WO2018006750A1 - 一种新型天然蛋白及其应用 - Google Patents
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- C07K2319/00—Fusion polypeptide
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- C12N2510/00—Genetically modified cells
Definitions
- the body In normal physiological activities, the body produces a large amount of junk protein, including oxidized proteins, glycosylated proteins and some abnormally cut proteins (polypeptides).
- the body retains a variety of mechanisms to eliminate junk protein to maintain normal physiological functions, but aging or disease can lead to the transition of junk protein production or the body's ability to remove junk protein, thereby causing a large amount of junk protein accumulation.
- Abnormal accumulation of junk protein in or outside the cell is a key mechanism leading to a range of diseases, including chronic kidney disease, Alzheimer's disease (AD), Huntington's disease, diabetes complications, etc. [1-5] .
- the accumulation of oxidized proteins, glycated proteins or other junk proteins in the circulatory system is also one of the important causes of aging [6-7] .
- sDSS1 secret DSS1
- sDSS1 secret DSS1
- sDSS1 protein to the culture medium can shield cytotoxicity caused by oxidized proteins, A ⁇ oligomers, amylin oligomers, glycosylated proteins, and protect cell viability. Therefore, we judged that this novel protein sDSS1 has the potential to be used as a drug for the prevention and treatment of diseases caused by oxidized proteins, glycosylated proteins, A ⁇ , amylin and other similarly characteristic pathogenic proteins.
- the anthropoids include chimpanzees, bonobos, gorillas, orangutans, white-cheeked gibbons, golden monkeys, rhesus monkeys, East African baboons, Angora simian monkeys, white-headed white-browed macaques, sneaky owls, and porpoises.
- the amino acid sequence of the natural protein sDSS1 of the chimpanzee is SEQ ID NO: 5
- the amino acid sequence of the natural protein sDSS1 of the chimpanzee is SEQ ID NO: 6
- the amino acid sequence of the natural protein sDSS1 of gorilla is SEQ ID NO: 7.
- the amino acid sequence of the native protein sDSS1 of the orangutan is SEQ ID NO: 8
- the amino acid sequence of the native protein sDSS1 of the white-cheeked gibbon is SEQ ID NO: 9
- the amino acid sequence of the native protein sDSS1 of Rhinopithecus roxie is SEQ ID NO: 10
- the amino acid sequence of the natural protein sDSS1 of rhesus monkey is SEQ ID NO: 11
- the amino acid sequence of the native protein sDSS1 of East African scorpion is SEQ ID NO: 12
- the amino acid sequence of the natural protein sDSS1 of Angora simian is SEQ ID NO: 13.
- amino acid classification is as follows:
- Neutral amino acids threonine, glycine, serine, histidine, glutamine;
- Acidic amino acids glutamic acid, aspartic acid, proline, asparagine.
- the sDSS1 protein of the human scorpion animal comprises a C-terminal amino acid of the formula:
- X1 is a neutral amino acid
- X2 is a hydrophobic amino acid
- X3, X4 are each a hydrophobic amino acid
- X5 is a hydrophobic amino acid
- X6 is a hydrophobic amino acid
- X7 is a hydrophobic amino acid
- X8 is a hydrophobic amino acid
- X9 is a hydrophobic amino acid
- X10 is an acidic amino acid; It is a neutral amino acid
- X12 is a hydrophobic amino acid
- X13 is a hydrophobic amino acid
- X14 is a neutral amino acid
- X15 is a hydrophobic amino acid
- X16 is a hydrophobic amino acid
- X17 is a hydrophobic amino acid
- X18 is a hydrophobic amino acid
- X19 is a basic amino acid
- X20 is an acidic Amino acid
- X21 is a basic amino acid
- X22 is
- a fusion protein comprising the complete sequence or partial sequence of the novel native protein sDSS1 described in the above scheme and the polypeptide sequence described in the above scheme.
- the fusion protein is a protein complex formed by linking the protein itself, a carrier protein, an antibody or any other amino acid sequence.
- a complex comprising the complete sequence or partial sequence of the novel natural protein sDSS1 described in the above scheme, the polypeptide sequence described in the above scheme, and some or all of the fusion proteins described in the above scheme.
- the cell is a stem cell, a precursor cell or an adult cell of any mammal.
- the mammal is a human, an orangutan, a monkey, a horse, a cow, a sheep, a pig, a donkey, a dog, a rabbit, a cat, a rat or a mouse.
- the cells comprise embryonic stem cells, induced pluripotent stem cells, or stem cells derived from primary culture, pluripotent or pluripotent stem cells differentiated from mother cells.
- the expression system comprises a eukaryotic expression plasmid vector, an adenovirus, a lentivirus, a retrovirus, a CRISPR/Cas technology, and other viable gene editing techniques.
- the organism is a human, an orangutan, a monkey, a horse, a cow, a sheep, a pig, a donkey, a dog, a rabbit, a cat, a rat, a mouse, a chicken, a duck or a goose.
- a drug comprising the protein of the above-mentioned protocol or the polypeptide sequence of the above-mentioned protocol as a main target, and after administration, may affect the expression level of the protein of the above-mentioned protocol or the polypeptide sequence of the above-mentioned protocol in the body after administration.
- the drug is a chemical small molecule drug, a protein/polypeptide drug or a nucleic acid drug, a nano drug.
- the nucleic acid drug comprises one or more of siRNA, microRNA, antisense oligonucleotide, triple-stranded DNA and ribozyme.
- a method of producing a protein comprising the steps of:
- a nucleotide sequence encoding the polypeptide of the above scheme or the polypeptide sequence described in the above scheme is inserted into a plasmid and introduced into a bacterial or yeast cell, or the protein described in the above scheme may be encoded or The nucleotide sequence of the polypeptide sequence described in the above scheme is inserted into the genome of an insect cell or a mammalian cell;
- the pathogenic protein/polypeptide is an oxidized, glycosylated protein product, amyloid precursor protein and its splicing, amylin and its splicing, and other oxidized proteins, glycosylation A pathogenic protein/polypeptide similar in character to proteins, amyloids, and amylin.
- the prevention includes one or more of genetic modification, nucleic acid introduction, drug injection/administration, cell transplantation, and tissue transplantation.
- the present invention has been verified by molecular experiments that the sDSS1 protein can form a polymer with an oxidized protein in serum or an oxidized protein in a buffer, or bind to an A ⁇ protein to reduce A ⁇ oligomer formation.
- the sDSS1 gene is a new subtype of the DSS1 gene.
- the human DSS1 gene cDNA (NM_006304.1, 509 bp) and the human sDSS1 gene cDNA (AK309241.1, 1195 bp) are shown to have overlapping regions, and the overlapping region nucleic acid sequence is The analysis encodes an N-terminal 58 amino acid sequence.
- FIG. 5A - Figure 5D Biochemical experiments and cell experiments demonstrate that sDSS1 protein can bind to oxidized proteins and screen Oxidation of oxidized proteins.
- Figure 5A The sDSS1 protein was mixed with 0.72 ⁇ g of purified sDSS1 protein mixed with different proportions of serum proteins, and the sDSS1 protein was detected by V5 binding protein (V5-HRP). The results showed that the sDSS1 protein forms a macromolecular protein complex with the oxidized protein of serum.
- Figure 5B Advanced oxidation protein (AOPP, 200 ⁇ g/mL) was incubated with different concentrations of purified sDSS1 protein at 4 °C overnight.
- AOPP Advanced oxidation protein
- NCBI National Center of Biotechnology Information
- NCBI Nucleotide blast tool
- NCBI Align sequences nucleotide blast tool
- SIB Bioinformatics Resource Portal SIB Bioinformatics Resource Portal
- Clustal X2.1 Multiple Sequence Alignment (EMBL-EBI); SecretomeP 2.0 (CBS prediction service); WoLF PSORT II.
- the secretory protein analysis software Wolf PSORT and SecretomeP 2.0 were used to analyze the amino acid sequence of sDSS1 protein.
- the predicted results showed that sDSS1 protein was localized to the outside of the cell, similar to many identified secreted proteins, and was predicted to be a secreted protein (Table 2). ).
- the signal peptide cleavage site of sDSS1 protein is located between amino acids 58-59.
- the sDSS1 protein is a type of secreted protein that can be synthesized in cells and secreted outside the cell.
- the C-terminal 31 amino acid sequence of sDSS1 protein functions as a signal peptide.
- the positive cloned E. coli strain was selected, expanded, and the bacterial cells were stimulated to express the target protein with I PTG in the log phase of bacterial growth.
- the bacteria were lysed and the expression level of the target protein was examined.
- the results showed that the level of sDSS1 protein expressed by the bacterial cells was significantly increased after I PTG stimulation, and a distinct protein band was observed on the gel map (Fig. 4A).
- the target protein in the concentrate is greatly concentrated (b channel), and the content of the heteroprotein is reduced; further purification can obtain the highly purified sDSS1 protein (a channel) (Fig. 4B), can be used in subsequent biological experiments.
- the purified sDSS1 protein was quantified by BCA protein at a final concentration of 0.72 mg/ml, and stored at 4 degrees for use.
- Oxidized serum reacted with sDSS1 protein Fresh human blood was centrifuged at 3500 g for 30 minutes, and the upper serum was taken for subsequent experiments. In the experiment, 10 ⁇ L of sDSS1 protein solution (0.72 mg/mL) was mixed with 10, 20, 50 and 100 ⁇ l of oxidized serum, respectively. The ratio of sDSS1 to serum protein was approximately 1:100, 1:200, 1:500,1. : 1000, additionally added 20 ⁇ M Fenton reagent (FeSO4 and H2O2 were mixed in a mass ratio of 1:1), mixed uniformly, and then incubated at 4 ° C overnight in the dark.
- Fenton reagent FeSO4 and H2O2 were mixed in a mass ratio of 1:1
- Oxidized fetal bovine serum and advanced oxidation protein Preparation 10 mL fetal bovine serum was added to 10 mM NaClO for 1 hour, and the oxidized serum was continuously dialyzed against PBS solution for 24 hours in a 3000 Da dialysis bag, and the solution was changed every 8 hours during dialysis. 1 mM Vitamin C (Vc) was added to the serum solution to completely remove the participating oxidants.
- the BCA protein quantification method detects protein concentration. Two methods were used to detect the content of oxidized protein.
- the chloramine T method was used to measure the double tyrosine value in the oxidized serum to 75.31 ⁇ M/mg protein (15.05 ⁇ mol/mg protein in untreated serum), and the carbonyl content was detected by dinitrophenylhydrazine method. It was 16.33 nmol/mg protein (untreated serum was 13.68 nmol/mg protein).
- AOPP advanced oxidation protein
- Advanced oxidation protein and sDSS1 protein reaction 30 ⁇ g AOPP protein (200 ⁇ g/mL) was added to 150 ⁇ L reaction system, and 15 ⁇ g (100 ⁇ g/mL), 30 ⁇ g (200 ⁇ g/mL), 60 ⁇ g (400 ⁇ g/mL) sDSS1 protein were added. The excess volume was replenished with sterile PBS solution. The solution was mixed and reacted overnight at 4 ° C. The sample after completion of the reaction was added with 5x loading buffer, heat-treated at 100 ° C for 10 minutes, and the treated sample was separated by SDS-PAGE and stained with Coomassie blue staining.
- Serum contains a lot of protein, mainly serum albumin. Under the action of Fenton's reagent, the protein in the serum is oxidized, and the oxidation product can react with sDSS1 to form a complex.
- sDSS1 protein monomer was observed, and no significant protein aggregation occurred; in the experimental group, incubation with serum resulted in the formation of a large number of high molecular weight protein complexes which could not be separated by SDS-PAGE (Fig. 5A).
- the results indicate that the sDSS1 protein can bind to oxidized proteins in serum.
- the addition of 10% oxidized protein significantly inhibited cell proliferation and cell viability compared with the control sera.
- the cytotoxicity of the oxidized protein was recovered by containing 20 ⁇ g/mL sDSS1 protein in the medium (Fig. 5B).
- sDSS1 and AOPP protein combine to form a complex, these complexes can not be separated by SDS-PAGE.
- concentration of sDSS1 protein in the reaction system increased, the complex significantly increased (Fig. 5C).
- AOPP caused significant cytotoxicity to cells and decreased cell viability, while equal concentrations of sDSS1 completely blocked the cytotoxicity of AOPP (Fig. 5D).
- sDSS1 protects cells from the cytotoxicity of oxidized serum or AOPP.
- the sDSS1 protein has an oxidized protein reaction, and both proteins can bind tightly to the oxidized protein. This binding ability can resist high concentrations of SDS, which is suspected to be covalent interactions.
- the difference is that the process by which DSS1 binds to oxidized proteins requires the assistance of an ATPase [Zhang et al, 2014], and our evidence suggests that sDSS1 binds tightly to oxidized proteins without the need to add ATP, ie, does not require ATPase involvement.
- Example 5 sDSS1 protein reduces A ⁇ oligomer formation and reduces cytotoxicity of A ⁇ oligomers
- a ⁇ and sDSS1 protein reaction A ⁇ protein (Human, 1-42) lyophilized powder was provided by Suzhou Qiang Yao Biotechnology Co., Ltd. 2 mg of A ⁇ lyophilized powder was dissolved in 20 ⁇ L of DMSO and further diluted to 2 mg/mL with PBS. Store at -20 degrees. The reaction system was firstly added with 10 ⁇ g of A ⁇ and sDSS1 protein in a 300 ⁇ L PBS solution at a mass ratio of 1:1, 1:5, 1:10, mixed, and incubated at 4 ° C overnight. 5 ⁇ loading buffer was added to the incubated reaction and treated at 100 ° C for 10 minutes for Western blot analysis.
- Cell line culture Human neuroblastoma cells (SH-SY5Y) were grown in basal medium DMEM supplemented with 10% fetal bovine serum; cells were passaged every two days.
- amylin protein pretreatment amylin protein (Human) lyophilized powder was provided by Suzhou Qiang Yao Biotechnology Co., Ltd. 2 mg of amylin lyophilized powder was dissolved in 2 mg/mL with 10 mM sodium acetate solution (pH 5.5), and stored frozen at -20 degrees. The amylin stock solution was diluted to 1 mg/mL with a basal medium (pH 7.2), and the oligomer formed after the amylin dilution was incubated at 4 ° C for 48 hours was used for the cell experiment. The concentration of amylin in subsequent experiments was always labeled as the protein concentration before incubation.
- Adding 10 ⁇ M of incubated amylin protein to the cell culture medium can cause significant cytotoxicity, and the addition of sDSS1 protein can block the cytotoxicity caused by amylin oligomer, and the cell viability is even higher than that of the control group (Fig. 7), indicating that the sDSS1 protein can be effective. Block the cytotoxicity of amylin protein.
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Abstract
提供了一种高等灵长类动物的分泌型蛋白sDSS1，其可以在人血清和脑脊液中检测到。sDSS1蛋白在非酶促条件下可与氧化蛋白结合形成聚合物或与Aβ多肽结合降低Aβ寡聚物形成，在培养基中可以屏蔽氧化蛋白、Aβ寡聚物、Amylin寡聚物、糖基化蛋白引起的细胞毒性。sDSS1蛋白可以显著延长早衰型小鼠生存期。该蛋白可用于预防和治疗氧化蛋白、糖化蛋白、Aβ蛋白累积、Amylin蛋白累积或其他相似特征致病蛋白过渡生成或积累引起的疾病。
Description
本发明涉及一种新型天然蛋白及其应用，该蛋白可用于预防和治疗因垃圾蛋白过渡生成或过渡积累导致的疾病。
在正常的生理活动中，机体产生大量垃圾蛋白，其中包括氧化蛋白、糖基化蛋白和一些异常剪切蛋白(多肽)等。机体保留多种清除垃圾蛋白的机制以维持正常的生理功能，但是衰老或疾病会导致垃圾蛋白过渡生成或机体清除垃圾蛋白的能力下降，由此引起大量垃圾蛋白累积。垃圾蛋白在细胞内或细胞外异常积累是导致一系列疾病的关键机制，典型疾病包括慢性肾病、阿尔兹海默症(Alzheimer's disease，AD)、亨廷顿氏症、糖尿病并发症等[1-5]。循环系统中氧化蛋白、糖化蛋白或其他垃圾蛋白的积累还是机体衰老的重要原因之一[6-7]。研究证明，血清中晚期氧化蛋白(Advanced oxidation protein，AOPP)损伤肾细胞，是慢性肾病的主要发病机制；血清中AOPP也会诱导胰岛β细胞的程序性凋亡[3,8]。β淀粉样蛋白假说认为Aβ蛋白在组织中产生和清除失衡导致的毒性蛋白进行性堆积，由此引起的突触功能紊乱、神经元死亡是导致AD的主要原因[9]。Amylin蛋白不仅在部分糖尿病患者的胰岛组织发生异常聚集，也会在脑组织的斑块中存在，与糖尿病和神经退行性疾病的进展密切相关[10,11]。基于这些研究结果，在一些疾病模型上，例如利用抗体[12]、多肽药物[13]或小分子药物[14]阻断AD模型动物中Aβ聚集或生成可以减少神经组织斑块形成，提高动物认知水平。这些结果说明，通过药物抑制这些致病蛋白的生成、聚集或者促进致病蛋白清除，减少致病蛋白累积，是预防或治疗这些疾病的重要方法。
前期的研究表明，当细胞内发生氧化应激时，DSS1(deleted split hand/split foot 1)蛋白作为一种在真核生物中高度保守的小蛋白，能够在酶促反应并消耗ATP的条件下被共价修饰到氧化蛋白上，这种修饰将介导氧化蛋白在细胞内的降解[15]。DSS1基因敲除导致细胞死亡；高表达DSS1蛋白的细胞对
氧化应激或抗肿瘤药物诱导的细胞凋亡表现显著的抗性[16]。这些结果显示DSS1蛋白在细胞清除氧化蛋白过程中的重要作用，对细胞的生存非常关键。
上述中涉及的参考文献如下：
1.Dobson CM(1999)Proteinmisfolding,evolution and disease.Trends Biochem Sci 24:329–332.
2.Liang M,Wang J,Xie C,Yang Y,Tian JW,Xue YM,Hou FF(2014)Increased plasma advanced oxidation protein products is an early marker of endothelial dysfunction in type 2 diabetes patients without albuminuria 2.J Diabetes 6(5):417-26.
3.Cao W,Hou FF,Nie J(2014)AOPPs and the progression of kidney disease.Kidney Int Suppl(2011)4(1):102-106.
4.Sadigh-Eteghad S,Sabermarouf B,Majdi A,Talebi M,Farhoudi M,Mahmoudi J(2015)Amyloid-beta:a crucial factor in Alzheimer's disease.Med Princ Pract 24(1):1-10.
5.Choe YJ,Park SH,Hassemer T,
rner R,Vincenz-Donnelly L,Hayer-Hartl M,Hartl FU(2016)Failure of RQC machinery causes protein aggregation and proteotoxic stress.Nature 531(7593):191-5.
6.Ott C,Grune T(2014)Protein oxidation and proteolytic signalling in aging.Curr Pharm Des 20(18):3040-51.
7.Simm A,Müller B,Nass N,Hofmann B,Bushnaq H,Silber RE,Bartling B(2015)Protein glycation-Between tissue aging and protection.Exp Gerontol 68:71-5.
8.Liang M,Li A,Lou A,Zhang X,Chen Y,Yang L,Li Y,Yang S,Hou FF(2017)Advanced oxidation protein products promote NADPH oxidase-dependent β-cell destruction and dysfunction through the Bcl-2/Bax apoptotic pathway.Lab Invest 24.[Epub ahead of print].
9.Zhao LN,Long H,Mu Y,Chew LY(2012)The toxicity of amyloid βoligomers.Int J Mol Sci 13(6):7303-27.
10.Fernández MS (2014) Human IAPP amyloidogenic properties and pancreatic β-cell death. Cell Calcium 56(5):416-27.
11.Lim YA,Rhein V,Baysang G,Meier F,Poljak A,Raftery MJ, Guilhaus M,Ittner LM, Eckert A,
J (2010) Abeta and human amylin share a common toxicity pathway via mitochondrial dysfunction. Proteomics 10(8):1621–33.
12.Winblad B, Andreasen N,Minthon L,Floesser A,Imbert G,Dumortier T,Maguire RP,Blennow K,Lundmark J,Staufenbiel M,Orgogozo JM,Graf A(2012)Safety,tolerability,and antibody response of active A βimmunotherapy with CAD106 in patients with Alzheimer's disease:randomised,double-blind,placebo-controlled,first-in-human study.Lancet Neurol 11(7):597-604.
13.Chang L,Cui W, Yang Y,Xu S,Zhou W,Fu H,Hu S,Mak S,Hu J,Wang Q,Ma VP,Choi TC, Ma ED,Tao L,Pang Y,Rowan MJ,Anwyl R,Han Y,Wang Q(2015)Protection against β-amyloid-induced synaptic and memory impairments viaaltering β-amyloid assembly by bis(heptyl)-cognitin.Sci Rep 5:10256.
14.Kim HY,Kim HV,Jo S,Lee CJ,Choi SY,Kim DJ,Kim Y(2015)EPPS rescues hippocampus-dependent cognitive deficits in APP/PS1 mice by disaggregation of amyloid-β oligomers and plaques. Nat Commun 6:8997.
15.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 proteinmodification targets proteins induced by oxidative stress,and facilitates their degradation in cells.Protein Cell 5(2):124-40.
16.Rezano A, Kuwahara K, Yamamoto-Ibusuki M,Kitabatake M,Moolthiya P,Phimsen S,Suda T,Tone S,Yamamoto Y,Iwase H,Sakaguchi N(2013)Breast cancers with high DSS1 expression that potentially maintains BRCA2 stability have poor prognosis in the relapse-free survival.BMC Cancer 13:562.
发明内容
在最新的研究中，我们(发明人)发现在高等灵长类(类人猿亚科)基因组中存在一类新亚型DSS1蛋白，命名为分泌型DSS1蛋白(secretary DSS1；sDSS1)。sDSS1是首次发现的一种DSS1蛋白亚型，在序列和性质和功能上与DSS1高度相似，但是可以分泌到血液和脑脊液中，性质更活泼，不需要通过耗能的酶促反应就可以与血清或缓冲液中的氧化蛋白形成聚合体，或者与Aβ蛋白结合并抑制Aβ寡聚体的形成。sDSS1蛋白添加到培养基中可以屏蔽氧化蛋白、Aβ寡聚物、amylin寡聚物、糖基化蛋白引起的细胞毒性，保护细胞活力。因而我们判断这种新型蛋白sDSS1有潜力成为药物用于预防和治疗氧化蛋白、糖基化蛋白、Aβ、amylin以及其他相似特征致病蛋白导致的疾病。
具体的技术方案如下：
一种新型天然蛋白sDSS1，包括人在内的类人猿亚目动物存在天然蛋白sDSS1，其中人源天然蛋白sDSS1的氨基酸序列如SEQ ID NO：1。
优选地，所述类人猿亚目动物还包括黑猩猩、倭黑猩猩、大猩猩、红毛猩猩、白颊长臂猿、川金丝猴、恒河猴、东非狒狒、安哥拉疣猴、白顶白眉猴、鬼狒、豚尾猴；其中黑猩猩的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：5，倭黑猩猩的天然蛋白sDSS1的氨基酸序列如SEQ I D NO：6，大猩猩的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：7，红毛猩猩的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：8，白颊长臂猿的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：9，川金丝猴的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：10,恒河猴的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：11，东非狒狒的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：12，安哥拉疣猴的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：13，白顶白眉猴的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：14，鬼狒的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：15，豚尾猴的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：16。
优选地，所述天然蛋白sDSS1分为N端58个氨基酸序列和C端31个氨基酸序列，其中人源sDSS1蛋白N端58个氨基酸序列如SEQ ID NO：3，人源sDSS1蛋白C端的31氨基酸序列如SEQ ID NO：2；两段序列的特征分别在于，N端58个氨基酸序列包含3个或3个以上连续排列的酸性氨基酸序列，每个连续排列
的酸性氨基酸序列片段包含的酸性氨基酸数目不高于10个，不同的连续排列的酸性氨基酸序列之间的间隔长度不超过4个氨基酸，每个这样的间隔中存在至少一个疏水氨基酸，pH值不高于4.5，N端58个氨基酸组成的序列酸性氨基酸数量不低于10个；以N端58个氨基酸序列为基础的第58位氨基酸以后的C端氨基酸序列，总体偏疏水性质，C端31个氨基酸组成的氨基酸序列中疏水氨基酸数量不低于10个；
氨基酸分类的界定如下：
疏水氨基酸:丙氨酸、异亮氨酸、亮氨酸、缬氨酸、半胱氨酸、苯丙氨酸，甲硫氨酸、色氨酸、酪氨酸；
中性氨基酸：苏氨酸、甘氨酸，丝氨酸、组氨酸、谷氨酰胺；
酸性氨基酸：谷氨酸、天冬氨酸、脯氨酸、天冬酰胺。
碱性氨基酸:精氨酸，赖氨酸。
优选地，类人猿亚目动物的sDSS1蛋白包含的C端氨基酸通式为：
X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19X20X21X22X23X24X25X26X27X28X29X30X31；
X1为中性氨基酸；X2为疏水氨基酸；X3，X4分别为疏水氨基酸；X5为疏水氨基酸；X6为疏水氨基酸；X7为疏水氨基酸；X8为疏水氨基酸；X9为疏水氨基酸；X10为酸性氨基酸；X11为中性氨基酸；X12为疏水氨基酸；X13为疏水氨基酸；X14为中性氨基酸；X15为疏水氨基酸；X16为疏水氨基酸；X17为疏水氨基酸；X18为疏水氨基酸；X19为碱性氨基酸；X20为酸性氨基酸；X21为碱性氨基酸；X22为中性氨基酸；X23为碱性氨基酸；X24为疏水氨基酸；X25为疏水氨基酸；X26为中性氨基酸；X27为疏水氨基酸；X28为疏水氨基酸；X29为疏水氨基酸；X30为疏水氨基酸；X31为疏水氨基酸；
与C端31个氨基酸序列中有40％或40％以上同源性氨基酸序列，该序列与人源sDSS1蛋白C端的氨基酸序列在多肽中的性质和发挥的功能相同或相似。
一种多肽序列，以上述方案所述的新型天然蛋白sDSS1的N端58个氨基酸序列和C端31个氨基酸序列为基础构建的多肽序列，其是：
1)该多肽序列的N端58个氨基酸组成的序列有40％或40％以上相似性，C端氨基酸序列有40％或40％以上相似性的蛋白，该蛋白与人源天然蛋白的性质和功能相同或相似；或者，
2)以人源天然蛋白中多肽序列的N端58个氨基酸组成的序列，或以其40％或40％以上相似序列为基础，在C端或N端融合其他氨基酸序列，用于融合的氨基酸序列能实现与人源天然蛋白中C端31个氨基酸序列相同或相似的性质并且发挥相同或相似的功能，修饰后的蛋白能发挥与人源天然蛋白sDSS1相同或相似功能的蛋白；或者，
3)用上述方案所述的新型天然蛋白sDSS1中的C端多肽序列融合其他天然或非天然多肽序列得到的蛋白。
一种融合蛋白，所述融合蛋白包括上述方案所述的新型天然蛋白sDSS1的完整序列或部分序列和上述方案所述的多肽序列。
优选地，所述融合蛋白是与该蛋白自身、载体蛋白、抗体或其他任意氨基酸序列连接形成的蛋白复合体。
一种复合体，所述复合体包括上述方案所述的新型天然蛋白sDSS1的完整序列或部分序列、上述方案所述的多肽序列、上述方案所述的融合蛋白中的部分或全部复合。
优选地，所述复合体是多肽/蛋白与医药上可应用的药物载体连接形成的复合体。
优选地，所述载体包含微球/囊、脂质体、微乳液、纳米颗粒、磁颗粒和凝胶中的一种或一种以上。
一种核苷酸序列，所述核苷酸序列可以编码上述方案所述的蛋白或上述方案所述的多肽序列。
优选地，所述核苷酸序列包括DNA序列和RNA序列。
一种细胞，所述细胞表达上述方案所述的蛋白或上述方案所述的多肽序列。
优选地，所述细胞是任意一种哺乳动物的干细胞、前体细胞或成体细胞。
优选地，所述哺乳动物是人、猩猩、猴、马、牛、羊、猪、驴、狗、兔、猫、大鼠或小鼠。
优选地，所述细胞包括胚胎干细胞、诱导多能干细胞，或者来源于原代培养的干细胞、由母细胞分化得到的多能或单能干细胞。
一种表达体系，其把编码上述方案所述蛋白或上述方案所述的多肽序列的核苷酸序列导入生物体，并在生物体中表达上述方案所述的蛋白或上述方案所述的多肽序列。
优选地，所述表达体系包括真核表达质粒载体、腺病毒、慢病毒、逆转录病毒、CRISPR/Cas技术及其他可行的基因编辑技术。
优选地，所述生物体是人、猩猩、猴、马、牛、羊、猪、驴、狗、兔、猫、大鼠、小鼠、鸡、鸭或鹅。
一种药物，所述药物以上述方案所述蛋白或上述方案所述的多肽序列为主要靶点，并在施用后可以影响机体内上述方案所述蛋白或上述方案所述的多肽序列的表达水平。
优选地，所述药物是化学小分子药物、蛋白/多肽药物或核酸药物、纳米药物。
优选地，所述的核酸药物包括siRNA，microRNA，反义寡核苷酸，三链DNA和核酶的一种或一种以上。
一种蛋白的生产方法，所述方法包括以下步骤：
S1.表达载体构建：将可编码上述方案所述蛋白或上述方案所述的多肽序列的核苷酸序列插入质粒中并导入到细菌或酵母菌细胞中，或将可编码上述方案所述蛋白或上述方案所述的多肽序列的核苷酸序列插入昆虫细胞、哺乳动物细胞基因组中；
S2.蛋白表达：将S1中所述改造细菌、酵母菌、昆虫细胞或哺乳动物细胞进行扩大培养，收集包含上述方案所述蛋白或上述方案所述的多肽序列的培养液或细胞裂解物；
S3.蛋白纯化：将S2中得到的培养液或细胞裂解物经过粗过滤和纯化得到蛋白。
一种蛋白的生产方法，利用化学合成技术生产上述方案所述蛋白或上述方案所述的多肽序列。
一种蛋白的生产方法，利用体外核糖体表达系统生产上述方案所述蛋白或上述方案所述的多肽序列。
一种上述方案所述的蛋白、多肽序列、融合蛋白、复合体、核苷酸序列、细胞、表达体系、药物在疾病诊断、预防和治疗中的应用。
优选地，所述疾病指因致病蛋白/多肽过渡生成或积累导致的疾病。
优选地，所述致病蛋白/多肽是氧化、糖基化的蛋白产物、淀粉样前体蛋白及其剪切体、胰岛淀粉样多肽及其剪切体、以及其他具备氧化蛋白、糖基化蛋白、淀粉样蛋白、胰岛淀粉样多肽相似特征的致病蛋白/多肽。
优选地，所述疾病诊断包括检测上述方案所述蛋白全部或部分氨基酸序列表达水平、mRNA水平、基因拷贝数量的一种或一种以上。
优选地，所述预防包括基因改造、核酸导入、药物注射/服用、细胞移植、组织移植中的一种或一种以上。
优选地，所述治疗包括基因改造、核酸导入、药物注射/服用、细胞移植、组织移植中的一种或一种以上。
本发明的特点和/或有益效果有：
1.本发明提供的新型天然蛋白sDSS1，典型的人源sDSS1蛋白多肽序列为MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNWDDDNVEDDFSNQLRATVLLMILVCETPYGCYVLHQKGRMCSAFLCC(参见SEQ ID NO：1)。经过生物信息学分析发现该蛋白是类人猿亚科动物具有的一种天然蛋白。
2.本发明根据生物信息学分析和细胞实验验证，多肽sDSS1碳端31个氨基酸序列是信号肽，它们对该蛋白质的性质和分泌特性有关键作用。C端31个氨基酸序列为：TVLLMILVCETPYGCYVLHQKGRMCSAFLCC(参见SEQ ID NO：2)。
3.本发明所述的sDSS1蛋白可以与氧化蛋白、糖基化蛋白、Aβ蛋白、amylin蛋白结合并屏蔽这些毒性蛋白聚集诱导的细胞毒性，因而具有治疗这些毒性蛋白及其他具备相似特征的致病蛋白过渡生成或过渡积累导致的疾病的重要潜力。
4.本发明的sDSS1蛋白通过大肠杆菌发酵生产。将编码sDSS1蛋白的核苷酸序列导入pET151D质粒中，sDSS1表达的同时，其氮端融合了一个6-his标签和一个V5标签用于纯化和免疫印迹检测。蛋白在大肠杆菌中表达，利用镍-NTA凝胶柱进行初步纯化，然后利用SDS-PAGE进行切胶纯化。将切出的含有His-V5-sDSS1蛋白的胶条装入含有转膜液的透析袋中，在电场的驱动下，蛋白从凝胶中移出，收集在透析袋中。SDS聚丙烯酰胺凝胶电泳分析蛋白纯度达到可以用于生物实验的水平。
5.本发明经过分子实验验证，sDSS1蛋白可以与血清中氧化蛋白、缓冲液中的氧化蛋白结合形成聚合体，或与Aβ蛋白结合从而减少Aβ寡聚物形成。
6.本发明通过细胞实验证明sDSS1蛋白可以有效屏蔽培养基中氧化蛋白、糖基化蛋白、Aβ寡聚物、amylin寡聚物引起的细胞毒性，维持细胞活力。
综上，本发明提供了一种新型天然sDSS1蛋白，通过生物信息学、分子生物学、细胞生物学三个层次的研究证明sDSS1蛋白的生物学性质和活性。sDSS1蛋白可以有效降低培养基中氧化蛋白、糖基化蛋白、Aβ寡聚物、amylin寡聚物引起的细胞毒性，维持细胞活力。由于sDSS1蛋白是高等灵长类动物本身具有的蛋白，在临床使用时可以免除免疫反应的问题。因而，本发明提供了一种新型预防和治疗氧化蛋白、糖基化蛋白、Aβ蛋白、amylin多肽及其他具备相似特征的致病蛋白过渡生成或过渡积累导致的相关疾病的候选药物，在生物医学上有重要的应用前景。
下面结合附图，对本发明做进一步详细的阐述，以使本发明能够清楚、完整，但不是为了限制本发明的保护范围。
图1A.sDSS1基因是DSS1基因的新亚型，人DSS1基因cDNA(NM_006304.1，509bp)与人sDSS1基因cDNA(AK309241.1，1195bp)比对显示二者有重叠区，重叠区域核酸序列经分析可编码N端58个氨基酸序列。
图1B.用Clustal X2.1软件对灵长目13个物种的sDSS1蛋白氨基酸序列进
行比对，结果显示sDSS1蛋白氨基酸序列高度保守，N端58个氨基酸序列完全相同，C端31个氨基酸序列仅有少量位点出现点突变。
图2A.质粒转染293T细胞，24h后观察GFP蛋白分布。对照细胞(GFP)内绿色荧光清晰明亮，溶液中背景暗淡。sDSS1与GFP螯合蛋白(sDSS1-GFP)或sDSS1蛋白C端31个氨基酸序列与GFP螯合蛋白(sDSS1-c-GFP)的培养物溶液中观察到明显的绿色荧光信号，而细胞内荧光弥散且强度下降，说明GFP蛋白随着sDSS1蛋白或sDSS1蛋白C端序列分泌而被带到细胞外。
图2B.点膜免疫印迹方法检测转染细胞培养基，结果显示在sDSS1-GFP和sDSS1-c-GFP组培养基中检测到GFP信号，在空白对照(blank)和GFP对照组没有检测到明显的信号，证明sDSS1蛋白是一种分泌蛋白，而C端31个氨基酸序列是信号肽。
图3A.利用sDSS1蛋白C端多肽序列特异性抗体(抗原序列：sDSS1蛋白的C端31个氨基酸序列)在人血清(human serum)或脑脊液(human cerebral spinal fluid,CSF)样本中可以检测到sDSS1信号。Human CSF样本来源于老年人，a血清样本来源剧烈运动后青年人血液，b，c，d血清样本来源保持静息状况下的青年人血液。
图3B.利用PCR方法可以在人脑星形胶质母细胞瘤(U-87MG)检测到sDSS1基因特异的mRNA序列(扩增产物为293bp),DSS1基因的作为对照(扩增产物为238bp)。
图4A-图4B.考马斯亮蓝染色显示sDSS1蛋白生产和纯化过程中目的蛋白的含量。图4A.挑选阳性大肠杆菌克隆菌株扩大培养，加入IPTG后可以诱导sDSS1蛋白的表达，没有诱导的细胞中目的蛋白的表达量极低。图4B.检测镍-NTA凝胶柱初步纯化后浓缩的裂解物和纯化之后目的蛋白含量，a通道显示纯化的sDSS1蛋白，b通道显示初步纯化的细胞裂解液。
图5A-图5D.生化实验和细胞实验证明sDSS1蛋白可以与氧化蛋白结合并屏
蔽氧化蛋白的毒性。图5A.用0.72μg纯化的sDSS1蛋白与不同比例的血清蛋白混合后孵育，用V5结合蛋白(V5-HRP)检测sDSS1蛋白，结果显示sDSS1蛋白与血清的氧化蛋白形成大分子的蛋白复合体。图5B.晚期氧化蛋白(AOPP，200μg/mL)与不同浓度纯化的sDSS1蛋白在4℃共同孵育过夜，产物用SDS-PAGE分离后考马斯亮蓝染色显示，sDSS1蛋白与AOPP可以形成大分子的复合体，随着sDSS1蛋白浓度增加复合体含量增加。图5C.在培养基中加入10％氧化的血清显著降低细胞增殖，在培养基中sDSS1蛋白可以屏蔽氧化的血清带来的细胞毒性。图5D.在无血清培养基中添加100μg/mL AOPP蛋白降低细胞活力，加入等浓度sDSS1蛋白可以挽回细胞活力，100μg/mL BSA用于对照组。数据用t-test双尾检验分析并用ANOVE分析验证。**，p-value<0.01。
图6A-图6E.sDSS1蛋白减少Aβ寡聚体形成，并且降低Aβ寡聚体引起的细胞毒性和细胞凋亡。图6A.不同比例的sDSS1蛋白与10μg Aβ蛋白混合后孵育，经Aβ抗体检测显示，sDSS1蛋白与Aβ形成共价结合的高分子量蛋白复合体，这种结合可以减少有细胞毒性的Aβ寡聚体的形成。图6B.V5结合蛋白(V5-HRP)检测sDSS1蛋白，结果显示sDSS1蛋白与Aβ形成蛋白复合体。图6C.培养基中添加Aβ寡聚体导致细胞毒性，从而降低细胞活力，sDSS1蛋白可以完全屏蔽Aβ寡聚物导致的细胞毒性。图6D.细胞凋亡实验显示，sDSS1蛋白降入到培养液中显著降低Aβ寡聚体导致的SH-SY5Y细胞的早期凋亡和晚期凋亡，从而降低毒性蛋白对细胞的影响。图6E.sDSS1蛋白也可以屏蔽Aβ寡聚物对小鼠神经干细胞(NSCs)的毒性。数据用t-test双尾检验分析并用ANOVE分析验证。**，p-value<0.01。
图7.Amylin寡聚物添加到培养基中引起细胞毒性降低细胞活力，添加sDSS1蛋白能挽回细胞活力，促进细胞存活。数据用t-test双尾检验分析并用ANOVE分析验证。**，p-value<0.01。
图8.400μg/mL糖基化蛋白引起的细胞活力下降可以被sDSS1蛋白挽回，随着sDSS1蛋白浓度增加(100μg/mL到200μg/mL)挽回效应更加强烈。400μg/mL BSA蛋白用于对照组。数据用t-test双尾检验分析并用ANOVE分析验证。**，p-value<0.01。
图9A.示意图显示在SAMP8小鼠的侧脑室(Ventricle)注射病毒的操作方法和病毒注射位点。
图9B.在5月龄早衰型小鼠SAMP8小鼠的大脑侧脑室中注射腺病毒后(左右半脑各1μL病毒液)，连续观察显示动物存活情况。结果显示随着时间延长，注射表达sDSS1蛋白腺病毒的小鼠术后生存率明显高于对照小鼠(表达GFP蛋白)。数据用ANOVE分析分析。**，p-value<0.01。
以下内容将结合实例对本发明中的优选方案进行说明和验证，不是对本发明的范围进行限定。本发明的所有范围限定以权利要求书中的限定为准。
下述实施案例中所用的实验方法如无特殊说明，均为常规实验方法。
下述实施案例中sDSS1蛋白由自己生产并检验纯度达到生物实验的水平，其他材料和试剂均可以通过商业途径获取。
实施例1、sDSS1蛋白是一种灵长目动物具有的分泌型蛋白
生物信息学分析工具：
美国国家生物技术信息中心(National Center of Biotechno logy Information，NCBI)基因组数据库；Nucleotide blast tool(NCBI)；Align sequences nucleotide blast tool(NCBI)，Translate tool(SIB Bioinformatics Resource Portal)；Clustal X2.1：Multiple Sequence Alignment(EMBL-EBI)；SecretomeP 2.0(CBS prediction service)；WoLF PSORT II。
生物实验方法：
1.细胞培养293T细胞购自美国模式培养物集存库(American type culture collection，ATCC)，细胞培养在含有90％基础培养基(Dulbecco's modified eaglemedium，DMEM)(Life technology公司C#12500062)和10％胎牛血清(Fetal bovine serum，FBS)(Gibco公司C#10100-147)的细胞培养基中，放在在细胞培养箱(温度37℃，湿度95％，CO2浓度5％)中培养，每两天传代一次。
2.细胞转染293T细胞按照3x105每孔接种到6孔板中，添加1.5mL细胞培养基，细胞贴壁12小时后开始转染质粒。采用真核表达质粒pCMV-C-Flag，插入的表达sDSS1蛋白核酸序列如参见SEQ ID NO：17所示。质粒2500ng用750μL
Medium(Life technology公司C#31985062)稀释并混匀，10μL转染试剂Lip2000(Invitrogen公司C#12566014)用750μL 
Medium稀释并混匀，将稀释的质粒溶液逐滴滴入稀释的转染试剂中，混匀后常温孵育5分钟。吸去6孔板中的细胞培养基，用PBS清洗细胞一遍，然后换成孵育好的转染工作液。细胞继续放在培养箱中培养，在24至48小时期间观察细胞中荧光蛋白表达。
3.点膜蛋白免疫印迹实验PVDF膜用甲醇活化后干燥，分别把对照培养基和不同转染细胞培养基滴加到膜上。等待膜干燥后，PVDF膜依次完成1％BSA封闭、第一级抗体(Rabbit-anti-GFP)(Cell signal technology公司C#2956)孵育，第二级抗体(Goat-anti-rabbit HRP抗体)(中杉金桥，ZDR-5403)孵育。膜用PBST清洗三次，用发光液(中杉金桥，ZLI-9017)显影并用X光胶片曝光条带。
结果分析：
在对人基因组中shfm1基因进行生物信息学分析时，我们发现该基因有多条转录本(参考NCBI数据库中shfm1基因信息，http://www.ncbi.nlm.nih.gov/gene/7979)。除了共同编码DSS1蛋白序列的一条mRNA序列(NM_006304.1，509bp)，还有一条更长的mRNA序列(AK309241.1，1195bp)。短mRNA序列与长mRNA序列仅有256bp重复序列(图1A)，经核酸序列分析，利用Translate tool得知重复序列可编码DSS1蛋白N端58个氨基酸序列。长mRNA序列编码89个氨基酸的多肽序列。通过多肽序列比对可知，长mRNA编码的多肽序列与DSS1多肽序列有N端58个氨基酸的重叠区，另有31个氨基酸的变异区。我们命名这条新的多肽为分泌型DSS1蛋白(secretory DSS1，sDSS1)，多肽序列如下面所示：
DSS1(Homo sapiens):
MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNWDDDNVEDDFSNQLRAELEKHG
YKMETS(参见SEQ ID NO：4)
s-DSS1(Homo sapiens):
MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNWDDDNVEDDFSNQLRATVLLMI
LVCETPYGCYVLHQKGRMCSAFLCC(参见SEQ ID NO：1)
通过对NCB I数据库中已测序的灵长目动物基因组和其他模式动物基因组的筛查，我们发现只有在类人猿亚目动物的基因组中存在类似的长mRNA序列并且得到与人源sDSS1蛋白相似的多肽序列，如表1所示。这些多肽序列比对结果显示，sDSS1蛋白序列高度保守，N端59个氨基酸序列完全相同，C端其余氨基酸序列仅有少量点突变(图1B)。
表1
利用两种分泌蛋白分析预测软件Wolf PSORT和SecretomeP 2.0分析sDSS1蛋白氨基酸序列，预测结果显示sDSS1蛋白定位于细胞外，与多种已鉴定的分泌型蛋白相似，被预测是一种分泌蛋白(表2)。其中，根据Wolf PSORT软件的分析结果，sDSS1蛋白的信号肽剪切位点(Cleavage site)位于58-59位氨基酸之间。
表2
[根据细则26改正14.09.2017]
[根据细则26改正14.09.2017]
根据生物信息学分析结果，分别把该蛋白全序列或C端31个氨基酸序列(58位氨基酸之后的31个氨基酸序列)与绿色荧光蛋白(Green fluorescent protein,GFP)连接并表达到293T细胞中(sDSS1-GFP，sDSS1-c-GFP)，结果可以观察到溶液中出现绿色荧光，背景发光，细胞内荧光模糊暗淡；而对照组(GFP)溶液中没有荧光，背景很暗，细胞内荧光清晰明亮(图2A)。用点膜免疫印迹方法检测细胞培养基，发现在sDSS1-GFP和sDSS1-c-GFP组的细胞培养基中检测到GFP信号，在对照组(GFP)中没有检测到信号(图2B)。总结这些结果，说明sDSS1蛋白是一类分泌蛋白，可以在细胞内合成并被分泌到细胞外，sDSS1蛋白的C端31个氨基酸序列发挥了信号肽的功能。
实施例2、sDSS1蛋白是一类天然蛋白质
1.人血清和脑脊液样品处理。收集新鲜的人全血，室温静置10-20分钟后，3500g离心30分钟，取上清即为人血清。向血清中加入100mMβ巯基乙醇，混匀后沸水浴处理10分钟，冷却后12000g高速离心10分钟，取上清与1/5体积的5x上样缓冲液混合。从医院得到新鲜脑脊液放置于冰盒中运输，当天处理。新鲜脑脊液直接与5x loading buffer混合后直接制成样品用于上样。
2.蛋白免疫印迹实验(westernblotting)15μL制备的上样样品加入上样孔，4-12％预制胶(Life technology公司C#NP0321BOX)分离蛋白后转移到PVDF膜上。膜依次经过第一级抗体(Rabbit-anti-sDSS1)(抗原序列：sDSS1蛋白的
C端31个氨基酸序列)孵育，PBST溶液清洗三遍；第二级抗体(Goat-anti-rabbit HRP抗体)孵育。用PBST清洗三遍，完成后用发光液显影并用X光片显示条带。
3.细胞培养人脑胶质瘤细胞(U87-MG细胞)购自ATCC，细胞培养在含有90％基础培养基DMEM和10％胎牛血清FBS的完全细胞培养基中，放在在细胞培养箱(温度37℃，湿度95％，CO2浓度5％)中培养，每两天传代一次。
4.PCR实验收集U87-MG细胞后迅速裂解细胞，用一个总RNA提取试剂盒(QIAGEN,51304)从细胞裂解液中提取总RNA，RNA样品继续用1U/μL DNaseⅠ在室温处理15分钟除去残余的基因组DNA。获得的RNA样品用一个cDNA合成试剂盒(全式金生物技术有限公司，AT301)全部转化合成为cDNA并被用作模板样本进行后续PCR实验。PCR反应采用20μL反应体系，其中包括10μL PCR预混试剂(PCR Taq Mixture)(Omega bio-tek，TQ2200)，0.5μL cDNA模板(3.5μg/mL)，0.5μL引物，9μL超纯水，混匀后开始PCR反应。DSS1 cDNA引物：前向引物：GCAGACAGTCGAGATGTCAGAG，反向引物：TTCTTCTGGATGCTATGAAGTCTCC；sDSS1 cDNA引物：前向引物：GCAGACAGTCGAGATGTCAGAG，反向引物：TGATGATCTGTTAACAGCAGAGG。PCR反应程序：94℃10分钟，开始循环反应40次：包括94℃10s、62℃20s、72℃20s，循环结束后继续72℃10分钟然后在4℃保存并用3％琼脂糖凝胶[0.05％SYBR Green Stain(Thermo Fisher,4472903)]电泳检测PCR产物中的DNA含量。
结果分析：
利用sDSS1特异性抗体，可以在人脑脊液或血清中检测到sDSS1蛋白的信号，不同个体来源的血清样本表现出不同的信号模式，运动之后的个体血清中sDSS1信号明显高于静息状态下的个体(图3A)。在U87-MG细胞内，可以检测到sDSS1基因的mRNA信号，PCR扩增产物的基因测序结果与数据库的序列完全相同(图3B)。这些结果说明，sDSS1蛋白是一种天然蛋白质，存在于人脑脊液和血清。
实施例3、sDSS1蛋白的少量制备
实验方法
1.sDSS1蛋白制备：全基因合成编码人sDSS1蛋白的核苷酸片段(参见SEQ ID NO：17)，插入到pET151D中Hisx6-V5标签后面。然后将质粒转入表达菌株
BL21(DE3)中。大肠杆菌中融合表达Hisx6-V5-sDSS1蛋白，利用镍-NTA凝胶柱进行初步纯化，然后利用SDS-PAGE进行切胶纯化。将切出的含有His-V5-sDSS1蛋白的胶条装入含有转膜液的透析袋中，在电场的驱动下，蛋白从凝胶中移出，收集在透析袋中。浓缩蛋白至500微升左右，在PBS溶液中4度透析4次，每次200毫升。
2.SDS聚丙烯酰胺凝胶电泳纯化制备的sDSS1蛋白或细菌裂解液蛋白与5x上样缓冲液混合后，沸水浴处理10分钟，12000g高速离心10分钟，取上清液用于分析。用4-12％预制胶分离蛋白后，考马斯亮蓝染液染色胶块1小时，然后用脱色液室温下脱色过夜。脱色完成后，观察胶块上的条带并拍照保存。
结果分析：
挑选阳性克隆的大肠杆菌菌株，扩大培养，在细菌生长的对数期开始用I PTG刺激细菌细胞表达目的蛋白。裂解细菌，检测目的蛋白的表达水平，结果显示I PTG刺激后，细菌细胞表达sDSS1蛋白的水平显著上升，在胶图上可以看到明显的蛋白条带(图4A)。细菌裂解液经过镍-NTA凝胶柱初步纯化之后，浓缩液中目的蛋白得到极大的浓缩(b通道)，杂蛋白含量减少；继续纯化可以得到纯度极高的sDSS1蛋白(a通道)(图4B)，可以用于后续的生物实验。纯化之后的sDSS1蛋白经BCA蛋白定量，终浓度为0.72mg/ml，4度保存备用。
实施例4、sDSS1蛋白与氧化蛋白反应并屏蔽氧化蛋白的细胞毒性
实验方法
1.氧化血清与sDSS1蛋白反应新鲜人血液经3500g离心30分钟，取上层血清用于后续实验。实验中在10μL sDSS1蛋白溶液(0.72mg/mL)中分别与10，20，50和100微升氧化血清混合，sDSS1与血清蛋白质量比大概为1：100，1：200，1：500，1：1000，另外添加20μM Fenton试剂(FeSO4和H2O2按照1：1的质量比混合)，混合均匀后放在4℃避光孵育过夜。第二天，用10微升Ni-NTA磁珠，将各反应中的His-V5-sDSS1分离出来。反应液与磁珠4度混合2小时，用磁力架将磁珠吸附在管壁上，吸走液体，加入1ml PBST，将管子从磁力架上取下，反复震荡洗涤后，磁力架吸附磁珠，吸走PBST，如此反复4次，最后用50微升含50mM EDTA的TBS洗脱蛋白，洗脱液加入等体积2xSDS溶液，100℃
处理10分钟，12000g离心10分钟，取上清液用于检测。上清液与5X上样缓冲液混合，100℃加热处理10分钟，制备的样品用于蛋白质免疫印迹实验。
2.氧化胎牛血清和晚期氧化蛋白制备10mL胎牛血清加入10mM NaClO处理1小时，氧化的血清用3000Da透析袋在PBS溶液中连续透析24小时，透析期间每隔8小时换液一次，处理完成的血清溶液中加入1mM维生素C(Vc)以彻底清除参与的氧化剂。BCA蛋白定量方法检测蛋白浓度。利用两种方法检测氧化蛋白的含量，氯胺T方法测量氧化血清中双酪氨酸值为75.31μM/mg蛋白(未处理血清为15.05μmol/mg蛋白)，二硝基苯肼法检测羰基含量为16.33nmol/mg蛋白(未处理血清为13.68nmol/mg蛋白)。
10mg血清白蛋白首先用160mM NaClO处理1小时，氧化蛋白用3000Da透析袋在PBS溶液中连续透析24小时，透析期间每隔8小时换液一次。处理完成的晚期氧化蛋白(AOPP)首先用BCA蛋白定量方法测定蛋白浓度。氯胺T方法测AOPP样品中双酪氨酸值为54.21μmol/mg蛋白(未处理的BSA为14.55μmol/mg蛋白)，二硝基苯肼法检测羰基含量为1042.57nmol/mg蛋白(未处理的BSA为10.26nmol/mg蛋白)。
3.晚期氧化蛋白与sDSS1蛋白反应在150μL反应体系中加入30μg AOPP蛋白(200μg/mL)，另外分别加入15μg(100μg/mL)、30μg(200μg/mL)、60μg(400μg/mL)sDSS1蛋白，用无菌PBS溶液补充多余的体积。溶液混匀后在4℃环境下反应过夜。反应完成后的样品中加入5x上样缓冲液，100℃加热处理10分钟，处理好的样品用SDS-PAGE分离并用考马斯亮蓝染色显示条带。
4.蛋白免疫印迹实验反应完成的蛋白混合物与5x loading buffer混合后，沸水浴10分钟并用于western blotting分析。具体方法与前述相同。所用的抗体为V5-HRP抗体(1：5000稀释)。
5.细胞系培养人神经母细胞瘤细胞(SH-SY5Y)生长于基础培养基DMEM添加10％胎牛血清；细胞每两天传代一次。
6.细胞活力检测为了检测sDSS1蛋白对氧化血清的细胞毒性的效应，SH-SY5Y细胞按照104细胞每孔接种到96孔板中，200μL完全培养基。12小时后，完全培养基换成含0.5％BSA的无血清DMEM每孔200μL。继续处理24小时后，DMEM溶液换成10％氧化血清和含有20μg/mL sDSS1蛋白的10％氧化血清的
培养基，每孔200μL。继续处理48小时后，弃去96孔板中旧培养基，每孔加入100μL稀释的CCK-8工作液(1：20稀释)(同仁化学，CK04)检测细胞活力变化。加入等浓度BSA组用于对照组。
为了检测sDSS1蛋白对AOPP引起的细胞毒性的保护效应，SH-SY5Y细胞按照2Χ104细胞每孔接种到96孔板中，12小时贴壁后，培养基换成含有0.5％BSA的无血清培养基。继续处理24小时后，换成含有100μg/mL AOPP蛋白的无血清培养基，处理组同时加入100μg/mL sDSS1蛋白，每孔200μL。96孔板继续处理48小时后，用CCK-8试剂盒检测细胞活力变化。
结果分析：
血清中含有大量蛋白，主要是血清白蛋白。在Fenton试剂的作用下，血清中的蛋白被氧化，氧化产物可与sDSS1发生反应形成复合体。在对照组，可以看到sDSS1蛋白单体，没有发生明显的蛋白聚集；在实验组，与血清共同孵育导致大量高分子量蛋白复合体形成，这些复合体不能被SDS-PAGE分开(图5A)。结果说明，sDSS1蛋白可以与血清中氧化蛋白结合。在细胞毒性实验中，与对照血清相比，加入10％氧化蛋白可以明显抑制细胞增殖和细胞活力，培养基中包含20μg/mL sDSS1蛋白就可以挽回氧化蛋白的细胞毒性(图5B)。
继续把sDSS1与AOPP蛋白混合，可以看到sDSS1与AOPP蛋白结合形成复合体，这些复合体不能被SDS-PAGE分开。随着反应体系中sDSS1蛋白浓度增加，复合体明显增多(图5C)。在细胞实验中，AOPP对细胞造成显著的细胞毒性，降低细胞活力，而等浓度的sDSS1可以完全屏蔽AOPP的细胞毒性(图5D)。综合以上结果，sDSS1能保护细胞不受氧化血清或AOPP的细胞毒性的影响。
另外，总结sDSS1蛋白有氧化蛋白的反应，两种蛋白都能够和氧化蛋白发生紧密结合，这种结合力可以抵抗高浓度的SDS，疑似为共价相互作用。不同之处在于DSS1与氧化蛋白结合的过程需要一个ATP酶的协助[Zhang et al,2014],而我们的证据显示sDSS1与氧化蛋白发生紧密结合不需要添加ATP，即不需要ATP酶的参与。比较DSS1和sDSS1的氨基酸序列可知，两种蛋白1到58位的氨基酸序列完全相同，而DSS1的59到70位的氨基酸序列和sDSS1的59到89位的氨基酸序列完全不同。因而推断DSS1与sDSS1与氧化蛋白发生紧密结合的特性，应该是源于共享氨基酸序列的贡献，亦即前58氨基酸序列。sDSS1异于DSS1
的特性，亦即不依赖ATP酶介导与氧化蛋白发生紧密结合的特性，应该源于sDSS1独特的氨基酸序列，亦即59到89位的C端氨基酸序列。总而言之，sDSS1这种不依赖于ATP酶介导就能与氧化蛋白发生紧密结合的特性来源于前58个氨基酸序列和后31个氨基酸序列的有机组合。
实施例5、sDSS1蛋白降低Aβ寡聚物形成并降低Aβ寡聚物的细胞毒性
实验方法
1.细胞系培养人神经母细胞瘤细胞(SH-SY5Y)生长于基础培养基DMEM添加10％胎牛血清；细胞每两天传代一次。
2.神经干细胞培养神经干细胞(neural stem cell，NSC)来自P2小鼠脑组织，原代悬浮培养的神经干细胞经过两次传代后用于毒性检测实验，神经干细胞培养于干细胞培养基中，包含88％DMEM/F12基础培养基(Gibco，C#12500-062)，10％Proliferation supplementary添加物(Stem cell technology，C#05701)，2％BSA(Sigma，C#V900933)，10ng/mL Heparin(Sigma，C#H3149)，10ng/mL bFGF(Roche，C#11104616001)，20ng/mL EGF(BDBioscience，C#354010)。
3.Aβ与sDSS1蛋白反应Aβ蛋白(Human，1-42)冻干粉由苏州强耀生物科技有限公司提供。2mg Aβ冻干粉用20μL DMSO溶解L，继续用PBS稀释为2mg/mL。-20度冷冻保存。反应体系中首先加入300μL PBS溶液中10μg Aβ与sDSS1蛋白按摩尔质量比1：1，1：5，1：10混合，混合后放在4℃孵育过夜。孵育完成的反应物中加入5Χ上样缓冲液，100℃处理10分钟用于蛋白免疫印迹分析。
4.Aβ蛋白预处理Aβ用基础培养基(pH7.2)稀释Aβ储液至1000μg/mL，Aβ稀释液放在4℃孵育24小时后形成的寡聚物用于细胞实验。后续实验中Aβ的浓度始终按孵育前的蛋白浓度来标注。
5.蛋白免疫印迹实验处理好的反应物用SDS-PAGE分离，继续用于western blotting分析，具体方法与前述相同。所用的抗体为V5-HRP抗体(1：5000稀释)，Aβ抗体(Cell signal technology，9888)，二抗(Goat-anti-rabbit HRP抗体)。
6.细胞活性检测SH-SY5Y细胞按照2x104细胞每孔接种到96孔板中，贴壁12小时后，将旧培养基换成含有0.5％BSA的无血清培养基，继续处理24小时后。弃去旧培养基后换成包含Aβ或Aβ与sDSS1蛋白的DMEM溶液。细胞继续处理48小时后，用CCK-8试剂盒检测细胞活力水平。
7.细胞凋亡检测细胞凋亡检测试剂盒购自东仁化学科技(上海)有限公司(AD10)。SH-SY5Y细胞按照3x105细胞每孔接种到6孔板中，贴壁12小时后将旧培养基换成含0.5％BSA的无血清DMEM溶液，继续处理24小时后换成包含Aβ或Aβ与sDSS1蛋白的溶液。继续处理48小时后，用凋亡试剂盒检测细胞凋亡水平。收集所有溶液和细胞，离心弃去上清。用400μL凋亡试剂盒提供的染色缓冲液重悬细胞，吸取其中的185μL细胞悬液用于后续检测。在细胞悬液中加入5μL Annexin V染色液后混匀，细胞放在37℃孵育10分钟。继续加入10μL PI染液，混匀后用流式细胞仪检测细胞凋亡水平。
神经干细胞首先贴壁培养于6孔板中，孔板首先用0.025％Laminin处理至少两个小时，然后用无菌PBS清洗6遍备用。神经干细胞制成单细胞后，按照3x105每孔接种到6孔板中，细胞贴壁24h后用于后续实验。
结果分析
经过孵育，Aβ蛋白发生明显的聚集，在10-20KD之间形成大小不一的蛋白聚集体。根据文献报道，这些Aβ寡聚体是Aβ导致的细胞毒性的主要来源。加入sDSS1蛋白与Aβ共同孵育之后，sDSS1蛋白可以与Aβ蛋白聚集形成大分子量的复合体(分子量高于20KD)，10-20KD之间形成的寡聚体明显减少(图6A)。随着sDSS1蛋白浓度增加，Aβ寡聚物生成被明显抑制。检测sDSS1蛋白信号时，可以确定复合体是sDSS1蛋白与Aβ反应形成的(图6B)，并且不能被SDS-PAGE打开。
继续检测sDSS1蛋白对Aβ引起的细胞毒性的屏蔽效应。在细胞活力检测实验中，添加预处理的Aβ寡聚物之后，细胞活力显著下降。在培养液中加入sDSS1蛋白后，SH-SY5Y细胞活力得到显著的恢复并且细胞活力高于对照组(图6C)。在细胞凋亡检测实验中，Aβ寡聚物加入培养液后诱导SH-SY5Y细胞或神经干细胞的细胞凋亡，在培养液中添加sDSS1蛋白可以显著降低细胞的早期凋亡和晚
期凋亡水平(图6D,图6E)。根据以上结果，sDSS1蛋白可以与Aβ蛋白结合降低Aβ寡聚物形成，从而缓解Aβ蛋白引起的细胞毒性。
实施例6、sDSS1蛋白降低amylin寡聚物的细胞毒性
实验方法
1.细胞系培养人神经母细胞瘤细胞(SH-SY5Y)生长于基础培养基DMEM添加10％胎牛血清；细胞每两天传代一次。
2.amylin蛋白预处理amylin蛋白(Human)冻干粉由苏州强耀生物科技有限公司提供。2mg amylin冻干粉用10mM醋酸钠溶液(pH5.5)溶解为2mg/mL，-20度冷冻保存。用基础培养基(pH7.2)稀释amylin储液至1mg/mL，amylin稀释液放在4℃孵育48小时后形成的寡聚物用于细胞实验。后续实验中amylin的浓度始终按孵育前的蛋白浓度来标注。
3.细胞活性检测SH-SY5Y细胞按照2x104细胞每孔接种到96孔板中，贴壁12小时后，将旧培养基换成含有0.5％BSA的无血清培养基，继续处理24小时后。弃去旧培养基后换成包含amylin或amylin与sDSS1蛋白的DMEM溶液。细胞继续处理48小时后，用CCK-8试剂盒检测细胞活力水平。
结果分析
在细胞培养液中加入10μM孵育的amylin蛋白可以引起显著的细胞毒性，同时加入sDSS1蛋白可以屏蔽amylin寡聚物引起的细胞毒性，细胞活力甚至高于对照组(图7)，说明sDSS1蛋白可以有效屏蔽amylin蛋白的细胞毒性。
实施例7、sDSS1蛋白降低糖基化蛋白的细胞毒性
实验方法
1.细胞系培养人神经母细胞瘤细胞(SH-SY5Y)生长于基础培养基DMEM添加10％胎牛血清；细胞每两天传代一次。
2.糖基化蛋白制备10mg/mL血清白蛋白与2.5M核糖混合后在37℃环境下连续孵育7天，完成后用3000Da的透析袋在PBS中透析24小时，每隔8小时换液一次。糖基化蛋白制作完成后，BCA蛋白定量，样品放在-80℃保存备用。
3.细胞活性检测SH-SY5Y细胞按照2x104细胞每孔接种到96孔板中，贴壁12小时后，将旧培养基换成含有0.5％BSA的无血清培养基，继续处理24小时后。弃去旧培养基后换成包含糖基化蛋白或糖基化蛋白与不同浓度sDSS1蛋白的DMEM溶液，加入等浓度BSA组用于对照。细胞继续处理48小时后，用CCK-8试剂盒检测细胞活力水平。
结果分析
在细胞培养液中加入400μg/mL糖基化蛋白可以引起显著的细胞毒性，同时加入sDSS1蛋白可以降低糖基化蛋白引起的细胞毒性，随着sDSS1蛋白浓度的提高，细胞活力甚至高于对照组(图8)，说明sDSS1蛋白可以有效屏蔽糖基化蛋白的细胞毒性。
实施例8、sDSS1蛋白延长早衰型小鼠SAMP术后生存期
实验方法
1.动物饲养早衰型SAMP8小鼠(5月龄，雄性)购自北京维通利华实验动物技术有限公司，动物饲养在南方模式动物中心的洁净级实验动物饲养中心。给动物提供充足的无菌水和标准小鼠繁育饲料，12h/12h明暗交替光照，每月定期更换垫料和鼠笼，每天定期观察动物存活情况。
2.腺病毒合成表达sDSS1蛋白的腺病毒由我们(发明人)提供核酸序列(参见SEQ ID NO：17)，腺病毒构建和合成由赛业(广州)生物科技有限公司完成。腺病毒启动区和转录区序列构成：pAV[Exp]-UBC>EGFP:T2A:ORF_363bp，含泛素蛋白启动子序列。经测定，病毒滴度大于1010PFU/mL。经感染U87-MG细胞和小鼠神经母细胞瘤细胞(N2a)验证，腺病毒可以高效率感染细胞并表达sDSS1蛋白。表达GFP蛋白的腺病毒(pAV[Exp]-UBC>EGFP)作为对照。
3.脑立体定位注射早衰型SAMP8小鼠(6月龄，雄性)按照800mg/kg体重腹腔注射20％乌拉坦(溶解在生理盐水中，0.22μm滤器过滤除杂质和除菌)麻醉。小鼠麻醉后，固定到小鼠脑立体定位仪(Stoelting，51500)上，保持颅骨水平。切开头部皮肤，暴露颅骨，以前囟(Bregma)为坐标起点，定位侧脑室区域(0.58mm，1.25mm，1.75mm)，用牙科钻在标记点打孔。在微量注射器(Hamilton 600-2.5μL，针头直径0.2mm)中吸去2μL病毒液，按照相同坐标数值重新定位侧脑室区域。按照指定坐标值快速进针到侧脑室区域，平均
200nL/min缓慢注射病毒液，总计1000nL。注射完成后，每提针0.25mm，等待3分钟直至针头完全拔出脑组织。重新定位另一侧侧脑室区域，完成进针、注射和提针步骤，注射1μL病毒液。注射完成的小鼠，首先用100U/mL的氨苄青霉素/链霉素擦拭颅骨及周边组织，再用缝合线缝合皮肤。给小鼠腹腔注射150μL抗生素并腹部朝上放置到鼠笼中，直至小鼠苏醒。
结果分析
示意图指示小鼠侧脑室病毒注射的基本操作和腺病毒注射位点(图9A)。其中，注射表达sDSS1腺病毒小鼠共两批次，共10只小鼠；注射表达GFP腺病毒小鼠两个批次，共14只。注射腺病毒1个月内，对照组小鼠死亡3只，实验组小鼠死亡1只；8个月内，对照组小鼠陆续出现死亡，而实验组仅仅死亡一只(图9B)。总结结果，注射表达sDSS1蛋白腺病毒SAMP8小鼠的术后生存期显著长于仅仅注射对照病毒的小鼠，ANOVA分析二者呈现显著差异(P-value<0.01)。
上文所列出的一系列的详细说明仅仅是针对本发明的可行性实施例的具体说明，它们并非用以限制本发明的保护范围，凡未脱离本发明精神所作的等效实施例或变更均应包含在本发明的保护范围之内。
Claims (31)
- 一种新型天然蛋白sDSS1，其特征在于，包括人在内的类人猿亚目动物存在天然蛋白sDSS1，其中人源天然蛋白sDSS1的氨基酸序列如SEQ ID NO：1。
- 根据权利要求1所述的新型天然蛋白sDSS1，其特征在于，所述类人猿亚目动物还包括黑猩猩、倭黑猩猩、大猩猩、红毛猩猩、白颊长臂猿、川金丝猴、恒河猴、东非狒狒、安哥拉疣猴、白顶白眉猴、鬼狒、豚尾猴；其中黑猩猩的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：5，倭黑猩猩的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：6，大猩猩的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：7，红毛猩猩的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：8，白颊长臂猿的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：9，川金丝猴的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：10,恒河猴的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：11，东非狒狒的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：12，安哥拉疣猴的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：13，白顶白眉猴的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：14，鬼狒的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：15，豚尾猴的天然蛋白sDSS1的氨基酸序列如SEQ ID NO：16。
- 根据权利要求2所述的新型天然蛋白sDSS1，其特征在于，所述天然蛋白sDSS1分为N端58个氨基酸序列和C端31个氨基酸序列，其中人源sDSS1蛋白N端58个氨基酸序列如SEQ ID NO：3，人源sDSS1蛋白C端的31氨基酸序列如SEQ ID NO：2；两段序列的特征分别在于，N端58个氨基酸序列包含3个或3个以上连续排列的酸性氨基酸序列，每个连续排列的酸性氨基酸序列片段包含的酸性氨基酸数目不高于10个，不同的连续排列的酸性氨基酸序列之间的间隔长度不超过4个氨基酸，每个这样的间隔中存在至少一个疏水氨基酸，pH值不高于4.5，N端58个氨基酸组成的序列酸性氨基酸数量不低于10个；以N端58个氨基酸序列为基础的第58位氨基酸以后的C端氨基酸序列，总体偏疏水性质，C端31个氨基酸组成的氨基酸序列中疏水氨基酸数量不低于10个；氨基酸分类的界定如下：疏水氨基酸:丙氨酸、异亮氨酸、亮氨酸、缬氨酸、半胱氨酸、苯丙氨酸，甲硫氨酸、色氨酸、酪氨酸；中性氨基酸：苏氨酸、甘氨酸，丝氨酸、组氨酸、谷氨酰胺；酸性氨基酸：谷氨酸、天冬氨酸、脯氨酸、天冬酰胺。碱性氨基酸:精氨酸，赖氨酸。
- 根据权利要求1-3任一所述的新型天然蛋白sDSS1，其特征在于，类人猿亚目动物的sDSS1蛋白包含的C端氨基酸通式为：X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19X20X21X22X23X24X25X26X27X28X29X30X31；X1为中性氨基酸；X2为疏水氨基酸；X3，X4分别为疏水氨基酸；X5为疏水氨基酸；X6为疏水氨基酸；X7为疏水氨基酸；X8为疏水氨基酸；X9为疏水氨基酸；X10为酸性氨基酸；X11为中性氨基酸；X12为疏水氨基酸；X13为疏水氨基酸；X14为中性氨基酸；X15为疏水氨基酸；X16为疏水氨基酸；X17为疏水氨基酸；X18为疏水氨基酸；X19为碱性氨基酸；X20为酸性氨基酸；X21为碱性氨基酸；X22为中性氨基酸；X23为碱性氨基酸；X24为疏水氨基酸；X25为疏水氨基酸；X26为中性氨基酸；X27为疏水氨基酸；X28为疏水氨基酸；X29为疏水氨基酸；X30为疏水氨基酸；X31为疏水氨基酸；与C端31个氨基酸序列中有40％或40％以上同源性氨基酸序列，该序列与人源sDSS1蛋白C端的氨基酸序列在多肽中的性质和发挥的功能相同或相似。
- 一种多肽序列，其特征在于，以权利要求1-4任一所述的新型天然蛋白sDSS1的N端58个氨基酸序列和C端31个氨基酸序列为基础构建的多肽序列，其是：1)该多肽序列的N端58个氨基酸组成的序列有40％或40％以上相似性，C端氨基酸序列有40％或40％以上相似性的蛋白，该蛋白与人源天然蛋白的性质和功能相同或相似；或者，2)以人源天然蛋白中多肽序列的N端58个氨基酸组成的序列，或以其40％或40％以上相似序列为基础，在C端或N端融合其他氨基酸序列，用于融合的氨基酸序列能实现与人源天然蛋白中C端31个氨基酸序列相同或相似的性质并且发挥相同或相似的功能，修饰后的蛋白能发挥与人源天然蛋白sDSS1相同或相似功能的蛋白；或者，3)用权利要求4中所述的新型天然蛋白sDSS1中的C端多肽序列融合其他天然或非天然多肽序列得到的蛋白。
- 一种融合蛋白，其特征在于，所述融合蛋白包括权利要求1-4任一所述的新型天然蛋白sDSS1的完整序列或部分序列和权利要求5所述的多肽序列。
- 根据权利要求6所述的融合蛋白，其特征在于，其是与该蛋白自身、载体蛋白、抗体或其他任意氨基酸序列连接形成的蛋白复合体。
- 一种复合体，其特征在于，所述复合体包括权利要求1-4任一所述的新型天然蛋白sDSS1的完整序列或部分序列、权利要求5任一所述的多肽序列、权利要求6-7任一所述的融合蛋白中的部分或全部复合。
- 根据权利要求8所述的复合体，其特征在于，其是多肽/蛋白与医药上可应用的药物载体连接形成的复合体。
- 根据权利要求9所述的复合体，其特征在于，所述载体包含微球/囊、脂质体、微乳液、纳米颗粒、磁颗粒和凝胶中的一种或一种以上。
- 一种核苷酸序列，其特征在于，所述核苷酸序列可以编码权利要求1-4任一所述的蛋白或权利要求5所述的多肽序列。
- 根据权利要求11所述的核苷酸序列，其特征在于，所述核苷酸序列包括DNA序列和RNA序列。
- 一种细胞，其特征在于，所述细胞表达权利要求1-4任一所述的蛋白或权利要求5所述的多肽序列。
- 根据权利要求13所述的细胞，其特征在于，所述细胞是任意一种哺乳动物的干细胞、前体细胞或成体细胞。
- 根据权利要求14所述的细胞，其特征在于，所述哺乳动物是人、猩猩、猴、马、牛、羊、猪、驴、狗、兔、猫、大鼠或小鼠。
- 根据权利要求13所述的细胞，其特征在于，所述细胞包括胚胎干细胞、诱导多能干细胞，或者来源于原代培养的干细胞、由母细胞分化得到的多能或单能干细胞。
- 一种表达体系，其特征在于，其把编码权利要求1-4任一所述蛋白或权利要求5所述的多肽序列的核苷酸序列导入生物体，并在生物体中表达权利要求1-4任一所述的蛋白或权利要求5所述的多肽序列。
- 根据权利要求17所述的表达体系，其特征在于，所述表达体系包括真核表达质粒载体、腺病毒、慢病毒、逆转录病毒、CRISPR/Cas技术及其他可行的基因编辑技术。
- 根据权利要求17所述的表达体系，其特征在于，所述生物体是人、猩猩、猴、马、牛、羊、猪、驴、狗、兔、猫、大鼠、小鼠、鸡、鸭或鹅。
- 一种药物，其特征在于，所述药物以权利要求1-4任一所述蛋白或权利要求5所述的多肽序列为主要靶点，并在施用后可以影响机体内权利要求1-4任一所述蛋白或权利要求5所述的多肽序列的表达水平。
- 根据权利要求20所述的药物，其特征在于，所述药物是化学小分子药物、蛋白/多肽药物或核酸药物、纳米药物。
- 根据权利要求21所述的药物，其特征在于，所述的核酸药物包括siRNA，microRNA，反义寡核苷酸，三链DNA和核酶的一种或一种以上。
- 一种蛋白的生产方法，其特征在于，所述方法包括以下步骤：S1.表达载体构建：将可编码权利要求1-4任一所述蛋白或权利要求5所述的多肽序列的核苷酸序列插入质粒中并导入到细菌或酵母菌细胞中，或将可编码权利要求1-4任一所述蛋白或权利要求5所述的多肽序列的核苷酸序列插入昆虫细胞、哺乳动物细胞基因组中；S2.蛋白表达：将S1中所述改造细菌、酵母菌、昆虫细胞或哺乳动物细胞进行扩大培养，收集包含权利要求1-4任一所述蛋白或权利要求5所述的多肽序列的培养液或细胞裂解物；S3.蛋白纯化：将S2中得到的培养液或细胞裂解物经过粗过滤和纯化得到蛋白。
- 一种蛋白的生产方法，其特征在于，利用化学合成技术生产权利要求1-4任一所述蛋白或权利要求5所述的多肽序列。
- 一种蛋白的生产方法，其特征在于，利用体外核糖体表达系统生产权利要求1-4任一所述蛋白或权利要求5所述的多肽序列。
- 一种权利要求1-4任一所述的蛋白、权利要求5所述的多肽序列、权利要求6或7所述的融合蛋白、权利要求8-10任一所述的复合体、权利要求11 或12所述的核苷酸序列、权利要求13-16任一所述的细胞、权利要求17-19任一所述的表达体系、权利要求20-22任一所述的药物在疾病诊断、预防和治疗中的应用。
- 根据权利要求26所述的应用，其特征在于，所述疾病指因致病蛋白/多肽过渡生成或积累导致的疾病。
- 根据权利要求27所述的应用，其特征在于，所述致病蛋白/多肽是氧化、糖基化的蛋白产物、淀粉样前体蛋白及其剪切体、胰岛淀粉样多肽及其剪切体、以及其他具备氧化蛋白、糖基化蛋白、淀粉样蛋白、胰岛淀粉样多肽相似特征的致病蛋白/多肽。
- 根据权利要求26所述的应用，其特征在于，所述疾病诊断包括检测权利要求1-4任一所述蛋白全部或部分氨基酸序列表达水平、mRNA水平、基因拷贝数量的一种或一种以上。
- 根据权利要求26所述的应用，其特征在于，所述预防包括基因改造、核酸导入、药物注射/服用、细胞移植、组织移植中的一种或一种以上。
- 根据权利要求26所述的应用，其特征在于，所述治疗包括基因改造、核酸导入、药物注射/服用、细胞移植、组织移植中的一种或一种以上。
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| WO2019134498A1 (zh) * | 2018-01-05 | 2019-07-11 | 上海清流生物医药科技有限公司 | 一种蛋白在细胞培养中的应用 |
| WO2020052570A1 (zh) * | 2018-09-10 | 2020-03-19 | 上海清流生物医药科技有限公司 | 预防和治疗动脉粥样硬化及相关疾病的方法和组合物 |
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| CN109913483A (zh) * | 2017-12-12 | 2019-06-21 | 上海清流生物医药科技有限公司 | 一种蛋白药物的发酵生产工艺 |
| CN109985231A (zh) * | 2018-01-02 | 2019-07-09 | 上海清流生物医药科技有限公司 | 一种蛋白在制备预防和治疗痴呆症的药物中的应用 |
| CN109985230B (zh) * | 2018-01-02 | 2023-10-17 | 上海普佑生物医药有限公司 | 一种蛋白在制备预防和治疗肾病药物中的应用 |
| CN110302362B (zh) * | 2018-03-20 | 2024-04-05 | 上海普佑生物医药有限公司 | 一种蛋白在制备预防和治疗糖尿病并发症的药物中的应用 |
| IL311448A (en) * | 2021-09-15 | 2024-05-01 | Shanghai Puyou Biomedical Co Ltd | polypeptide |
| CN115287276A (zh) * | 2022-03-07 | 2022-11-04 | 兰州大学 | Sem1蛋白、表达sem1蛋白工程化益生菌在制备治疗和/或预防心脏病药物中的应用 |
| WO2024056028A1 (en) * | 2022-09-15 | 2024-03-21 | Shanghai Puyou Biochemical Co., Ltd. | Analgesic polypeptide |
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| EP2510361A4 (en) * | 2009-12-11 | 2013-07-17 | Purdue Research Foundation | DETECTION OF OXIDIZED POLYPEPTIDES |
| CN102755317A (zh) * | 2011-04-28 | 2012-10-31 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | 一类2-吲哚酮衍生物及其药用盐用于制备抗氧化药物的用途 |
| CN107573412B (zh) | 2016-07-04 | 2021-04-27 | 上海清流生物医药科技有限公司 | 一种新型天然蛋白及其应用 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US10815282B2 (en) | 2016-07-04 | 2020-10-27 | Shanghai Clear Fluid Biomedical Science Co., Ltd. | Secretory protein |
| US11198712B2 (en) | 2016-07-04 | 2021-12-14 | Shanghai Clear Fluid Biomedical Science Co., Ltd. | Secretory protein |
| WO2019134498A1 (zh) * | 2018-01-05 | 2019-07-11 | 上海清流生物医药科技有限公司 | 一种蛋白在细胞培养中的应用 |
| CN111770988A (zh) * | 2018-01-05 | 2020-10-13 | 上海清流生物医药科技有限公司 | 一种蛋白在细胞培养中的应用 |
| WO2020052570A1 (zh) * | 2018-09-10 | 2020-03-19 | 上海清流生物医药科技有限公司 | 预防和治疗动脉粥样硬化及相关疾病的方法和组合物 |
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