty T,Gibson B,Heyer WD(2010)Human BRCA2protein promotes RAD51 filament formation on RPA-covered singlestranded DNA.Nat Struct Mol Biol 17:1260–1262.

17.Zhou Q,Kojic M,Cao Z,Lisby M,Mazloum NA,Holloman WK(2007)Dss1 interaction with Brh2 as a regulatory mechanism for recombinational repair.Mol Cell Biol 2:2512–2526.

18.Kragelund,B.B.,S.M.,Rebula,C.A.,Panse,V.G.,&Hartmann-Petersen,R(2016)DSS1/Sem1,a multifunctional and intrinsically disordered protein.Trends in biochemical sciences41:446-459.

19.Zhang Y,Chang FM,Huang J,Junco JJ,Maffi SK,Pridgen HI,Catano G,Dang H,Ding X,Yang F,Kim DJ,Slaga TJ,He R,Wei SJ(2014)DSSylation,a novel protein modification targets proteins induced by oxidative stress,and facilitates their degradation in cells.Protein Cell 5(2):124-40.

disclosure of Invention

In the invention, sDSS1 protein provided by the inventor can be combined with AOPPs, so that cytotoxicity and active oxygen generation caused by the AOPPs are reduced. sDSS1 protein can reduce the level of AOPPs and improve the kidney function obviously. Therefore, sDSS1 protein has potential for clinically preparing medicines for preventing and treating kidney diseases and improving kidney disease nursing equipment.

The specific technical scheme is as follows:

an application of protein in preparing a medicament for preventing and treating kidney diseases, wherein the application is that sDSS1 protein is used for preparing the medicament for preventing and treating kidney diseases.

Preferably, the kidney disease comprises the chronic kidney disease GFR of stage 1 not less than 90mL/min/1.73m ² The GFR of the 2 nd stage chronic kidney disease is 60-89 mL/min/1.73m ² The GFR of the 3 rd stage chronic kidney disease is 30-59 mL/min/1.73m ² The GFR of the 4 th stage chronic kidney disease is 15-29 mL/min/1.73m ² Or GFR of stage 5 chronic kidney disease<15mL/min/1.73m ² 。

Preferably, the kidney disease has an increased AOPP signature, including diabetic nephropathy, igA nephropathy, membranous nephropathy, acute kidney injury, lupus nephritis, glomerulonephritis, polycystic kidney disease, or chronic glomerulonephritis.

Preferably, the sDSS1 protein comprises a basic protein formed by any sDSS1 protein sequence of human, chimpanzee, bonobo, gorilla, red chimpanzee, white cheek gibbon, sichuan golden monkey, rhesus monkey, yunnan golden monkey, east non baboon, angora monkey, white top white brow monkey, spanish, and ragweed monkey, wherein the amino acid sequence of human sDSS1 is shown as SEQ ID NO:1, the amino acid sequence of chimpanzee sDSS1 is shown in SEQ ID NO:2, the amino acid sequence of the bonobo sDSS1 is shown in SEQ ID NO:3, the amino acid sequence of gorilla sDSS1 is shown in SEQ ID NO:4, the amino acid sequence of the chimpanzee sDSS1 is shown in SEQ ID NO:5, the amino acid sequence of the ape of the cheek gibbon sDSS1 is shown in SEQ ID NO:6, the amino acid sequence of the Sichuan golden monkey sDSS1 is shown as SEQ ID NO:7, the amino acid sequence of the rhesus sDSS1 is shown as SEQ ID NO:8, the amino acid sequence of the Yunnan golden monkey sDSS1 is shown as SEQ ID NO:9, the amino acid sequence of the baboon sDSS1 is shown in SEQ ID NO:10, the amino acid sequence of the angora wart monkey sDSS1 is shown in SEQ ID NO:11, the amino acid sequence of the white top white brow sDSS1 is shown in SEQ ID NO:12, the amino acid sequence of the spanish baboon sDSS1 is shown as SEQ ID NO:13, the amino acid sequence of the sDSS1 of the pigtail monkey is shown in SEQ ID NO:14.

preferably, the sDSS1 protein is any first protein with a similarity of more than 70% with the basic protein.

Preferably, the sDSS1 protein is a second protein based on 58 amino acids at the nitrogen end of the basic protein, and other polypeptide fragments are fused at the nitrogen end or the carbon end, and the structural characteristics or amino acid sequence characteristics of the polypeptide fragments used for fusion are the same as or similar to those of 31 sequences at the carbon end of the basic protein.

Preferably, the sDSS1 protein is a third protein which is based on 58 amino acids at the nitrogen end of the basic protein, and is fused with other amino acid fragments at the nitrogen end or the carbon end, and the fused protein can realize a transmembrane transport function.

Preferably, the sDSS1 protein is a fusion protein formed by connecting the basic protein, the first protein, the second protein or the third protein with the protein itself, a carrier protein, an antibody or other amino acid fragments with any length.

Preferably, the sDSS1 protein is a polypeptide/protein modification based on modification of the base protein, first protein, second protein, third protein or fusion protein.

Preferably, the polypeptide/protein modification is a specific or non-specific 1-20 site chemical modification of amino acid side chains, carbonyl groups on amino acid side chains, nitrogen terminal amino groups, carbon terminal carbonyl groups, cysteines, tyrosines, serines, tryptophan.

Preferably, the modification method of the polypeptide/protein modifier comprises one or more of glycosylation modification, fatty acid modification, acylation modification, fc fragment fusion, albumin fusion, polyethylene glycol modification, dextran modification, heparin modification, polyvinylpyrrolidone modification, polyamino acid modification, polysialic acid modification, chitosan and derivatives thereof modification, lectin modification, sodium alginate modification, carbomer modification, polyvinylpyrrolidone modification, hydroxypropyl methylcellulose modification, hydroxypropyl cellulose modification, acetylation modification, formylation modification, phosphorylation modification, methylation modification, sulfonation modification and other pharmaceutically usable polypeptide/protein drug modification methods.

Preferably, the sDSS1 protein is an unnatural amino acid substitution protein with 1-31 arbitrary amino acid substitutions made with amino acids other than 20 basic amino acids based on the amino acid sequence of the base protein, first protein, second protein, third protein, or fusion protein.

Preferably, the amino acid substitutions of the unnatural amino acid substitution protein include substitutions to hydroxyproline, hydroxylysine, selenocysteine, D-amino acids, or synthetic unnatural amino acids and derivatives thereof.

Preferably, the sDSS1 protein is a partial or complete complex of the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/protein modification or the unnatural amino acid replacement protein with a pharmaceutically acceptable carrier.

Preferably, the pharmaceutical carrier of the complex comprises one or more of enteric coated formulations, capsules, microspheres/vesicles, liposomes, microemulsions, multiple emulsions, nanoparticles, magnetic particles, gelatin and gels.

Preferably, the sDSS1 protein targets the individual's own sDSS1 protein, and the individual's own sDSS1 protein level is influenced by exogenous drugs.

Preferably, the medicament takes sDSS1 protein, a gene of the sDSS1 protein, a regulatory element of the gene of the sDSS1 and a transcription product of the gene of the sDSS1 as medicament action targets.

Preferably, the agent modulates the amount of sDSS1 protein in the blood by affecting protease/peptidase activity in the blood.

Preferably, the drug is a first drug formed from a chemical small molecule drug, an antibody, a polypeptide/protein drug, a nucleic acid drug, or a nano-drug.

Preferably, the sDSS1 protein is a second drug formed by a combination of two or more of any one of the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/protein modification, the complex, the first drug.

Preferably, the sDSS1 protein is a third drug formed by one, two or more of any one component of the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/protein modification, the complex, the first drug and pharmaceutically acceptable excipients.

Preferably, the sDSS1 protein is a fourth protein obtained by introducing a nucleotide sequence encoding the basic protein, the first protein, the second protein, the third protein or the fusion protein into the body through an expression system and expressing the fourth protein.

Preferably, the expression system is a eukaryotic expression plasmid vector, adenovirus, adeno-associated virus, lentivirus, retrovirus, baculovirus, herpesvirus, pseudorabies virus, ZFN gene editing technology, TALEN gene editing technology, CRISPR/Cas gene editing technology, or other medically available gene editing technology or viral vector.

Preferably, said sDSS1 protein is a fifth protein formed by said basic protein, first protein, second protein, third protein or fusion protein obtained by transplanting cells into an individual.

Preferably, the cell is any human stem cell, precursor cell or adult cell.

Preferably, the stem cells are embryonic stem cells, induced pluripotent stem cells, transdifferentiated cells, or pluripotent or monopotent stem cells derived from primary cultured stem cells, differentiated from blast cells.

Preferably, the sDSS1 protein is a sixth protein introduced into the individual by serum, interstitial fluid infusion.

Preferably, the sDSS1 protein is the seventh protein formed by the basic protein, the first protein, the second protein, the third protein or the fusion protein obtained by transplanting a tissue or organ in an individual.

Preferably, the tissue is a whole organ or a partial tissue mass of brain, liver, kidney, spleen, islets, or blood, fat, muscle, bone marrow, skin.

Preferably, the prophylactic agent is one or more of a basic protein, a first to seventh protein, a fusion protein, a polypeptide/protein modification, an unnatural amino acid-substituted protein, a complex, a pharmaceutical composition, an expression system, a cell, a tissue, an organ, a body fluid, a protein drug of a tissue fluid, a polypeptide drug, a nucleic acid drug, a chemical small molecule drug, a cell product, a commercial transplanted tissue, an injection, and a lyophilized powder.

Preferably, the therapeutic agent is one or more of a basic protein, a first to seventh protein, a fusion protein, a polypeptide/protein modification, an unnatural amino acid substitution protein, a complex, a first agent, a second agent, a third agent, a protein agent in an expression system, a cell, a tissue, an organ, a body fluid, a tissue fluid, a polypeptide agent, a nucleic acid agent, a chemical small molecule agent, a cell product, a commercial graft tissue, an injection, and a lyophilized powder.

The invention has the characteristics and/or beneficial effects that:

1. the sDSS1 protein provided by the invention is combined with the AOPPs protein, inhibits the combination ability of the AOPPs protein and receptor protein (advanced glycoprotein receptor, RAGE) thereof, and effectively relieves cytotoxicity caused by the AOPPs protein.

2. The sDSS1 protein provided by the invention can obviously improve and reduce the urine microalbumin excretion and improve the renal function on a SD rat model of chronic kidney disease.

3. The sDSS1 protein provided by the invention is a protein of human and other primates, has relatively small molecular weight and low immunogenicity, and has a natural protein degradation mechanism in vivo, so that the clinical application can not cause obvious immune response or other toxic and side effects, and is safe and reliable.

In conclusion, the invention provides an sDSS1 protein medicine for treating chronic kidney disease, and experiments on molecular level, cell level and animal level prove that the sDSS1 protein can be combined with AOPPs protein, so that the combination efficiency of the AOPPs protein and a receptor is reduced. In animal experiments, sDSS1 protein can effectively reduce the excretion of microalbumin in urine of rats with chronic kidney disease, improve kidney function and relieve disease symptoms. The sDSS1 protein has low immunogenicity and obvious drug effect, and has potential for clinically preparing and preventing and treating kidney diseases or improving the performance of kidney disease nursing equipment.

Drawings

The present invention will now be described in further detail with reference to the accompanying drawings, so that the invention will be clear and complete, but is not intended to limit the scope of the invention.

FIG. 1 shows that sDSS1 can interact with AOPPs. AOPPs proteins or products incubated with sDSS1 were separated by SDS-PAGE and stained with coomassie brilliant blue, which showed that AOPPs can interact with sDSS1 proteins to form complexes with molecular weights greater than 25KD, very pronounced between 50KD-250KD (L3, L5, L7), and that complex formation was proportional to sDSS1 protein concentration, exhibiting pronounced concentration dependence. The band of sDSS1 protein (molecular weight about 15 KD) in the AOPPs reaction system is significantly shallower than that of sDSS1 protein alone.

FIG. 2.SDSS1 protein can mask the interaction of AOPPs with its receptor protein. After incubation of the AOPPs with sDSS1 protein, they were incubated with glycosylated protein receptor protein (RAGE), and the incubated products were separated by SDS-PAGE and stained with Coomassie Brilliant blue. The results showed that the sDSS1 protein forms two distinct bands with the RAGE mixture, RAGE (48 KD) and sDSS1 (13.88 KD), respectively, suggesting no significant interaction (L4). AOPPs interact with the dss1 protein to form a diffuse tail (L5). The interaction of AOPPs with RAGE revealed a shallowing RAGE band with a pronounced tailing effect (L6). After sDSS1 protein is added into the reaction system, the RAGE band gradually deepens along with the increase of the concentration of the sDSS1 protein, and the diffuse tail gradually deepens (L7, L8 and L9).

FIGS. 3A-3B. SDSSS 1 protein reduces cytotoxicity caused by AOPPs.

FIG. 3A, the addition of 10. Mu.M AOPPs to rat kidney cell cultures significantly reduced the level of cell viability, which was recovered after the addition of different concentrations of sDSS1 protein and gradually increased with increasing sDSS1 protein concentration. 30. Mu.M sDSS1 protein can completely shield the decrease of cell viability caused by 10. Mu.M AOPPs.

FIG. 3B shows that detection of cytotoxic response levels in cells revealed that sDSS1 protein can reduce the level of cytotoxicity induced by AOPPs, and that this effect exhibited a typical dose-dependent effect. Each group n=6. Data were analyzed by ANOVA, #, AOPPs group vs alone with dss1, #, AOPPs group vs without AOPPs group; # p-value <0.05; #, # p-value <0.01; # #, p-value <0.001.

FIG. 4.SDSS1 protein reduces cellular oxidative stress induced to AOPPs. The addition of AOPPs to the culture medium stimulated a significant increase in cellular ROS levels, which decreased upon addition of sDSS1 protein. The decrease is more and more pronounced as the concentration of sDSS1 increases.

Fig. 5A-5 b. Prolonged injection of dss1 protein alleviates disease symptoms in chronic kidney disease model rats.

Fig. 5A, chronic kidney disease model rat urine, due to kidney injury, has significantly higher urine albumin content than the control group. Animals in the protein administration group had significantly reduced urine protein content, while the urine protein content in the saline control group remained high, by 3 weeks of continuous sDSS1 protein injection.

Fig. 5B, rats were sacrificed and examined for kidney index, and as a result, it was found that the kidney index of the rats in the protein administration group was significantly recovered, and there was no significant difference from the rats in the control group, whereas the kidney index of the rats in the normal saline injection group was significantly higher. Each group n=5. Data analyzed by ANOVE, p-value <0.05; # and p-value <0.01.

Detailed Description

The following description will illustrate and verify the preferred embodiments of the present invention with reference to examples, and is not intended to limit the scope of the present invention. The full scope of the invention is defined by the claims.

The experimental methods used in the following examples are conventional experimental methods unless otherwise specified.

The sDSS1 protein used in the following embodiment is self-produced and quality controlled by the company, the purity of the detected protein is more than 95%, endotoxin (endotoxin content is less than 3EU/mg protein) and other impurity residues meet the standards, and the protein can be used for animal experiments without causing obvious animal toxicity reaction.

Materials and reagents in the following examples, except for sDSS1 protein, are commercially available.

Example 1. Interaction of sDSS1 protein with AOPPs.

1.1. Experimental materials and methods

Materials: bovine serum albumin (aledine, a 104912), sodium hypochlorite (aledine, S01636).

The method comprises the following steps: and (3) carrying out light-shielding reaction on bovine serum albumin and sodium hypochlorite for 30 minutes, and dialyzing the reaction product in PBS solution overnight to obtain the AOPPs protein. The AOPPs proteins were quantified using the BCA kit (ThermoFisher Scientific, 23252). Then 2. Mu.g of AOPPs, 1. Mu.g of sDSS1 with 2. Mu.g of AOPPs, 2. Mu.g of sDSS1 with 2. Mu.g of AOPPs, 4. Mu.g of sDSS1 with 2. Mu.g of AOPPs were added to a 1.5mL EP tube, respectively, and reacted overnight at 4 ℃. After the incubation products are added into the loading buffer solution, the mixture is uniformly mixed, and the sample is prepared by denaturation treatment at 100 ℃ for 10 minutes. Samples were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and the PAGE gels were stained with Coomassie blue to reveal protein bands.

1.2. Experimental results

As can be seen on the stained PAGE gel, the molecular weight of the prepared AOPPs protein is different from 1KD-37KD, a diffuse band (L2) is formed, and the molecular weight of the sDSS1 protein is 13.88KD (L1, L4 and L6). When AOPPs were incubated in admixture with sDSS1 protein, the diffuse bands of AOPPs protein were seen to be substantially disappeared, while large molecular weight diffuse bands (L3, L5, L7) were formed between 50KD-250KD, indicating that AOPPs interacted with sDSS1 protein to form a complex. Moreover, as the proportion of sDSS1 increases, the large molecular weight complex increases progressively, showing a darker band color (FIG. 1). These results indicate that sDSS1 proteins can interact with AOPPs and form complexes that cannot be separated by SDS-PAGE.

Example 2.SDSS1 protein reduces interaction of AOPPs with receptor proteins.

2.1. Experimental materials and methods

Materials: AOPPs (prepared according to the method of example 1), terminally glycosylated protein receptor proteins (RAGE) (Yinqiao Shenzhou; cat. No. 50489-M08H-100), sDSS1 proteins.

The method comprises the following steps: firstly, mixing a certain mass of AOPPs with sDSS1 proteins with different concentrations, and incubating for 24 hours at 4 ℃. RAGE protein was added to the post-incubation product and incubation was continued for 24 hours at 4 ℃. After the reaction, a 5 Xloading buffer (non-denaturing loading buffer) was added to the product to prepare a loading sample. Mu.l of the sample was subjected to SDS-PAGE under 200V for 90 minutes for electrophoresis. PAGE gels were stained with coomassie brilliant blue to reveal protein bands.

2.2. Experimental results

On PAGE gels, the sDSS1 protein did not interact with the receptor protein RAGE, and two distinct bands were shown on the gel, sDSS1 protein (13.88 KD) and RAGE (48 KD), respectively, with no other diffuse bands (L4). When AOPPs are mixed with RAGE, the two can interact, with the result that the RAGE band becomes significantly shallower, while the AOPPs band substantially disappears (L6). However, when sDSS1 protein was present in the reaction system, it was seen that as the concentration of sDSS1 protein increased, the RAGE band concentration was increasingly stronger, indicating that sDSS1 protein inhibited the interaction of AOPPs with RAGE (L7, L8, L9). Correspondingly, the higher the concentration of the complex formed by the sDSS1 protein and the AOPPs, the more and more intense the color of the diffuse band is shown (FIG. 2). These results demonstrate that the reaction of sDSS1 protein with AOPPs to form complexes can reduce the interaction of AOPPs with their receptor proteins.

Example 3.SDSS1 protein masks cytotoxicity caused by AOPPs.

3.1. Experimental materials and methods

Materials: NRK-52E cell line (China academy of sciences typical culture Collection Committee cell Bank, catalog number: GNR 8), DMEM medium (Hyclone, AC 10210629), carbon dioxide cell incubator (ThermoFisher), spectraMax Plus384 multifunctional enzyme-labeled instrument (Molecular Devices company), cell proliferation/toxicity assay kit (Dojindo, CK 04), lactate dehydrogenase cytotoxicity assay kit (Biyunshii Biotechnology Co., C0017).

The method comprises the following steps: NRK-52E cells were cultured in DMEM complete medium containing 10% peptide bovine serum and passaged every two days. When seeding cells, the cells are first digested with pancreatin to single cells, at 2 x 10 ⁴ Cells were seeded into 96-well plates per well. Cells were attached overnight, and after completion, replaced with serum-free medium, starved for 24 hours. Cells were divided into 7 groups of 6 duplicate wells according to experimental requirements, each with blank medium, 10 μM AOPPs, 10 μMAOPPs and 0.3 μM sDSS1, 10 μM AOPPs and 1 μM sDSS1, 10 μMAOPPs and 3 μM sDSS1, 10 μM AOPPs and 10 μM sDSS1, 10 μMAOPPs and 30 μM sDSS1, respectively. After completion, the cells were further placed in a carbon dioxide cell incubator for 48 hours. The treated cells were subjected to cell viability level detection using a cell proliferation toxicity detection kit. Collecting culture medium, centrifuging 100g, collecting supernatant, and performing fine extraction with lactate dehydrogenase cytotoxicity detection kitCytotoxicity level detection.

3.2. Experimental results

The cell viability assay showed that when only 10 mu MAOPPs were added to NRK-52E cells, the level of cell activity was significantly reduced, only about 25% of the control cells. When sDSS1 protein was added to the medium, cell viability was gradually increased. In the range of 0.3 μm-30 μm, the enhancement of cell activity was more pronounced with increasing concentration of sDSS1 protein, exhibiting concentration dependence. The 30 mu M sDSS1 protein added into the culture medium can not only completely shield the decrease of the cell viability caused by the AOPPs, but also promote the increase of the cell viability, which is higher than that of the cells of the control group (figure 3A). The level of the cytotoxicity reaction is detected by using the lactic dehydrogenase cytotoxicity detection kit, and 10 mu M AOPPs can cause obvious cytotoxicity of cells, which is shown by a remarkable increase of the content of the lactic dehydrogenase in the culture solution. As the lactate dehydrogenase release gradually decreased after the sDSS1 protein was added to the medium, the effect of the sDSS1 protein exhibited concentration dependence (fig. 3B). These results demonstrate that sDSS1 protein can shield cytotoxicity caused by AOPPs and protect cell viability.

Example 4 sDSS1 protein reduces the cellular oxidative stress induced by AOPPs.

4.1. Experimental materials and methods

Experimental materials: NRK-52E cells; reactive oxygen species detection kit (Biyun Tian, cat# S0033); bovine serum albumin component v (aladine a104912-100 g); DMEM medium (HyClone, AC 10210629); flow cytometer (Millipore, guava easy cyto)

The method comprises the following steps: digestion of NRK-52E cells into single cells was followed by 1X 10 ⁵ The density of the cells/well was plated on a 6-well plate for cell culture and the cells were attached overnight. After cell attachment is completed, the cells are replaced by a serum-free culture medium (containing 0.1% bovine serum albumin) for starvation treatment for 24 hours; after starvation, sDSS1 at concentrations of 3. Mu.M, 30. Mu.M, 100. Mu.M, respectively, was added to 6 well plates, while AOPPs at final concentrations of 10. Mu.M were added, and Control, AOPPs (10. Mu.M) and BSA (10. Mu.M) wells were additionally set as controls for 48 hours; after 48 hours, 1mL of diluted DCFH-DA (10. Mu.M) was added to each well, and incubated in a 37℃cell incubator for 20 minutes; washing with serum-free cell culture solutionCells 2 times to sufficiently remove DCFH-DA that did not enter the cells; flow cytometry detection, using 488nm excitation wavelength, 525nm emission wavelength, detecting the fluorescence intensity of DCFH, reacting to cellular ROS levels;

4.2. experimental results

The addition of AOPPs to the cell culture medium significantly stimulated oxidative stress in the cells, resulting in a significant increase in cellular ROS levels, which was manifested by higher DCFH fluorescence intensities than in the control and 10 μm BSA-containing groups. After adding sDSS1 protein to the culture medium, the cellular oxidative stress induced by AOPPs gradually decreased, and the cellular ROS level of 30. Mu.M sDSS1 protein group was substantially the same as that of 10. Mu.M BSA, lower than that of control group (FIG. 4). These results demonstrate that sDSS1 protein can reduce the cellular oxidative stress response caused by AOPPs.

EXAMPLE 5 sDSS1 protein alleviates disease symptoms in SD rats in the CKD model

5.1. Experimental materials and methods

5.1.1. Experimental animal

SD male rats weighing 180-220g, purchased from Shanghai Laike laboratory animal center, and placed in animal house for one week before the experiment.

5.1.2. Experimental reagent

Experimental medicine: sDSS1 protein

Reagent: rat urinary Microalbumin (MAU) ELISA kit (E-EL-R0025 c, WU, irelett biosciences Co., ltd.); doxorubicin for injection (Pfizer); high protein feed (Shanghai Proteus Biotechnology Co., ltd.).

5.2. Grouping, modeling and administration of animals

And (3) molding: CKD model rats are a model of chronic kidney disease. And (3) adopting tail vein injection of doxorubicin, assisting high-protein diet, collecting urine of the rats after continuous high-protein diet is carried out for 3 weeks, detecting the urine protein level, confirming that the urine protein level of the rats is obviously improved, and proving that the CKD model of the rats is successfully induced and the rats with the urine protein level not obviously improved are eliminated.

Grouping: rats with modeling effort are divided into 2 groups, including CKD model group and sDSS1 dosing group, 5 each. The same-cage rats without molding served as a negative control group for 5 rats.

Administration: sDSS1 group administration was performed by intraperitoneal injection of sDSS1 protein solution 1 time per day at 50mg/kg, with a dose of 3mL, and continuous injection for 3 weeks. CKD model and negative control groups were injected with an equal volume of physiological saline daily.

5.3. Detection index and detection method

The urine of the rat was collected once each 4 hours before and after the end of the administration, and the amount of microalbuminuria was measured for 24 hours using an enzyme-linked immunosorbent assay kit.

Elisa detects urine protein levels: 100. Mu.L of standard or sample was added to each well and incubated at 37℃for 90 minutes; pouring out the liquid in the hole, adding 100 mu L of biotinylated antibody working solution, and incubating at 37 ℃ for 60 minutes; washing for 3 times; adding 100 mu L of enzyme binding working solution, and incubating for 30 minutes at 37 ℃; washing for 5 times; adding 90 mu L of substrate solution, and incubating for 15 minutes at 37 ℃; add 50. Mu.L of stop solution and immediately measure OD at 450nm wavelength; and calculating a result.

Kidney index: after completion of administration, the kidneys were removed, weighed and the kidney index (kidney index=kidney weight (g)/body weight (g) ×100%) was calculated, and the results are shown in fig. 5B.

5.4. Experimental results

According to the urine protein detection result, the urine protein content of the CKD model mice is obviously improved before starting administration, and the average urine protein content is more than 2 times higher than that of negative control mice, so that the CKD model is proved to be successful to be manufactured. After 3 weeks of continuous injection of sDSS1 protein, urine protein levels were found to be significantly reduced in the dosing group, compared to the CKD model group (FIG. 5A), indicating that sDSS1 protein effectively restored kidney function in the CKD model rats. Continuing to examine the kidney index of the rats, the results showed that the kidney index of the rats in the group to which the sDSS1 protein was administered was substantially recovered to the level of the rats in the control group, which was significantly better than that of the rats in the CKD model group (FIG. 5B). These results demonstrate that sDSS1 can effectively improve disease symptoms in CKD model rats.

Sequence listing

<110> Shanghai Qingdao biological medicine technologies Co., ltd

Application of <120> protein in preparing medicament for preventing and treating kidney disease

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 89

<212> PRT

<213> human (Homosapiens)

<400> 1

Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp

1 5 10 15

Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu

20 25 30

Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val

35 40 45

Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile

50 55 60

Leu Val Cys Glu Thr Pro Tyr Gly Cys Tyr Val Leu His Gln Lys Gly

65 70 75 80

Arg Met Cys Ser Ala Phe Leu Cys Cys

85

<210> 2

<211> 89

<212> PRT

<213> chimpanzee (Pantroglymes)

<400> 2

Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp

1 5 10 15

Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu

20 25 30

Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val

35 40 45

Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile

50 55 60

Leu Val Cys Glu Thr Pro Tyr Gly Cys Tyr Val Leu His Gln Lys Gly

65 70 75 80

Arg Met Cys Ser Ala Phe Leu Cys Cys

85

<210> 3

<211> 89

<212> PRT

<213> bonbonobe (Panpanicut)

<400> 3

Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp

1 5 10 15

Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu

20 25 30

Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val

35 40 45

Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile

50 55 60

Leu Val Cys Glu Thr Pro Tyr Gly Cys Tyr Val Leu His Gln Lys Gly

65 70 75 80

Arg Met Cys Ser Ala Phe Leu Cys Cys

85

<210> 4

<211> 89

<212> PRT

<213> gorilla (nomascusleuceogenys)

<400> 4

Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp

1 5 10 15

Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu

20 25 30

Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val

35 40 45

Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Val

50 55 60

Leu Val Cys Glu Thr Pro Tyr Gly Cys Tyr Val Leu His Gln Lys Glu

65 70 75 80

Arg Met Cys Ser Ala Phe Leu Cys Cys

85

<210> 5

<211> 89

<212> PRT

<213> Red chimpanzee (Gorillagorilla)

<400> 5

Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp

1 5 10 15

Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu

20 25 30

Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val

35 40 45

Glu Asp Asp Phe Ser Asn Gln Leu Arg Val Thr Val Leu Leu Met Ile

50 55 60

Leu Val Cys Glu Thr Leu Tyr Gly Cys Tyr Val Leu His Gln Lys Gly

65 70 75 80

Arg Met Cys Ser Ala Phe Leu Cys Cys

85

<210> 6

<211> 89

<212> PRT

<213> white cheek gibbon (Pongoabelii)

<400> 6

Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp

1 5 10 15

Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu

20 25 30

Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val

35 40 45

Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Ile Leu Leu Met Ile

50 55 60

Leu Val Cys Glu Thr Pro Tyr Gly Cys Tyr Val Leu His Gln Lys Gly

65 70 75 80

Arg Met Cys Ser Ala Phe Leu Cys Cys

85

<210> 7

<211> 89

<212> PRT

<213> Sichuan golden monkey (Rhinopithecus roxellana)

<400> 7

Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp

1 5 10 15

Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu

20 25 30

Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val

35 40 45

Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile

50 55 60

Lys Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly

65 70 75 80

Arg Met Cys Ser Ala Phe Leu Cys Cys

85

<210> 8

<211> 89

<212> PRT

<213> rhesus monkey (Macacamulatata)

<400> 8

Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp

1 5 10 15

Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu

20 25 30

Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val

35 40 45

Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile

50 55 60

Lys Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly

65 70 75 80

Arg Met Cys Ser Ala Phe Leu Cys Cys

85

<210> 9

<211> 89

<212> PRT

<213> Yunnan golden monkey (Rhinophitecus bieti)

<400> 9

Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp

1 5 10 15

Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu

20 25 30

Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val

35 40 45

Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile

50 55 60

Lys Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly

65 70 75 80

Arg Met Cys Ile Ala Phe Leu Cys Cys

85

<210> 10

<211> 89

<212> PRT

<213> Baboon (Colobusangolensis)

<400> 10

Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp

1 5 10 15

Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu

20 25 30

Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val

35 40 45

Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Lys

50 55 60

Lys Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly

65 70 75 80

Arg Met Cys Ser Ala Phe Leu Cys Cys

85

<210> 11

<211> 89

<212> PRT

<213> Angola warts monkey (Cercocebustics)

<400> 11

Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp

1 5 10 15

Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu

20 25 30

Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val

35 40 45

Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile

50 55 60

Lys Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly

65 70 75 80

Arg Met Cys Ser Ala Phe Leu Cys Cys

85

<210> 12

<211> 89

<212> PRT

<213> white-top white-eyebrow monkey (M. Leucophaeus)

<400> 12

Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp

1 5 10 15

Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu

20 25 30

Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val

35 40 45

Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile

50 55 60

Lys Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly

65 70 75 80

Arg Met Cys Ser Ala Phe Leu Cys Cys

85

<210> 13

<211> 89

<212> PRT

<213> Bidentis (Macacanmestrina)

<400> 13

Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp

1 5 10 15

Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu

20 25 30

Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val

35 40 45

Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile

50 55 60

Lys Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly

65 70 75 80

Arg Met Cys Ser Ala Phe Leu Cys Cys

85

<210> 14

<211> 89

<212> PRT

<213> Dolphin tail monkey (Papioanusis)

<400> 14

Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp

1 5 10 15

Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu

20 25 30

Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val

35 40 45

Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile

50 55 60

Lys Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly

65 70 75 80

Arg Met Cys Ser Ala Phe Leu Cys Cys

85

Claims

1. Use of a sDSS1 protein in the manufacture of a medicament for treating chronic kidney disease, wherein the sDSS1 protein is a basic protein formed by any one of the sDSS1 protein sequences of human, chimpanzee, bonobo, gorilla, red chimpanzee, buckyape, golden monkey, rhesus, yunnan golden monkey, eastern baboon, angora wart, white top white brow, spanish, and ragtail monkey, wherein the amino acid sequence of human sDSS1 is as set forth in SEQ ID NO:1, the amino acid sequence of chimpanzee sDSS1 is shown in SEQ ID NO:2, the amino acid sequence of the bonobo sDSS1 is shown in SEQ ID NO:3, the amino acid sequence of gorilla sDSS1 is shown in SEQ ID NO:4, the amino acid sequence of the chimpanzee sDSS1 is shown in SEQ ID NO:5, the amino acid sequence of the ape of the cheek gibbon sDSS1 is shown in SEQ ID NO:6, the amino acid sequence of the Sichuan golden monkey sDSS1 is shown as SEQ ID NO:7, the amino acid sequence of the rhesus sDSS1 is shown as SEQ ID NO:8, the amino acid sequence of the Yunnan golden monkey sDSS1 is shown as SEQ ID NO:9, the amino acid sequence of the baboon sDSS1 is shown in SEQ ID NO:10, the amino acid sequence of the angora wart monkey sDSS1 is shown in SEQ ID NO:11, the amino acid sequence of the white top white brow sDSS1 is shown in SEQ ID NO:12, the amino acid sequence of the spanish baboon sDSS1 is shown as SEQ ID NO:13, the amino acid sequence of the sDSS1 of the pigtail monkey is shown in SEQ ID NO:14.

2. the use according to claim 1, wherein the chronic kidney disease is chronic kidney disease stage 1, chronic kidney disease stage 2, chronic kidney disease stage 3, chronic kidney disease stage 4 or chronic kidney disease stage 5, wherein GFR of chronic kidney disease stage 1 is not less than 90mL/min/1.73m ² The GFR of the 2 nd stage chronic kidney disease is 60-89 mL/min/1.73m ² The GFR of the 3 rd stage chronic kidney disease is 30-59 mL/min/1.73m ² The GFR of the 4 th stage chronic kidney disease is 15-29 mL/min/1.73m ² GFR for stage 5 chronic kidney disease<15mL/min/1.73m ² 。

3. The use according to claim 1, wherein said chronic kidney disease is characterized by increased AOPP.

4. The use according to claim 3, wherein the chronic kidney disease is diabetic nephropathy, igA nephropathy, membranous nephropathy, lupus nephritis, polycystic kidney disease or chronic glomerulonephritis.

5. The use according to any one of claims 1-4, wherein the sDSS1 protein forms part or all of a complex with a pharmaceutically applicable pharmaceutical carrier.

6. The use according to claim 5, wherein the pharmaceutical carrier of the complex comprises one or more of enteric coated formulations, capsules, microspheres/vesicles, liposomes, microemulsions, multiple emulsions, nanoparticles, magnetic particles, gelatin and gels.

7. The use according to any one of claims 1 to 4, wherein said sDSS1 protein is a sDSS1 protein obtained by introducing a nucleotide sequence encoding said sDSS1 protein into the body via an expression system and expressing.

8. The use of claim 7, wherein the expression system is a eukaryotic expression plasmid vector, adenovirus, adeno-associated virus, lentivirus, retrovirus, baculovirus, herpes virus, pseudorabies virus, ZFN gene editing technology, TALEN gene editing technology, CRISPR/Cas gene editing technology or other medically useful gene editing technology or viral vector.