U.S. patent application number 17/521286 was filed with the patent office on 2022-05-05 for secretory protein.
The applicant listed for this patent is Shanghai Clear Fluid Biomedical Science Co., Ltd.. Invention is credited to Jingpeng Fu, Jia Wan, Yinghao Zhang.
Application Number | 20220135631 17/521286 |
Document ID | / |
Family ID | 1000006082728 |
Filed Date | 2022-05-05 |
United States Patent
Application |
20220135631 |
Kind Code |
A1 |
Zhang; Yinghao ; et
al. |
May 5, 2022 |
SECRETORY PROTEIN
Abstract
The disclosure of the present application relates to a secretory
deleted split hand/split foot 1 (sDSS1) protein, the amino acid
sequence thereof, the nucleic acid sequence thereof, and the
applications of the same. The sDSS1 protein is a secretory protein
from higher primate, and can be detected in human serum and
cerebral spinal fluid (CSF). The sDSS1 protein can form conjugate
with oxidized protein under nonenzymatic condition or with
amyloid-beta (A.beta.) polypeptide to reduce formation of A.beta.
oligomer. The addition of sDSS1 protein to culture medium can
shield the cytotoxicity induced by oxidized protein, A.beta.
oligomer, amylin oligomer and glycosylated protein, so as to
protect the cells against these toxoproteins. The sDSS1 protein can
prolong survival time of senescence-accelerated mice significantly.
The protein can be used to prevent and treat the diseases induced
by oxidized protein, glycated protein, A.beta. protein
accumulation, amylin protein accumulation or excessive formation or
accumulation of other pathogenic proteins with similar features,
and has important potential in biological medicine.
Inventors: |
Zhang; Yinghao; (Shanghai,
CN) ; Fu; Jingpeng; (Shanghai, CN) ; Wan;
Jia; (Shanghai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Shanghai Clear Fluid Biomedical Science Co., Ltd. |
Shanghai |
|
CN |
|
|
Family ID: |
1000006082728 |
Appl. No.: |
17/521286 |
Filed: |
November 8, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17028769 |
Sep 22, 2020 |
11198712 |
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17521286 |
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16145251 |
Sep 28, 2018 |
10815282 |
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17028769 |
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PCT/CN2017/090785 |
Jun 29, 2017 |
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16145251 |
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
C12N 15/867 20130101;
A61K 48/00 20130101; C12N 15/861 20130101; A61K 45/00 20130101;
C07K 14/47 20130101; C12N 5/10 20130101; C12N 15/85 20130101; A61P
25/28 20180101; C07K 19/00 20130101; A61K 38/17 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; A61P 25/28 20060101 A61P025/28; C07K 19/00 20060101
C07K019/00; C12N 15/861 20060101 C12N015/861; A61K 38/17 20060101
A61K038/17; C12N 15/85 20060101 C12N015/85; C12N 5/10 20060101
C12N005/10; A61K 45/00 20060101 A61K045/00; A61K 48/00 20060101
A61K048/00; C12N 15/867 20060101 C12N015/867 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 4, 2016 |
CN |
201610519038.9 |
Claims
1-18. (canceled)
19. A composition comprising: (i) a polypeptide having an amino
acid sequence at least 80% identical to any one of SEQ ID NO. 1,
SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO.
8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ
ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16; or (ii)
a nucleic acid encoding said polypeptide of (i), which composition
has been formulated for administration to a subject.
20. The composition of claim 19, wherein the polypeptide comprises
an amino acid sequence having at least 80% sequence identity to SEQ
ID NO: 1.
21. The composition of claim 19, wherein the polypeptide comprises
the amino acid sequence of SEQ ID NO: 1.
22. The composition of claim 19, wherein the nucleic acid comprises
a polynucleotide sequence at least 80% identical to SEQ ID NO:
17.
23. The composition of claim 19, wherein the polypeptide comprises
a fusion protein.
24. The composition of claim 23, wherein the fusion protein
comprises the polypeptide fused to itself or a fragment
thereof.
25. The composition of claim 23, wherein the fusion protein
comprises the polypeptide fused to a carrier protein.
26. The composition of claim 23, wherein the fusion protein
comprises the polypeptide fused to an antibody.
27. The composition of claim 19, wherein the polypeptide is
complexed with a pharmaceutically acceptable carrier.
28. The composition of claim 27, wherein the pharmaceutically
acceptable carrier comprises one or more of microspheres, vesicles,
liposomes, microemulsions, nanoparticles, magnetic particles, or
gels.
29. The composition of claim 19, wherein the polypeptide further
comprises a pharmaceutically acceptable excipient.
30. The composition of claim 19, wherein the polypeptide is
complexed with a pathogenic polypeptide selected from the group
consisting of advanced oxidation protein products (AOPP),
amyloid-.beta., amylin, and glycosylated protein.
31. The composition of claim 30, wherein the pathogenic polypeptide
is advanced oxidation protein products (AOPP).
32. The composition of claim 30, wherein the pathogenic polypeptide
is amyloid-.beta..
33. The composition of claim 30, wherein the pathogenic polypeptide
is amylin.
34. The composition of claim 30, wherein the pathogenic polypeptide
is glycosylated protein.
35. The composition of claim 19, wherein the polypeptide is
complexed with a pathogenic polypeptide, wherein the pathogenic
polypeptide is glycated protein.
36. A method for treating or preventing dementia in a subject, the
method comprising administering a composition of claim 19 in an
effective amount to the subject in need thereof.
37. The method of claim 36, wherein the dementia is Alzheimer's
Disease (AD).
38. A method for treating or preventing type II diabetes in a
subject, the method comprising administering a composition of claim
19 in an effective amount to the subject in need thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation of U.S.
Non-Provisional patent application Ser. No. 17/028,769, filed Sep.
22, 2020, which is a division of U.S. Non-Provisional patent
application Ser. No. 16/145,251, filed Sep. 28, 2018, now U.S. Pat.
No. 10,815,282, which is continuation-in-part application of PCT
Application No. PCT/CN2017/090785 filed on Jun. 29, 2017, which
claims the benefit of Chinese Patent Application No. 201610519038.9
filed on Jul. 4, 2016, the disclosure of which is hereby
incorporated by reference in their entirety.
REFERENCE TO SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy created
on Nov. 5, 2021, is named 57275701301 SL.txt and is 14,615 bytes in
size.
BACKGROUND
[0003] The present application relates generally to a secretory
protein, the secretory protein can be used to prepare the drugs for
preventing and treating the diseases induced by excessive formation
or excessive accumulation of junk proteins.
DESCRIPTION OF RELATED ART
[0004] In normal physiological activities, the organism generates
lots of junk proteins, including oxidized protein, glycosylated
protein and some abnormal spliced proteins (polypeptide). The
organism retains multiple mechanisms for removing junk proteins to
maintain normal physiological function. However, the aging or
diseases will induce excessive formation of junk proteins or
degrade the organism's ability to remove junk proteins, so that
lots of junk proteins accumulate. The abnormal accumulation of junk
proteins inside or outside the cells is the key mechanism inducing
a series of diseases. The typical diseases include chronic kidney
disease, Alzheimer's disease (AD), Huntington's disease, diabetes
complications and so on.sup.[1-5]. The accumulation of oxidized
protein, glycated protein or other junk proteins in the circulatory
system is one of the key causes for the aging of
organism.sup.[6-7]. It is proved by research that the advanced
oxidation protein products (AOPP) in the serum damages renal cells,
and it is the main pathogenesis of chronic kidney disease. The AOPP
in serum can induce the programmed apoptosis of islet .beta.
cells.sup.[3-8]. The .beta. amyloid hypothesis indicates that the
synaptic dysfunction and neuron death resulted from progressive
accumulation of toxoprotein induced by unbalance of generation and
removal of A.beta. protein in tissues are the first causes of AD
.sup.[9]. The amylin protein not only performs abnormal aggregation
in the insular tissues of partial diabetics, but also exists in the
plaques of brain tissue, and it is closely related to the progress
of diabetes and neurodegenerative diseases.sup.[10, 11]. Based on
these findings, in some disease models, the A.beta. aggregation or
formation in the AD animal pattern is blocked by using
antibody.sup.[12], polypeptide drug.sup.[13] or micromolecular
drug.sup.[14], the formation of neuronal tissue plaques can be
reduced, and the animal cognition level is increased. These results
show that using drugs to depress the formation and aggregation of
these pathogenic proteins or to promote the removal of pathogenic
proteins to reduce the accumulation of pathogenic proteins is an
important method to prevent or treat these diseases.
[0005] Previous research indicates that when the oxidative stress
occurs in the cell, the DSS1 (deleted split hand/split foot 1)
protein, as a highly conservative small protein in eukaryote, can
be covalently modified to oxidized protein under the conditions of
enzymatic reaction and ATP consumption, such a modification will
mediate the oxidized protein to degrade in the cell.sup.[15]. The
DSS1 gene knockout leads to cell death; the cells with high
expression of DSS1 protein manifest significant resistance to the
oxidative stress or antineoplastic-induced cell apoptosis.sup.[16].
These results show the vital function of DSS1 protein in the course
of removing oxidized protein from cells, and it is the key to the
existence of cells.
[0006] The related references are described below: [0007] 1. Dobson
C M (1999) Protein misfolding, evolution and disease. Trends
Biochem Sci 24:329-332. [0008] 2. Liang M, Wang J, Xie C, Yang Y,
Tian J W, Xue Y M, Hou F F (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. [0009] 3. Cao W, Hou F F, Nie J (2014) AOPPs
and the progression of kidney disease. Kidney Int Suppl (2011)
4(1):102-106. [0010] 4. Sadigh-Eteghad S, Sabermarouf B, Maj di A,
Talebi M, Farhoudi M, Mahmoudi J (2015) Amyloid-beta: a crucial
factor in Alzheimer's disease. Med Princ Pract 24(1):1-10. [0011]
5. Choe Y J, Park S H, Hassemer T, Korner R, Vincenz-Donnelly L,
Hayer-Hartl M, Hartl F U (2016) Failure of RQC machinery causes
protein aggregation and proteotoxic stress. Nature 531(7593):191-5.
[0012] 6. Ott C, Grune T (2014) Protein oxidation and proteolytic
signalling in aging. Curr Pharm Des 20(18):3040-51. [0013] 7. Simm
A, Muller B, Nass N, Hofmann B, Bushnaq H, Silber R E, Bartling B
(2015) Protein glycation--Between tissue aging and protection. Exp
Gerontol 68:71-5. [0014] 8. Liang M, Li A, Lou A, Zhang X, Chen Y,
Yang L, Li Y, Yang S, Hou F F (2017) Advanced oxidation protein
products promote NADPH oxidase-dependent .beta.-cell destruction
and dysfunction through the Bcl-2/Bax apoptotic pathway. Lab Invest
24. [Epub ahead of print]. [0015] 9. Zhao L N, Long H, Mu Y, Chew L
Y (2012) The toxicity of amyloid .beta. oligomers. Int J Mol Sci
13(6):7303-27. [0016] 10. Fernandez M S (2014) Human IAPP
amyloidogenic properties and pancreatic .beta.-cell death. Cell
Calcium 56(5):416-27. [0017] 11. Lim Y A, Rhein V, Baysang G, Meier
F, Poljak A, Raftery M T, Guilhaus M, Ittner L M, Eckert A, Gotz J
(2010) Abeta and human amylin share a common toxicity pathway via
mitochondrial dysfunction. Proteomics 10 (8): 1621-33. [0018] 12.
Winblad B, Andreasen N, Minthon L, Floesser A, Imbert G, Dumortier
T, Maguire R P, Blennow K, Lundmark J, Staufenbiel M, Orgogozo J M,
Graf A (2012) Safety, tolerability, and antibody response of active
A.beta. immunotherapy with CAD106 in patients with Alzheimer's
disease: randomised, double-blind, placebo-controlled,
first-in-human study. Lancet Neurol 11(7):597-604. [0019] 13. Chang
L, Cui W, Yang Y, Xu S, Zhou W, Fu H, Hu S, Mak S, Hu J, Wang Q, Ma
V P, Choi T C, Ma E D, Tao L, Pang Y, Rowan M J, Anwyl R, Han Y,
Wang Q (2015) Protection against .beta.-amyloid-induced synaptic
and memory impairments via altering .beta.-amyloid assembly by
bis(heptyl)-cognitin. Sci Rep 5:10256. [0020] 14. Kim H Y, Kim H V,
Jo S, Lee C J, Choi S Y, Kim D J, Kim Y (2015) EPPS rescues
hippocampus-dependent cognitive deficits in APP/PS1 mice by
disaggregation of amyloid-.beta. oligomers and plaques. Nat Commun
6:8997. [0021] 15. Zhang Y, Chang F M, Huang J, Junco J J, Maffi S
K, Pridgen H I, Catano G, Dang H, Ding X, Yang F, Kim D J, Slaga T
J, He R, Wei S J (2014) DSSylation, a novel protein modification
targets proteins induced by oxidative stress, and facilitates their
degradation in cells. Protein Cell 5(2):124-40. [0022] 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.
SUMMARY OF THE APPLICATION
[0023] In the latest study, we (inventors) have found that there is
a new subtype of DSS1 protein in higher primate (anthropoid
subfamily) genome, named secretory DSS1 protein (sDSS1). The sDSS1
is the first DSS1 protein subtype discovered, and its sequence,
properties and function are highly similar to DSS1. However, it can
be secreted into blood and cerebral spinal fluid, its properties
are more active, and can form a conjugate with the oxidized protein
in serum or buffer solution without energy-consuming enzymatic
reaction or combine with A.beta. protein and reduce the formation
of A.beta. oligomer. The sDSS1 protein added to the culture medium
can shield the cytotoxicity induced by oxidized protein, A.beta.
oligomer, amylin oligomer and glycosylated protein to protect cell
viability. Therefore, we identify this new type of protein sDSS1 as
a promising drug for preventing and treating the diseases induced
by oxidized protein, glycosylated protein, A.beta., amylin and
other pathogenic proteins with similar features.
[0024] The specific technical solution is described below:
[0025] A sDSS1 protein is provided, which may have an amino acid
sequence of human protein sDSS1 as shown in SEQ ID NO: 1. A protein
having the same or similar amino acid sequence as SEQ ID NO: 1
exists in the Anthropoidea animals.
[0026] Preferably, the Anthropoidea animals may further be
chimpanzee, bonobo, gorilla, orangutan, white-cheeked gibbon,
golden snub-nosed monkey, rhesus macaque, olive baboon, Angola
colobus, sooty mangabey, drill and northern pigtail macaque;
wherein Pan troglodytes (a chimpanzee) sDSS1 protein has an amino
acid as set forth in SEQ ID NO: 5, Pan paniscus (a bonobo) sDSS1
protein has an amino acid as set forth in SEQ ID NO: 6, Gorilla
gorilla (a gorilla) sDSS1 protein has an amino acid as set forth in
SEQ ID NO: 7, Pongo abelii (an orangutan) sDSS1 protein has an
amino acid as set forth in SEQ ID NO: 8, Nomascus leucogenys (a
white-cheeked gibbon) sDSS1 protein has an amino acid as set forth
in SEQ ID NO: 9, Rhinopithecus roxellana (a golden snub-nosed
monkey) sDSS1 protein has an amino acid as set forth in SEQ ID NO:
10, Macaca mulatta (a rhesus macaque) sDSS1 protein has an amino
acid as set forth in SEQ ID NO: 11, Papio anubis (an olive baboon)
sDSS1 protein has an amino acid as set forth in SEQ ID NO: 12,
Colobus angolensis (a Angola colobus sDSS1 protein has an amino
acid as set forth in SEQ ID NO: 13, Cercocebus atys (a sooty
mangabey) sDSS1 protein has an amino acid as set forth in SEQ ID
NO: 14, Mandrillus leucophaeus (a drill) sDSS1 protein has an amino
acid as set forth in SEQ ID NO: 15, Macaca nemestrina (a northern
pigtail macaque) sDSS1 protein has an amino acid as set forth in
SEQ ID NO: 16.
[0027] Preferably, the sDSS1 protein includes a N-terminal amino
acid sequence of 58 amino acids and a C-terminal amino acid
sequence of 31 amino acids, wherein the human sDSS1 protein has a
N-terminal amino acid sequence of 58 amino acids as set forth in
SEQ ID NO: 3, the human sDSS1 protein has a C-terminal amino acid
sequence of 31 amino acids as set forth in SEQ ID NO: 2; wherein
the N-terminal amino acid sequence of the 58 amino acids includes 3
or more amino acid sequences with consecutive acidic amino acids,
each of amino acid sequences with consecutive acidic amino acids
includes no more than 10 acidic amino acids, any two adjacent amino
acid sequences of the amino acid sequences with consecutive acidic
amino acids have a spacing of no more than 4 amino acids, and the
spacing includes at least one hydrophobic amino acid, a pH value is
not higher than 4.5, the N-terminal amino acid sequence of the 58
amino acids includes no less than 10 acidic amino acids; the
C-terminal amino acid sequence following position 58 of the
N-terminal amino acid sequence of the 58 amino acids are relatively
hydrophobic overall, the C-terminal amino acid sequence of the 31
amino acids includes no less than 10 hydrophobic amino acids;
[0028] wherein the hydrophobic amino acid is selected from the
group consisting of alanine, isoleucine, leucine, valine, cysteine,
phenylalanine, methionine, tryptophan, and tyrosine,
[0029] the neutral amino acid is selected from the group consisting
of threonine, glycine, serine, histidine, and glutamine;
[0030] the acidic amino acid is selected from the group consisting
of glutamic acid, aspartate, proline, and asparaginate; and
[0031] the basic amino acids is selected from the group consisting
of arginine, and lysine.
[0032] Preferably, the sDSS1 protein in the Anthropoidea animals
includes a C-terminal amino acid sequence of: [0033]
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.-
sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19X.su-
b.20X.sub.21X.sub.22X.sub.23X.sub.24X.sub.25X.sub.26X.sub.27X.sub.28X.sub.-
29X.sub.30X.sub.31;
[0034] X.sub.1 is a neutral amino acid; X.sub.2 is a hydrophobic
amino acid; X.sub.3 and X.sub.4 are hydrophobic amino acids;
X.sub.5 is a hydrophobic amino acid; X.sub.6 is a hydrophobic amino
acid; X.sub.7 is a hydrophobic amino acid; X.sub.8 is a hydrophobic
amino acid; X.sub.9 is a hydrophobic amino acid; X.sub.10 is an
acidic amino acid; X.sub.11 is a neutral amino acid; X.sub.12 is a
hydrophobic amino acid; X.sub.13 is a hydrophobic amino acid;
X.sub.14 is a neutral amino acid; X.sub.15 is a hydrophobic amino
acid; X.sub.16 is a hydrophobic amino acid; X.sub.17 is a
hydrophobic amino acid; X.sub.18 is a hydrophobic amino acid;
X.sub.19 is a basic amino acid; X.sub.20 is an acidic amino acid;
X.sub.21 is a basic amino acid; X.sub.22 is a neutral amino acid;
X.sub.23 is a basic amino acid; X.sub.24 is a hydrophobic amino
acid; X.sub.25 is a hydrophobic amino acid; X.sub.26 is a neutral
amino acid; X.sub.27 is a hydrophobic amino acid; X.sub.28 is a
hydrophobic amino acid; X.sub.29 is a hydrophobic amino acid;
X.sub.30 is a hydrophobic amino acid; and X.sub.31 is a hydrophobic
amino acid;
[0035] an amino acid sequence having 40% or more homology to the
C-terminal amino acid sequence of the 31 amino acids, wherein the
amino acid sequence has a same or similar property and function to
a C-terminal amino acid sequence of a human sDSS1 protein.
[0036] A polypeptide comprising an amino acid sequence constructed
based on the N-terminal 58 amino acid sequence and the C-terminal
31 amino acid sequence of the sDSS1 protein as described
hereinabove, wherein
[0037] 1) the polypeptide sequence has a N-terminal having 40% or
more homology to the N-terminal amino acid sequence of the 58 amino
acids, and the polypeptide sequence has a C-terminal having 40% or
more homology to the C-terminal amino acid sequence of the 31 amino
acids, a protein encoded by the polypeptide sequence has a same or
similar property and function to a human sDSS1 protein; or
[0038] 2) a N-terminal of the polypeptide sequence is based on a
N-terminal amino acid sequence of 58 amino acids of a human sDSS1
protein, or is a sequence having 40% or more homology to the
N-terminal amino acid sequence of the 58 amino acids of the human
sDSS1 protein, wherein a C-terminal or the N-terminal of the
polypeptide is fused with other amino acid sequence, the other
amino acid sequence for fusion has an identical or similar property
to a C-terminal amino acid sequence of 31 amino acids of the human
sDSS1 protein and perform the same or similar functions, a modified
protein encoded by the polypeptide sequence performs an identical
or similar function to the human sDSS1 protein; or
[0039] 3) the peptide sequence is constructed by fusing the
C-terminal amino acid sequence of the 31 amino acids in the sDSS1
protein, such as is described hereinabove, with other polypeptide
sequence.
[0040] The fusion protein includes a full sequence or a partial
sequence of the sDSS1 protein such as is described hereinabove, and
the polypeptide sequence such as is described hereinabove.
[0041] Preferably, the fusion protein is a protein complex formed
by linking the protein sDSS1 protein, a carrier protein, an
antibody or other arbitrary amino acid sequence.
[0042] A complex includes a full sequence or a partial sequence of
the sDSS1 protein such as is described hereinabove, the polypeptide
sequence such as is described hereinabove, or a full sequence or a
partial sequence of the fusion protein such as is described
hereinabove.
[0043] Preferably, the complex is a complex formed by linking the
sDSS1 protein to a pharmaceutically acceptable drug carrier.
[0044] Preferably, the pharmaceutically acceptable drug carrier
includes one or more of a microsphere/capsule, liposome,
micro-emulsion, nanoparticle, magnetic particle and gel.
[0045] A nucleotide encodes the sDSS1 protein such as is described
hereinabove, or the polypeptide such as is described
hereinabove.
[0046] Preferably, the nucleotide includes DNA and RNA.
[0047] A cell expresses the sDSS1 protein such as is described
hereinabove or the polypeptide such as is described
hereinabove.
[0048] Preferably, the cell is a stem cell, a precursor cell or an
adult cell of a mammal.
[0049] Preferably, the mammal is a human, an orangutan, a monkey, a
horse, a cattle, a sheep, a pig, a donkey, a dog, a rabbit, a cat,
a rat or a mouse.
[0050] Preferably, the cell includes an embryo stem cell, an
induced multipotential stem cell or a stem cell derived from a
primary culture, a multipotential or monopotential stem cell
derived from a mother cell differentiation.
[0051] An expression system, wherein a nucleotide sequence encoding
the sDSS1 protein such as is described hereinabove or the
polypeptide such as is described hereinabove is introduced into an
organism, and the sDSS1 protein such as is described hereinabove or
the polypeptide such as is described hereinabove is expressed in
the organism.
[0052] Preferably, the expression system is selected from the group
consisting of eukaryotic expression plasmid vector, adenovirus,
slow virus, retrovirus, CRISPR/Cas technique and other feasible
gene-editing techniques.
[0053] Preferably, the organism is a human, an orangutan, a monkey,
a horse, a cattle, a sheep, a pig, a donkey, a dog, a rabbit, a
cat, a rat, a mouse, a chicken, a duck or a goose.
[0054] A drug primarily targets the sDSS1 protein such as is
described hereinabove or the polypeptide such as is described
hereinabove, wherein the drug can affect an expression level of the
sDSS1 protein such as is described hereinabove or the polypeptide
such as is described hereinabove in the organism upon
administration.
[0055] Preferably, the drug is a chemical micromolecular drug, a
protein/polypeptide drug, a nucleic acid drug, or a nanodrug.
[0056] Preferably, the nucleic acid drug includes one or more of a
siRNA, a microRNA, an antisense oligonucleotide, a triple strand
DNA and a ribozyme.
[0057] A method of producing a protein, includes the following
steps:
[0058] S1. constructing an expression vector: inserting a
nucleotide sequence coding the sDSS1 protein such as is described
hereinabove or the polypeptide such as is described hereinabove
into a plasmid and introducing the plasmid into bacteria or yeast
cell, or inserting the nucleotide sequence coding the sDSS1 protein
such as is described hereinabove or the polypeptide such as is
described hereinabove into genome of an insect cell or a mammalian
cell;
[0059] S2. expressing the sDSS1 protein: expanding a culture of the
bacteria, yeast cell, insect cell or mammalian cell as modified in
S1, and collecting a culture medium or cell lysate containing the
sDSS1 protein such as is described hereinabove or the polypeptide
such as is described hereinabove;
[0060] S3. purifying the sDSS1 protein: coarse filtering and
purifying the culture medium or cell lysate obtained in S2 to
obtain the sDSS1 protein.
[0061] A method of producing a protein, includes using chemical
synthesis technique to produce the sDSS1 protein such as is
described hereinabove or the polypeptide such as is described
hereinabove.
[0062] A method of producing a protein, includes using in vitro
ribosome expression system to produce the sDSS1 protein such as is
described hereinabove or the polypeptide such as is described
hereinabove.
[0063] A method of diagnosing, preventing or treating disease,
includes preparing a diagnostic reagent, a preventive drug, or a
therapeutic drug using the sDSS1 protein, polypeptide, fusion
protein, complex, nucleotide sequence, cell, expression system, or
drug such as is described hereinabove, and administering the
diagnostic reagent, preventive drug, or therapeutic drug to a
subject in need thereof.
[0064] Preferably, the disease is a disease induced by excessive
formation or accumulation of pathogenic protein/polypeptide.
[0065] Preferably, the pathogenic protein/polypeptide is an
oxidized protein product, glycosylated protein product, an amyloid
precursor protein and a spliceosome thereof, an islet amyloid
polypeptide and a spliceosome thereof, or other pathogenic
protein/polypeptides having features similar to an oxidized protein
a glycosylated protein, an amyloid protein or an islet amyloid
polypeptide.
[0066] Preferably, the diagnosing of the disease includes detecting
one or more of an expression level of a full or partial sequence of
the amino acid sequence, mRNA level and number of gene copies of
the sDSS1 protein such as is described hereinabove.
[0067] Preferably, the preventing includes one or more of genetic
modification, nucleic acid introduction, drug
injection/administration, cellular transplantation and tissue
transplantation.
[0068] Preferably, the treating includes one or more of genetic
modification, nucleic acid introduction, drug
injection/administration, cellular transplantation and tissue
transplantation.
[0069] The characteristics and/or beneficial effects of the present
application are:
[0070] 1. The polypeptide sequence of the sDSS1 protein and typical
human sDSS1 protein provided by the present application is
MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNWDDDNVEDDFSNQLRATVLL
MILVCETPYGCYVLHQKGRMCSAFLCC (see SEQ ID NO: 1). According to
bioinformatic analysis, the protein is a protein of anthropoid
subfamily animals.
[0071] 2. According to bioinformatic analysis and cell experiment,
the 31-amino acid carbon terminal sequence of the sDSS1 protein is
a signal peptide and has critical effect on the properties and
secretion property of the protein. The C-terminal sequence of the
31 amino acids is TVLLMILVCETPYGCYVLHQKGRMCSAFLCC (see SEQ ID NO:
2).
[0072] 3. The sDSS1 protein defined in the present application can
be combined with oxidized protein, glycosylated protein, A.beta.
protein and amylin protein and shield the cytotoxicity induced by
aggregation of these toxoproteins, so it has important potential in
treating the diseases induced by excessive formation or excessive
accumulation of these toxoproteins and other pathogenic proteins
with similar features.
[0073] 4. The sDSS1 protein of the present application is produced
by fermentation of Escherichia coli. The nucleotide sequence coding
the sDSS1 protein is inserted into pET151D plasmid, during sDSS1
expression, the N-terminal is fused with a 6-his tag and a V5 tag
for purification and immunoblotting detection. The expression of
protein in Escherichia coli is preliminarily purified by using
Ni-NTA gel column, and then the SDS-PAGE is used for gel
purification. The cut strip of gel containing His-V5-sDSS1 protein
is put in a dialysis bag containing transfer buffer, the protein is
extracted from the gel under the drive of electric field and
collected in the dialysis bag. The protein purified by the SDS
polyacrylamide gel electrophoresis analysis can reach the level for
bioexperiment.
[0074] 5. According to molecular experiment, the sDSS1 protein of
the present application can be combined with the oxidized protein
in serum and the oxidized protein in buffer solution to form
conjugates, or combine with A.beta. protein to reduce the formation
of A.beta. oligomer.
[0075] 6. The cell experiment proves that the sDSS1 protein of the
present application can shield the cytotoxicity induced by oxidized
protein, glycosylated protein, A.beta. oligomer and amylin oligomer
in the culture medium effectively, so as to maintain the cell
viability.
[0076] To sum up, the present application provides a sDSS1 protein,
the biological property and activity of sDSS1 protein are proved by
the research in bioinformatics, molecular biology and cellular
biology. The sDSS1 protein can reduce the cytotoxicity induced by
oxidized protein, glycosylated protein, A.beta. oligomer and amylin
oligomer in culture medium effectively to maintain cell viability.
As the sDSS1 protein is a congenital protein of higher primate, it
is free of immunoreaction in clinical application. Therefore, the
present application provides a candidate drug for preventing and
treating the diseases induced by excessive formation or excessive
accumulation of oxidized protein, glycosylated protein, A.beta.
protein, amylin polypeptide and other pathogenic proteins with
similar features, and it has important application prospects in
biomedicine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0077] The present application is further explained by the
following attached figures, so as to make the present application
clear and complete, but not to limit the scope of protection of the
present application.
[0078] FIG. 1A. illustrates the comparison between human DSS1 gene
cDNA and human sDSS1 gene cDNA.
[0079] The sDSS1 gene is a new subtype of DSS1 gene, the comparison
between human DSS1 gene cDNA (NM 006304.1, 509 bp) and human sDSS1
gene cDNA (AK309241.1, 1195 bp) shows an overlapping area, the
nucleic acid sequence of the overlapping area can encode N-terminal
58 amino acid sequences according to analysis.
[0080] FIG. 1B. illustrates the comparison of The sDSS1 protein
amino acid sequences of 13 species of primates.
[0081] The sDSS1 protein amino acid sequences of 13 species of
primates were compared by using Clustal X2.1 software, the results
show that the sDSS1 protein amino acid sequence is highly
conservative, N-terminal 58 amino acid sequences are identical, and
the C-terminal 31 amino acid sequences have point mutation only at
a few sites.
[0082] FIG. 2A. illustrates the GFP protein distribution.
[0083] In plasmid transfected 293T cell, the GFP protein
distribution was observed 24 h later. The green fluorescence in
control cell (GFP) was clear and bright, and the background in
solution was dim. Obvious green fluorescence signal was observed in
the culture solution of sDSS1 and GFP chelated protein (sDSS1-GFP)
or sDSS1 protein C-terminal 31 amino acid sequences and GFP
chelated protein (sDSS1-c-GFP), and the intracellular fluorescence
disperses and the intensity declines, meaning that the GFP protein
was taken out of the cell with the sDSS1 protein or sDSS1 protein
C-terminal sequence secretion.
[0084] FIG. 2B. illustrates that the sDSS1 protein is a secretory
protein.
[0085] Point membrane immunoblotting tests for detecting
transfected cell culture medium, the results show that the GFP
signal was detected in sDSS1-GFP and sDSS1-c-GFP culture media, and
there was no obvious signal detected in blank control and GFP
control group, proving that the sDSS1 protein is a secretory
protein, and C-terminal 31 amino acid sequences are signal
peptide.
[0086] FIG. 3A. illustrates that the sDSS1 signal can be detected
in the human serum or human cerebral spinal fluid (CSF) sample.
[0087] The sDSS1 signal can be detected in the human serum or human
cerebral spinal fluid (CSF) sample by using specific antibody of
sDSS1 protein C-terminal polypeptide sequence (antigen sequence:
C-terminal 31 amino acid sequences of sDSS1 protein). The Human CSF
sample was from senior citizens, the serum sample a was from the
blood of a youth after strenuous exercise, the serum samples b, c
and d were from the blood of youths in resting state.
[0088] FIG. 3B. illustrates the specific mRNA sequence of sDSS1
gene.
[0089] The specific mRNA sequence of sDSS1 gene (amplified product
is 293 bp) can be detected in human astrocytomas glioblastoma (U-87
MG) by using PCR, the DSS1 gene is used as control (amplified
product is 238 bp).
[0090] FIG. 4A-FIG. 4B. illustrates that coomassie brilliant blue
staining shows the content of objective protein in the sDSS1
protein production and purification processes.
[0091] FIG. 4A. The positive Escherichia coli cloning strain was
selected to expand culture, the addition of IPTG can induce the
expression of sDSS1 protein, the expression level of objective
protein in the cell without induction was very low. FIG. 4B. The
concentrated lysate after preliminary purification of Ni-NTA gel
column and the objective protein content after purification were
tested, channel a shows the purified sDSS1 protein, channel b shows
the preliminarily purified cell lysis solution.
[0092] FIGS. 5A-5D. illustrates that biochemical experiment and
cell experiment prove that the sDSS1 protein can combine with
oxidized protein and shield the toxicity of oxidized protein.
[0093] FIG. 5A. The 0.72 .mu.g purified sDSS1 protein was mixed
with different proportions of serum protein for incubation, the
sDSS1 protein was tested by V5 conjugated protein (V5-HRP), the
result shows that the sDSS1 protein and oxidized protein of serum
formed macromolecular protein complex.
[0094] FIG. 5B. The AOPP (200 .mu.g/mL) and the purified sDSS1
proteins at different concentrations were incubated at 4.degree. C.
over night, the product was separated by SDS-PAGE, and the
Coomassie brilliant blue staining shows that the sDSS1 protein and
AOPP can form macromolecular complex, the complex content increases
with sDSS1 protein concentration. FIG. 5C. The culture medium is
mixed with 10% oxidized serum, the cell proliferation was reduced
significantly, the sDSS1 protein in culture medium can shield the
cytotoxicity derived from the oxidized serum. FIG. 5D. The culture
medium without serum was mixed with 100 .mu.g/mL AOPP protein to
reduce the cell viability, the addition of sDSS1 protein at
isoconcentration can retrieve cell viability, the 100 .mu.g/mL BSA
was used for control group. The data was analyzed by t-test
two-tailed test and validated by ANOVE. **, p-value<0.01.
[0095] FIGS. 6A-6E. illustrates that the sDSS1 protein reduces the
formation of A.beta. oligomer, and reduces the cytotoxicity and
cell apoptosis induced by A.beta. oligomer.
[0096] FIG. 6A. Different proportions of sDSS1 protein were mixed
with 10 .mu.g A.beta. protein before incubation, according to
A.beta. antibody test, the sDSS1 protein and A.beta. formed
covalently conjugated high molecular weight protein complex, such a
conjugation can reduce the formation of A.beta. oligomer with
cytotoxicity. FIG. 6B. V5 sDSS1 protein was tested by conjugated
protein (V5-HRP), the result shows that the sDSS1 protein and
A.beta. formed a protein complex. FIG. 6C. The addition of A.beta.
oligomer to the culture medium induced cytotoxicity, the cell
viability was degraded, the sDSS1 protein can shield the
cytotoxicity induced by A.beta. oligomer completely. FIG. 6D. The
cell apoptosis experiment shows that the sDSS1 protein added to the
culture medium reduced the early apoptosis and late apoptosis of
SH-SY5Y cells induced by A.beta. oligomer significantly, so as to
reduce the effect of toxoprotein on cells. FIG. 6E. The sDSS1
protein can shield the toxicity of A.beta. oligomer for mouse nerve
stem cells (NSCs). The data was analyzed by t-test two-tailed test
and validated by ANOVE. **, p-value<0.01.
[0097] FIG. 7. illustrates that the addition of sDSS1 protein can
retrieve the cell viability, promoting the cell survival.
[0098] The amylin oligomer added to the culture medium induces
cytotoxicity and reduces cell viability, the addition of sDSS1
protein can retrieve the cell viability, promoting the cell
survival. The data was analyzed by t-test two-tailed test and
validated by ANOVE. **, p-value<0.01.
[0099] FIG. 8. illustrates that the cell viability decline can be
retrieved by sDSS1 protein.
[0100] The cell viability decline induced by 400 .mu.g/mL
glycosylated protein can be retrieved by sDSS1 protein, the
retrieving effect increased with the sDSS1 protein concentration
(100 .mu.g/mL to 200 .mu.g/mL). The 400 .mu.g/mL BSA protein was
used for control group. The data was analyzed by t-test two-tailed
test and validated by ANOVE. **, p-value<0.01.
[0101] FIG. 9A. illustrates that Operating method of injecting
virus into the lateral ventricle of SAMP8 mouse and virus injection
site.
[0102] FIG. 9B. illustrates that the survival rate after operation
of the mouse injected with adenovirus expressing sDSS1 protein was
apparently higher than that of control mouse.
[0103] The adenovirus was injected into the lateral ventricle of a
5 months old senescence-accelerated mouse SAMP8 mouse (1 .mu.L,
virus into right and left brains respectively), the animal survival
was observed continuously. The result shows that the survival rate
after operation of the mouse injected with adenovirus expressing
sDSS1 protein was apparently higher than that of control mouse
(expressing GFP protein). The data was analyzed by ANOVE. **,
p-value<0.01.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0104] The preferred solutions of the present application are
described and validated with examples in the following text, not to
limit the scope of the present application. All scope of the
present application are subject to the scope of the Claims.
[0105] The experimental methods for the following cases are
conventional experimental methods unless otherwise specified.
[0106] In the following embodiments, the sDSS1 protein was produced
in-house and its purity reached the level for bioexperiment, the
other materials and reagents were commercially available.
Example 1, sDSS1 Protein is a Secretory Protein from Primate
[0107] Bioinformatic Analysis Tool:
[0108] National Center of Biotechnology Information (NCBI) genome
database; 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.
[0109] Bioexperimental Method:
[0110] 1. Cell culture, the 293T cells were bought from American
type culture collection (ATCC), the cells were cultured in the cell
culture medium containing 90% basal medium (Dulbecco's modified
eagle medium, DMEM) (Life technology C#12500062) and 10% Fetal
bovine serum (FBS) (Gibco C#10100-147), cultured in cell incubator
(temperature 37.degree. C., humidity 95%, CO2 concentration 5%),
subcultured once every two days.
[0111] 2. Cell transfection, the 293T cells were inoculated in a
6-well plate as per 3.times.10.sup.5 per well, mixed with 1.5 mL
cell culture medium, the plasmid was transfected when the cells
have been adhering to the wall for 12 hours. The eukaryotic
expression plasmid pCMV-C-Flag was used, the inserted nucleic acid
sequence expressing sDSS1 protein was expressed as SEQ ID NO: 17.
2500 ng of the plasmid was diluted and mixed with 750 uL
Opti-MEM.RTM.Medium (Life technology C#31985062) uniformly, 10
.mu.L transfection reagent Lip2000 (Invitrogen C#12566014) was
diluted and mixed with 7504, Opti-MEM.RTM.Medium uniformly, the
diluted plasmid solution was instilled into the diluted
transfection reagent drop by drop, mixed uniformly and incubated at
normal temperature for 5 minutes. The cell culture medium was
blotted from the 6-well plate, the cells were cleaned with PBS, and
then the incubated transfection working fluid was applied. The
cells were cultured in the incubator continuously, the fluorescent
protein expression of the cells 2 was observed during 24 to 48
hours.
[0112] 3. Western blotting, the PVDF membrane was activated by
methanol and dried, the control culture medium and different
transfection cell culture media were dripped onto the membrane.
When the membrane was dried, the PVDF membrane completed 1% BSA
sealing, primary antibody (Rabbit-anti-GFP) (Cell signal technology
C#2956) incubation, secondary antibody (Goat-anti-rabbit HRP
antibody) (Zsbio, ZDR-5403) incubation in turn. The membrane was
cleaned with PBST three times, developed by luminescent liquid
(Zsbio, ZLI-9017) and the bands were exposed by X-ray film.
[0113] Result Analysis:
[0114] In the bioinformatic analysis of shfml gene in human genome,
it was found that the gene has multiple transcripts (see shfml gene
information in NCBI database,
http://www.ncbi.nlm.nih.gov/gene/7979). Besides an mRNA sequence of
jointly coded DSS1 protein sequence (NM 006304.1, 509 bp), there is
a longer mRNA sequence (AK309241.1, 1195 bp). The short mRNA
sequence and long mRNA sequence only have 256 bp repeat sequence
(FIG. 1A). According to nucleic acid sequence analysis, it can be
seen from the Translate tool that the repeat sequence can encode
DSS1 protein N-terminal 58 amino acid sequences. The long mRNA
sequence coded for 89 amino acids. According to the alignment of
polypeptide sequences, the long mRNA encoded polypeptide sequence
and DSS1 polypeptide sequence have the overlapping area of
N-terminal 58 amino acids, and the variation area of 31 amino
acids. This new polypeptide was named secretory DSS1 protein
(sDSS1), the polypeptide sequences are expressed as follows:
TABLE-US-00001 DSS1 (Homo sapiens): (see SEQ ID NO: 4)
MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNWDD
DNVEDDFSNQLRAELEKHGYKMETS s-DSS1 (Homo sapiens): (see SEQ ID NO: 1)
MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNWDD
DNVEDDFSNQLRATVLLMILVCETPYGCYVLHQKGRMCSAFLCC
[0115] According to the screening of the sequenced primate genome
and other animal pattern genomes in NCBI database, only the genome
of Anthropoidea animals has similar long mRNA sequence and
polypeptide sequence similar to human sDSS1 protein, as shown in
Table 1. The polypeptide sequence alignment results show that the
sDSS1 protein sequence is highly conservative, the N-terminal 59
amino acid sequences are identical, the other C-terminal amino acid
sequences have a little point mutation (FIG. 1B).
TABLE-US-00002 TABLE 1 Primate Species Amino acid sequence
Haplorrhini Homo MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNWDDDNVE
sapiens DDFSNQLRATVLLMILVCETPYGCYVLHQKGRMCSAFL CC (see SEQ ID NO:
1) Pan MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW troglodytes
DDDNVEDDFSNQLRATVLLMILVCETPYGCYVLHQKGRMCSAFL CC (see SEQ ID NO: 5)
Pan MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW paniscus)
DDDNVEDDFSNQLRATVLLMILVCETPYGCYVLHQKGRMCSAFL CC (see SEQ ID NO: 6)
Gorilla MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW gorilla)
DDDNVEDDFSNQLRVTVLLMILVCETLYGCYVLHQKGRMCSAFL CC (see SEQ ID NO: 7)
Pongo MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW abelii
DDDNVEDDFSNQLRATILLMILVCETPYGCYVLHQKGRMCSAFLC C (see SEQ ID NO: 8)
Nomascus MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW leucogenys
DDDNVEDDFSNQLRATVLLMVLVCETPYGCYVLHQKERMCSAFL CC (see SEQ ID NO: 9)
Rhinopithecus MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW roxellana
DDDNVEDDFSNQLRATVLLMIKVYETPYGCYILHQKGRMCSAFL CC (see SEQ ID NO: 10)
Macaca MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW mulatta
DDDNVEDDFSNQLRATVLLMIKVYETPYGCYILHQKGRMCSAFL CC (see SEQ ID NO: 11)
Papio MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW anubis
DDDNVEDDFSNQLRATVLLMIKVYETPYGCYILHQKGRMCSAFL CC (see SEQ ID NO: 12)
Angola MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW colobus
DDDNVEDDFSNQLRATVLLMKKVYETPYGCYILHQKGRMCSAFL CC (see SEQ ID NO: 13)
Sooty MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW mangabey
DDDNVEDDFSNQLRATVLLMIKVYETPYGCYILHQKGRMCSAFL CC (see SEQ ID NO: 14)
Mandrillus MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW leucophaeus
DDDNVEDDFSNQLRATVLLMIKVYETPYGCYILHQKGRMCSAFL CC (see SEQ ID NO: 15)
Macaca MSEKKQPVDLGLLEEDDEFEEFPAEDWAGLDEDEDAHVWEDNW nemestrina
DDDNVEDDFSNQLRATVLLMIKVYETPYGCYILHQKGRMCSAFL CC (see SEQ ID NO:
16)
[0116] The sDSS1 protein amino acid sequence was analyzed by using
two kinds of secretory protein analysis and prediction software,
which are Wolf PSORT and SecretomeP 2.0. The prediction results
show that the sDSS1 protein is located outside the cells, similar
to multiple identified secretory proteins, it is estimated as a
secretory protein (Table 2). According to the analysis result of
Wolf PSORT software, the signal peptide cleavage site of sDSS1
protein is located between amino acids positions 58-59.
TABLE-US-00003 TABLE 2 SecretomeP 2.0 WoLF PS ORT Predicted
(Recommended threshold for (Numbers of similar protein Species Name
secreted protein: 0.6) secreted proteins) location Homo sapiens
0.85 28 Extracellular Pan troglodytes 0.85 28 Extracellular Pan
paniscus 0.85 28 Extracellular Nomascus leucogenys 0.85 27
Extracellular Gorilla gorilla 0.752 23 Extracellular Pongo abelii
0.86 27 Extracellular Rhinopithecus roxellana 0.836 29
Extracellular Macaca mulatta 0.836 29 Extracellular Angola colobus
0.823 28 Extracellular sooty mangabey 0.836 29 Extracellular M.
leucophaeus 0.836 29 Extracellular Macaca nemestrina 0.836 29
Extracellular Papio anubis 0.836 29 Extracellular
[0117] According to the bioinformatic analysis results, the
complete sequence or C-terminal 31 amino acid sequences (31 amino
acid sequences after amino acid position 58) of the protein are
connected to green fluorescent protein (GFP) and expressed in 293T
cells (sDSS1-GFP, sDSS1-c-GFP). The results show that the solution
had green fluorescence, the background emitted light, and the
fluorescence in the cells was dim. There was no fluorescence in the
control group (GFP) solution, the background was very dark, the
fluorescence in the cells was clear and bright (FIG. 2A). The cell
culture medium was tested by point membrane immunoblotting, the GFP
signal was detected in the cell culture media of sDSS1-GFP and
sDSS1-c-GFP groups, but the signal was not detected in the control
group (GFP) (FIG. 2B). To sum up these results, the sDSS1 protein
is a sort of secretory protein, it can be synthesized in the cells
and secreted out of the cells, the C-terminal 31 amino acid
sequences of sDSS1 protein perform the function of signal
peptide.
Example 2, sDSS1 Protein is a Naturally-Occurring Protein
[0118] 1. Human serum and CSF sample treatment. Fresh human whole
blood was collected, kept still at room temperature for 10-20
minutes, 3500 g centrifuged for 30 minutes, the supernatant was
human serum. The serum was mixed with 100 mM mercaptoethanol
uniformly and treated by boiling water bath for 10 minutes, 12000 g
high speed centrifuged for 10 minutes after cooling, the
supernatant and 1/5 of 5.times. loading buffer solution by volume
were mixed. The fresh CSF was obtained from hospital and placed in
ice box for transportation, treated on the day. The fresh CSF was
mixed with 5.times. loading buffer directly and made into samples
directly for loading.
[0119] 2. Western blotting, 15 .mu.L prepared loading sample was
put in the loading well, the protein was separated with 4-12%
prefabricated gel (Life technology C#NP0321BOX) and moved to PVDF
membrane. The membrane was subjected to primary antibody
(Rabbit-anti-sDSS1) (antigen sequence: C-terminal 31 amino acid
sequences of sDSS1 protein) incubation, PBST solution cleaning
three times; and secondary antibody (Goat-anti-rabbit HRP antibody)
incubation. It was cleaned with PBST three times, developed by
luminescent liquid and the bands were displayed by X-ray film.
[0120] 3. Cell culture, the human glioma cells (U87-MG cells) were
bought from ATCC, the cells were cultured in complete cell culture
medium containing 90% basal medium DMEM and 10% FBS, cultured in
cell incubator (temperature 37.degree. C., humidity 95%, CO2
concentration 5%), subcultured once every two days.
[0121] 4. PCR experiment, the U87-MG cells were collected and lysed
rapidly, the total RNA was extracted from cell lysis solution by
using a total RNA extraction kit (QIAGEN,51304), the RNA sample was
treated with 1 U/.mu.L DNase I at room temperature for 15 minutes
to remove residual genome DNA. The obtained RNA sample was all
converted and synthesized into cDNA by using a cDNA synthesis kit
(TransGen Biotech, AT301) and used as template sample for
subsequent PCR experiment. 20 .mu.L reaction system was used in the
PCR reaction, including 100 .mu.L, PCR premixed reagent (PCRTaq
Mixture) (Omega bio-tek, TQ2200), 0.54, cDNA template (3.5
.mu.g/mL), 0.54, primer, 94, ultrapure water, mixed uniformly
before PCR reaction. DSS1 cDNA primers: forward primer:
GCAGACAGTCGAGATGTCAGAG, reverse primer: TTCTTCTGGATGCTATGAAGTCTCC;
sDSS1 cDNA primers: forward primer: GCAGACAGTCGAGATGTCAGAG, reverse
primer: TGATGATCTGTTAACAGCAGAGG. PCR reaction procedure: 94.degree.
C. 10 minutes, cyclic reaction 40 times: including 94.degree. C. 10
s, 62.degree. C. 20 s, 72.degree. C. 20 s, 72.degree. C. 10 minutes
after the circulation is finished, stored at 4.degree. C. and the
DNA content in PCR product was tested by 3% sepharose [0.05% SYBR
Green Stain (Thermo Fisher, 4472903)] electrophoresis.
[0122] Result Analysis:
[0123] The signal of sDSS1 protein can be detected in CSF or serum
by using the specific antibody of sDSS1. The serum samples derived
from different individuals manifest different signal modes, the
sDSS1 signal in the serum of the individual after exercise was
apparently higher than that of the individual in resting state
(FIG. 3A). The mRNA signal of sDSS1 gene can be detected in U87-MG
cells, the gene sequencing result of PCR amplified product was
identical to the sequence of database (FIG. 3B). These results show
that the sDSS1 protein is a sort of protein, existing in CSF and
serum.
Example 3, Small-Amount Preparation of sDSS1 Protein
Experimental Method
[0124] 1. SDSS1 protein preparation: the nucleotide segment of
total gene synthesis coded human sDSS1 protein (see SEQ ID NO: 17)
was inserted into the back of Hisx6-V5 tag in pET151D. The plasmid
was transferred to the expression strain BL21 (DE3). The
Escherichia coli was fused with expression Hisx6-V5-sDSS1 protein,
the Ni-NTA gel column was used for preliminary purification, and
then the SDS-PAGE was used for gel purification. The cut strip
containing His-V5-sDSS1 protein was put in the bag filter with
transfer buffer. The protein was removed from the gel under the
drive of electric field and collected in the bag filter. The
protein was concentrated to about 500 dialyzed in PBS solution at
4.degree. C. four times, 200 ml each time.
[0125] 2. SDS polyacrylamide gel electrophoresis, the purified
sDSS1 protein or bacterial lysis solution protein was mixed with
5.times. loading buffer solution, treated by boiling water bath for
10 minutes, 12000 g high speed centrifuged for 10 minutes, the
supernatant was extracted for analysis. The protein was separated
by 4-12% prefabricated gel, the gel was stained for 1 hour using
Coomassie brilliant blue staining solution, and decolored by
destainer at room temperature over night. When the decolorization
was completed, the bands on the gel were observed and
photographed.
[0126] Result Analysis:
[0127] The positive cloned Escherichia coli strain was selected,
the culture was expanded, the bacterial cells were stimulated by
IPTG to express objective protein at the beginning of logarithmic
phase of bacterial growth. The bacteria were lysed, the objective
protein expression level was tested. The result shows after the
IPTG stimulation, the sDSS1 protein expression level of bacterial
cells was upgraded significantly. The protein bands were obvious in
the gel image (FIG. 4A). After the bacterial lysis solution was
preliminarily purified by Ni-NTA gel column, the objective protein
in concentrate was concentrated greatly (channel b), the impure
protein content decreased, very pure sDSS1 protein could be
obtained by further purification (channel a)(FIG. 4B), applicable
to subsequent bioexperiment. The purified sDSS1 protein was
quantified by BCA protein, the final concentration was 0.72 mg/ml,
stored at 4.degree. C. for future use.
Example 4, sDSS1 Protein Reacts with Oxidized Protein and Shields
Cytotoxicity of Oxidized Protein
Experimental Method
[0128] 1. Reaction between oxidized serum and sDSS1 protein, 3500 g
of fresh blood was centrifuged for 30 minutes, the upper serum was
extracted for subsequent experiment. The 10 .mu.L sDSS1 protein
solution (0.72 mg/mL) was mixed with 10, 20, 50 and 1004, oxidized
serums respectively, the mass ratios of sDSS1 to serum protein were
about 1:100, 1:200, 1:500 and 1:1000, mixed with 20 .mu.M Fenton
reagent (FeSO.sub.4 and H.sub.2O.sub.2 were mixed as per mass ratio
of 1:1), incubated in a dark place at 4.degree. C. over night. On
the next day, the reacting His-V5-sDSS1 was separated by using 10
.mu.L Ni-NTA beads. The reactant liquor was mixed with the beads at
4.degree. C. for 2 hours, the magnetic separation device adsorbed
the beads on the tube wall, the liquid was removed, 1 ml PBST was
applied, the tube was removed from the magnetic separation device,
after repeated oscillation cleaning, the magnetic separation device
adsorbed the beads, the PBST was sucked away, and the above steps
were repeated four times. Finally, the protein was eluted with 504,
TBS containing 50 mM EDTA, the eluent was mixed with isometric
2.times.SDS solution, treated at 100.degree. C. for 10 minutes,
12000 g centrifuged for 10 minutes, the supernatant was extracted
for test. The supernatant was mixed with 5.times. loading buffer
solution, heated at 100.degree. C. for 10 minutes, the prepared
sample was used for western blotting.
[0129] 2. Preparation of oxidized FBS and AOPP, 10 mL FBS was mixed
with 10 mM NaC10 and treated for 1 hour, the oxidized serum was
dialyzed continuously in PBS solution using 3000 Da bag filter for
24 hours, the solution was changed at intervals of 8 hours during
dialysis, the treated serum solution was mixed with 1 mM vitamin C
(Vc) to remove the participant oxidizer completely. The protein
concentration was tested by BCA protein quantification. The content
of oxidized protein was tested by using two methods, the dityrosine
value in the oxidized serum measured by chloramine-T was 75.31
.mu.M/mg protein (untreated serum was 15.05 .mu.mol/mg protein),
the carbonyl content detected by dinitrophenylhydrazine was 16.33
nmol/mg protein (untreated serum was 13.68 nmol/mg protein).
[0130] The 10 mg serum albumin was treated with 160 mM NaC10 for 1
hour, the oxidized protein was dialyzed continuously in PBS
solution using 3000 Da bag filter for 24 hours, the solution was
changed at intervals of 8 hours during dialysis. The protein
concentration of the treated AOPP was determined by BCA protein
quantification. The dityrosine value of AOPP sample measured by
chloramine-T was 54.21 .mu.mol/mg protein (untreated BSA was 14.55
.mu.mol/mg protein), the carbonyl content measured by
dinitrophenylhydrazine was 1042.57 nmol/mg protein (untreated BSA
was 10.26 nmol/mg protein).
[0131] 3. Reaction between AOPP and sDSS1 protein, the 1504,
reaction system was mixed with 30 .mu.g AOPP protein (200
.mu.g/mL), and mixed with 15 .mu.g (100 .mu.g/mL), 30 .mu.g (200
.mu.g/mL) and 60 .mu.g (400 .mu.g/mL) sDSS1 protein respectively,
the excess volume was supplemented by aseptic PBS solution. The
solution was stirred uniformly and reacted at 4.degree. C. over
night. The sample after reaction was mixed with 5.times. loading
buffer solution, heated at 100.degree. C. for 10 minutes, the
treated sample was separated by SDS-PAGE and the bands were
displayed by Coomassie brilliant blue staining.
[0132] 4. Western blotting, the protein mixture after reaction was
mixed with 5.times. loading buffer, treated by boiling water bath
for 10 minutes for western blotting analysis. The specific method
was the same as described above. The antibody was V5-HRP antibody
(1:5000 diluted).
[0133] 5. Cell line culture, the human neuroblastoma cells
(SH-SYSY) were grown in the basal medium DMEM with 10% FBS; the
cells were subcultured once every two days.
[0134] 6. Cell viability test, in order to test the effect of sDSS1
protein on the cytotoxicity of oxidized serum, the SH-SYSY cells
were inoculated to a 96-well plate as per 10.sup.4 cells per well,
2000, complete medium. 12 hours later, the complete medium was
changed to DMEM without serum containing 0.5% BSA 2004, per well.
After 24 hours of treatment, the DMEM solution was changed to 10%
oxidized serum and 10% oxidized serum containing 20 .mu.g/mL sDSS1
protein as culture medium, 2004, per well. After 48 hours of
treatment, the old culture medium was removed from the 96-well
plate, 1004, diluted CCK-8 working fluid (1:20 diluted) (DOJINDO,
CK04) was put in each well to test the changes in cell viability.
The group with BSA at isoconcentration was the control group.
[0135] In order to test the protective effect of sDSS1 protein on
the cytotoxicity induced by AOPP, the SH-SYSY cells were inoculated
to the 96-well plate as per 2.times.10.sup.4 cells per well, after
12 hours of adhesion, the culture medium was changed to culture
medium without serum containing 0.5% BSA. After 24 hours of
treatment, it was changed to culture medium without serum
containing 100 .mu.g/mL AOPP protein, and the treatment group was
provided with 100 .mu.g/mL sDSS1 protein, 2004, per well. After 48
hours of treatment of 96-well plate, the changes in cell viability
were tested by using CCK-8 kit.
[0136] Result Analysis:
[0137] The serum contained a lot of proteins, mainly being serum
albumin. Under the effect of Fenton reagent, the proteins in serum
were oxidized, the oxidation products reacted with sDSS1 to foam
complexes. In control group, the sDSS1 protein monomer had no
obvious protein aggregation. In the experimental group, the
co-incubation with serum led to the formation of lots of high
molecular weight protein complexes, these complexes cannot be
separated by SDS-PAGE (FIG. 5A). The result shows that the sDSS1
protein can combine with the oxidized protein in serum. In the
cytotoxicity experiment, compared with control serum, the addition
of 10% oxidized protein could depress cell proliferation and cell
viability obviously, and 20 .mu.g/mL sDSS1 protein in the culture
medium could retrieve the cytotoxicity of oxidized protein (FIG.
5B).
[0138] The sDSS1 was mixed with AOPP, the sDSS1 and AOPP were
combined to form complexes, these complexes cannot be separated by
SDS-PAGE. The number of complexes increased apparently with the
sDSS1 protein concentration in the reaction system (FIG. 5C). In
the cell experiment, the AOPP had significant cytotoxicity for
cells, reducing the cell viability, and the sDSS1 at
isoconcentration could shield the cytotoxicity of AOPP completely
(FIG. 5D). To sum up the results, the sDSS1 can protect the cells
from the cytotoxicity of oxidized serum or AOPP.
[0139] In addition, to sum up the reaction between sDSS1 protein
and oxidized protein, two proteins can combine with the oxidized
protein tightly, this binding force can resist high concentration
of SDS, which seems to be covalent interaction. The difference is
that the combination process of DSS1 and oxidized protein needs the
assistance of an ATP enzyme [Zhang et al, 2014]. Our evidence shows
that the tight coupling of sDSS1 and oxidized protein is free of
ATP, the ATP enzyme is not required. According to the amino acid
sequences of DSS1 and sDSS1, the sequences of amino acid positions
1 to 58 of the two proteins are identical, and the sequence of
amino acid positions 59 to 70 of DSS1 are completely different from
the sequence of amino acid positions 59 to 89 of sDSS1. The tight
coupling of DSS1 and sDSS1 with oxidized protein is supposed to be
derived from the shared amino acid sequences, i.e. the sequence of
the first 58 amino acids. The difference in characteristic between
sDSS1 and DSS1, which is the characteristic that the tight coupling
with oxidized protein is free of ATP enzyme mediation, should be
derived from the unique amino acid sequence of sDSS1, i.e.
C-terminal amino acid sequence of positions 59 to 89. Altogether,
the tight coupling with oxidized protein without ATP enzyme
mediation of sDSS1 is derived from the organic combination of the
sequences of the first 58 amino acids and the last 31 amino
acids.
Example 5, sDSS1 Protein Reduces the Formation of A.beta. Oligomer
and Reduces the Cytotoxicity of A.beta. Oligomer
Experimental Method
[0140] 1. Cell line culture, the human neuroblastoma cells
(SH-SY5Y) were grown in the basal medium DMEM with 10% FBS; the
cells were subcultured once every two days.
[0141] 2. Neural stem cell culture, the neural stem cells (NSCs)
were from P2 mouse brain tissue, the NSCs of primary suspension
culture were used for toxicity test after two subcultures, the NSCs
were cultured in the stem cell culture medium, including 88%
DMEM/F12 basal medium (Gibco, C#12500-062), 10% Proliferation
supplementary additive (Stem cell technology, C#05701), 2% BSA
(Sigma, C#V900933), 10 ng/mL Heparin (Sigma, C#H3149), 10 ng/mL
bFGF (Roche, C#11104616001), 20 ng/mL EGF (BD Bioscience,
C#354010).
[0142] 3. Reaction between A.beta. and sDSS1 proteins, the A.beta.
protein (Human, 1-42) freeze-dried powder was supplied from Suzhou
Qiangyao Biotechnology Co., Ltd. 2 mg A.beta. freeze-dried powder
was dissolved by 20 .mu.L DMSO, diluted with PBS to 2 mg/mL, stored
at -20.degree. C. The reaction system was provided with 300 .mu.L
PBS solution the 10 .mu.g A.beta. and sDSS1 proteins were mixed as
per molar mass ratios 1:1, 1:5 and 1:10, and then incubated at
4.degree. C. over night. The incubated reactant was mixed with
5.times. loading buffer solution, treated at 100.degree. C. for 10
minutes for western blotting analysis.
[0143] 4. A.beta. protein pretreatment, the A.beta. stock solution
was diluted with basal medium (pH7.2) to 1000 .mu.g/mL, the A.beta.
diluent was incubated at 4.degree. C. for 24 hours to form oligomer
for cell experiment. The A.beta. concentration in subsequent
experiment was always labeled according to the protein
concentration before incubation.
[0144] 5. Western blotting, the treated reactant was separated by
SDS-PAGE for western blotting analysis, the specific method was the
same as described above. The antibodies used were V5-HRP antibody
(1:5000 diluted), A.beta. antibody (Cell signal technology, 9888),
secondary antibody (Goat-anti-rabbit HRP antibody).
[0145] 6. Cell viability test, the SH-SY5Y cells were inoculated to
the 96-well plate as per 2.times.10.sup.4 cells per well, after 12
hours of adhesion, the old culture medium was changed to culture
medium without serum containing 0.5% BSA, after 24 hours of
treatment, the old culture medium was changed to DMEM solution
containing A.beta. or A.beta. and sDSS1 proteins. After 48 hours of
treatment of cells, the cell viability level was tested by CCK-8
kit.
[0146] 7. Cell apoptosis test, the cell apoptosis test kit was
bought from DOJINDO chemical technology (Shanghai) corp. (AD10).
The SH-SY5Y cells were inoculated to the 6-well plate as per
3.times.10.sup.5 cells per well, after 12 hours of adhesion, the
old culture medium was changed to DMEM solution without serum
containing 0.5% BSA. After 24 hours of treatment, it was changed to
solution containing A.beta. or A.beta. and sDSS1 proteins. After 48
hours of treatment, the cell apoptosis level was tested by
apoptosis kit. All of the solution and cells were collected, the
supernatant was removed by centrifugation. The cells were
resuspended in the 4004, staining buffer solution provided by the
apoptosis kit, 185 .mu.L cell suspension was extracted for
subsequent test. The cell suspension was mixed with 54, Annexin V
staining solution uniformly, the cells were incubated at 37.degree.
C. for 10 minutes. It was mixed with 10 .mu.L PI staining solution
uniformly, the cell apoptosis level was tested by flow
cytometer.
[0147] The NSCs were firstly adhered and cultured in the 6-well
plate, the plate was treated with 0.025% Laminin for at least two
hours and cleaned with aseptic PBS 6 times for future use. The NSCs
were made into unicells and inoculated to 6-well plate as per
3.times.10.sup.5 per well, the cells were adhered for 24 h for
subsequent experiment.
[0148] Result Analysis
[0149] The A.beta. protein had obvious aggregation after
incubation, there were protein aggregates of different sizes formed
within 10-20 KD. According to previous reports, these A.beta.
oligomers were the main source of the A.beta. induced cytotoxicity.
After co-incubation of sDSS1 protein and A.beta., the sDSS1 protein
and A.beta. protein aggregated to form high molecular weight
complex (molecular weight higher than 20 KD), the oligomers formed
within 10-20 KD were reduced obviously (FIG. 6A). As the sDSS1
protein concentration increased, the formation of A.beta. oligomer
was depressed apparently. According to the sDSS1 protein signal
detection, the complex was formed by the reaction between sDSS1
protein and A.beta. (FIG. 6B), and it could not be separated by
SDS-PAGE.
[0150] The shielding effect of sDSS1 protein on the A.beta. induced
cytotoxicity was tested. In the cell viability test, the cell
viability declined significantly after the pretreated A.beta.
oligomer was applied. When the culture medium was mixed with sDSS1
protein, the SH-SY5Y cell viability was recovered significantly and
the cell viability was higher than control group (FIG. 6C). In the
cell apoptosis test, the addition of A.beta. oligomer to the
culture medium induced the apoptosis of SH-SY5Y cells or NSCs. The
addition of sDSS1 protein to the culture medium can reduce the
early apoptosis and late apoptosis levels of cells significantly
(FIG. 6D, FIG. 6E). According to the results, the sDSS1 protein can
combine with A.beta. protein to reduce the A.beta. oligomer
formation, so as to mitigate the cytotoxicity induced by A.beta.
protein.
Example 6 sDSS1 Protein Reduces Cytotoxicity of Amylin Oligomer
Experimental Method
[0151] 1. Cell line culture, the human neuroblastoma cells
(SH-SY5Y) were grown in the basal medium DMEM with 10% FBS; the
cells were subcultured once every two days.
[0152] 2. Amylin protein pretreatment, the amylin protein (Human)
freeze-dried powder was supplied from Suzhou Qiangyao Biotechnology
Corp. The 2 mg amylin freeze-dried powder was dissolved to 2 mg/mL
in 10 mM sodium acetate solution (pH5.5), stored at -20.degree. C.
The amylin stock solution was diluted to 1 mg/mL with basal medium
(pH7.2). The amylin diluent was incubated at 4.degree. C. for 48
hours to form oligomer for cell experiment. The amylin
concentration in subsequent experiment was always labeled according
to the protein concentration before incubation.
[0153] 3. Cell viability test, the SH-SY5Y cells were inoculated to
96-well plate as per 2.times.10.sup.4 cells per well, after 12
hours of adhesion, the old culture medium was changed to culture
medium without serum containing 0.5% BSA. After 24 hours of
treatment, the old culture medium was removed and the DMEM solution
containing amylin or amylin and sDSS1 proteins was applied. After
48 hours of treatment of cells, the cell viability level was tested
by CCK-8 kit.
[0154] Result Analysis
[0155] The addition of 10 .mu.M inbubated amylin protein to the
cell culture medium can induce significant cytotoxicity, and the
addition of sDSS1 protein can shield the cytotoxicity induced by
amylin oligomer, the cell viability was even higher than control
group (FIG. 7), meaning the sDSS1 protein can shield the
cytotoxicity of amylin protein effectively.
Example 7 sDSS1 Protein Reduces Cytotoxicity of Glycosylated
Protein Experimental Method
[0156] 1. Cell line culture, the human neuroblastoma cells
(SH-SY5Y) were grown in the basal medium DMEM with 10% FBS; the
cells were subcultured once every two days.
[0157] 2. Glycosylated protein preparation, 10 mg/mL serum albumin
and 2.5M ribose were mixed and incubated at 37.degree. C. for 7
days, and then dialyzed in PBS using 3000 Da bag filter for 24
hours, the solution was changed at intervals of 8 hours. The
completed glycosylated protein was quantified by BCA, the sample
was stored at -80.degree. C. for future use.
[0158] 3. Cell viability test, the SH-SY5Y cells were inoculated to
96-well plate as per 2.times.10.sup.4 cells per well, after 12
hours of adhesion, the old culture medium was changed to culture
medium without serum containing 0.5% BSA. After 24 hours of
treatment, the old culture medium was removed and the DMEM solution
containing glycosylated protein or glycosylated protein and sDSS1
proteins at different concentrations was applied, the group with
BSA at isoconcentration was used for control. After 48 hours of
treatment of cells, the cell viability level was tested by CCK-8
kit.
[0159] Result Analysis
[0160] The addition of 400 .mu.g/mL glycosylated protein to the
cell culture medium can induce significant cytotoxicity, and the
addition of sDSS1 protein can reduce the cytotoxicity induced by
glycosylated protein. As the sDSS1 protein concentration increased,
the cell viability was even higher than control group (FIG. 8),
meaning the sDSS1 protein can shield the cytotoxicity of
glycosylated protein effectively.
Example 8, sDSS1 Protein Prolongs Postoperative Survival Time of
Senescence-Accelerated Mouse SAMP
Experimental Method
[0161] 1. Animal feeding, the senescence-accelerated SAMP8 mice (5
months old, male) were bought from Beijing Vital River Laboratory
Animal Technology Co., Ltd., the animals were fed at the clean
laboratory animal breeding center of Southern Model Organism
Center. The animals were provided with sufficient aseptic water and
standard mouse breeding feed, 12 h/12 h dark-and-bright alternate
illumination, the bedding and cage were changed monthly, the animal
survival was observed daily.
[0162] 2. Adenovirus synthesis, the nucleic acid sequence (see SEQ
ID NO: 17) of adenovirus expressing sDSS1 protein was provided by
us (inventors), the adenovirus construction and synthesis were
completed by Cyagen (Guangzhou) Biotechnology Co., Ltd. The
adenovirus promoter and transcription region sequence composition:
pAV[Exp]-UBC>EGFP:T2A:ORF_363 bp, including ubiquitin protein
promoter sequence. According to determination, the virus titer was
larger than 10.sup.10 PFU/mL. According to the validation by
infecting U87-MG cells and mouse neuroblastoma cells (N2a), the
adenovirus can infect cells and express sDSS1 protein efficiently.
The adenovirus expressing GFP protein (pAV[Exp]-UBC>EGFP) was
used as control.
[0163] 3. Stereotactic injection, the senescence-accelerated SAMP8
mouse (6 months old, male) was anaesthetized by intraperitoneal
injection with 20% urethane (dissolved in normal saline, impurities
and bacteria removed by 0.22 .mu.m filter) as per 800 mg/kg body
weight. When the mouse was anaesthetized, it was fixed to the mouse
stereotaxic apparatus (Stoelting, 51500), the cranial bone was kept
horizontal. The head skin was incised to expose the cranial bone,
the bregma was taken as the starting coordinate to locate the
lateral ventricle region (0.58 mm, 1.25 mm, 1.75 mm). The marking
point was perforated by dental drill. 24, of virus liquid was
sucked by the microinjector (Hamilton 600-2.5 .mu.L, syringe needle
diameter 0.2 mm), the lateral ventricle region was relocated
according to the same coordinate values. The needle was inserted
into the lateral ventricle region quickly according to the
specified coordinate values, the virus liquid was injected slowly
at 200 nL/min on average, for a total amount of 1000 nL. After the
injection was done, each time the needle was lifted for 0.25 mm, it
was waited for 3 minutes until the needle was drawn out of the
brain tissue completely. The lateral ventricle region on the
opposite side was relocated, the needle insertion, injection and
needle lifting were completed, 1 .mu.L virus liquid was injected.
For the mouse after injection, the cranial bone and peripheral
tissues were wiped with 100 U/mL ampicillin/streptomycin, and the
skin was sutured. The abdominal cavity of the mouse was injected
with 150 .mu.L antibiotics, and the mouse was put in the cage with
abdomen up until the mouse was awake.
[0164] Result Analysis
[0165] The schematic diagram indicates the basic operation of
injecting virus into the mouse's lateral ventricle and the
adenovirus injection site (FIG. 9A). Wherein there were two batches
of mice injected with adenovirus expressing sDSS1, 10 mice in
total; there were two batches of mice injected with adenovirus
expressing GFP, 14 mice in total. Within 1 month after the
injection of adenovirus, three mice of the control group died, and
one mouse of the experimental group died. Within 8 months, the mice
of the control group died successively, whereas only one mouse of
the experimental group died (FIG. 9B). To sum up the results, the
postoperative survival time of the SAMP8 mice injected with
adenovirus expressing sDSS1 protein was significantly longer than
that of the mice only injected with control virus. The significant
difference was analyzed by ANOVA (P-value<0.01).
[0166] The above detailed description only specifies the feasible
embodiments of the present application and is not intended to limit
the scope of protection of the present application. Any equivalent
embodiments or alterations not deviating from the gist of the
present application shall be covered in the scope of protection of
the present application.
Sequence CWU 1
1
17189PRTHomo sapiensThe SDSS1 amino acid sequence of Homo sapiens
1Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp1 5
10 15Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp
Glu 20 25 30Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp
Asn Val 35 40 45Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu
Leu Met Ile 50 55 60Leu Val Cys Glu Thr Pro Tyr Gly Cys Tyr Val Leu
His Gln Lys Gly65 70 75 80Arg Met Cys Ser Ala Phe Leu Cys Cys
85231PRTHomo sapiensC-terminal 31 amino acid sequences of the SDSS1
2Thr Val Leu Leu Met Ile Leu Val Cys Glu Thr Pro Tyr Gly Cys Tyr1 5
10 15Val Leu His Gln Lys Gly Arg Met Cys Ser Ala Phe Leu Cys Cys 20
25 30358PRTHomo sapiensN-terminal 58 amino acid sequences of the
SDSS1 3Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu
Asp1 5 10 15Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu
Asp Glu 20 25 30Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp
Asp Asn Val 35 40 45Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala 50
55470PRTHomo sapiensThe DSS1 amino acid sequence of Homo sapiens
4Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp1 5
10 15Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp
Glu 20 25 30Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp
Asn Val 35 40 45Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Glu Leu Glu
Lys His Gly 50 55 60Tyr Lys Met Glu Thr Ser65 70589PRTPan
troglodytesThe SDSS1 amino acid sequence of Pan troglodytes 5Met
Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp1 5 10
15Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu
20 25 30Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn
Val 35 40 45Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu
Met Ile 50 55 60Leu Val Cys Glu Thr Pro Tyr Gly Cys Tyr Val Leu His
Gln Lys Gly65 70 75 80Arg Met Cys Ser Ala Phe Leu Cys Cys
85689PRTPan paniscusThe SDSS1 amino acid sequence of Pan paniscus
6Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp1 5
10 15Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp
Glu 20 25 30Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp
Asn Val 35 40 45Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu
Leu Met Ile 50 55 60Leu Val Cys Glu Thr Pro Tyr Gly Cys Tyr Val Leu
His Gln Lys Gly65 70 75 80Arg Met Cys Ser Ala Phe Leu Cys Cys
85789PRTGorilla gorillaThe SDSS1 amino acid sequence of Gorilla
gorilla 7Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu
Glu Asp1 5 10 15Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly
Leu Asp Glu 20 25 30Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp
Asp Asp Asn Val 35 40 45Glu Asp Asp Phe Ser Asn Gln Leu Arg Val Thr
Val Leu Leu Met Ile 50 55 60Leu Val Cys Glu Thr Leu Tyr Gly Cys Tyr
Val Leu His Gln Lys Gly65 70 75 80Arg Met Cys Ser Ala Phe Leu Cys
Cys 85889PRTPongo abeliiThe SDSS1 amino acid sequence of Pongo
abelii 8Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu
Asp1 5 10 15Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu
Asp Glu 20 25 30Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp
Asp Asn Val 35 40 45Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Ile
Leu Leu Met Ile 50 55 60Leu Val Cys Glu Thr Pro Tyr Gly Cys Tyr Val
Leu His Gln Lys Gly65 70 75 80Arg Met Cys Ser Ala Phe Leu Cys Cys
85989PRTPongo Nomascus leucogenysThe SDSS1 amino acid sequence of
Pongo Nomascus leucogenys 9Met Ser Glu Lys Lys Gln Pro Val Asp Leu
Gly Leu Leu Glu Glu Asp1 5 10 15Asp Glu Phe Glu Glu Phe Pro Ala Glu
Asp Trp Ala Gly Leu Asp Glu 20 25 30Asp Glu Asp Ala His Val Trp Glu
Asp Asn Trp Asp Asp Asp Asn Val 35 40 45Glu Asp Asp Phe Ser Asn Gln
Leu Arg Ala Thr Val Leu Leu Met Val 50 55 60Leu Val Cys Glu Thr Pro
Tyr Gly Cys Tyr Val Leu His Gln Lys Glu65 70 75 80Arg Met Cys Ser
Ala Phe Leu Cys Cys 851089PRTRhinopithecus roxellanaThe SDSS1 amino
acid sequence of Rhinopithecus roxellana 10Met Ser Glu Lys Lys Gln
Pro Val Asp Leu Gly Leu Leu Glu Glu Asp1 5 10 15Asp Glu Phe Glu Glu
Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu 20 25 30Asp Glu Asp Ala
His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val 35 40 45Glu Asp Asp
Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile 50 55 60Lys Val
Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly65 70 75
80Arg Met Cys Ser Ala Phe Leu Cys Cys 851189PRTMacaca mulattaThe
SDSS1 amino acid sequence of Macaca mulatta 11Met Ser Glu Lys Lys
Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp1 5 10 15Asp Glu Phe Glu
Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu 20 25 30Asp Glu Asp
Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val 35 40 45Glu Asp
Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile 50 55 60Lys
Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly65 70 75
80Arg Met Cys Ser Ala Phe Leu Cys Cys 851289PRTPapio anubisThe
SDSS1 amino acid sequence of Papio anubis 12Met Ser Glu Lys Lys Gln
Pro Val Asp Leu Gly Leu Leu Glu Glu Asp1 5 10 15Asp Glu Phe Glu Glu
Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp Glu 20 25 30Asp Glu Asp Ala
His Val Trp Glu Asp Asn Trp Asp Asp Asp Asn Val 35 40 45Glu Asp Asp
Phe Ser Asn Gln Leu Arg Ala Thr Val Leu Leu Met Ile 50 55 60Lys Val
Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly65 70 75
80Arg Met Cys Ser Ala Phe Leu Cys Cys 851389PRTPapio Colobus
angolensisThe SDSS1 amino acid sequence of Papio Colobus angolensis
13Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu Asp1
5 10 15Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu Asp
Glu 20 25 30Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp Asp
Asn Val 35 40 45Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val Leu
Leu Met Lys 50 55 60Lys Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile Leu
His Gln Lys Gly65 70 75 80Arg Met Cys Ser Ala Phe Leu Cys Cys
851489PRTCercocebus atysThe SDSS1 amino acid sequence of Cercocebus
atys 14Met Ser Glu Lys Lys Gln Pro Val Asp Leu Gly Leu Leu Glu Glu
Asp1 5 10 15Asp Glu Phe Glu Glu Phe Pro Ala Glu Asp Trp Ala Gly Leu
Asp Glu 20 25 30Asp Glu Asp Ala His Val Trp Glu Asp Asn Trp Asp Asp
Asp Asn Val 35 40 45Glu Asp Asp Phe Ser Asn Gln Leu Arg Ala Thr Val
Leu Leu Met Ile 50 55 60Lys Val Tyr Glu Thr Pro Tyr Gly Cys Tyr Ile
Leu His Gln Lys Gly65 70 75 80Arg Met Cys Ser Ala Phe Leu Cys Cys
851589PRTMandrillus leucophaeusThe SDSS1 amino acid sequence of
Mandrillus leucophaeus 15Met Ser Glu Lys Lys Gln Pro Val Asp Leu
Gly Leu Leu Glu Glu Asp1 5 10 15Asp Glu Phe Glu Glu Phe Pro Ala Glu
Asp Trp Ala Gly Leu Asp Glu 20 25 30Asp Glu Asp Ala His Val Trp Glu
Asp Asn Trp Asp Asp Asp Asn Val 35 40 45Glu Asp Asp Phe Ser Asn Gln
Leu Arg Ala Thr Val Leu Leu Met Ile 50 55 60Lys Val Tyr Glu Thr Pro
Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly65 70 75 80Arg Met Cys Ser
Ala Phe Leu Cys Cys 851689PRTMacaca nemestrinaThe SDSS1 amino acid
sequence of Macaca nemestrina 16Met Ser Glu Lys Lys Gln Pro Val Asp
Leu Gly Leu Leu Glu Glu Asp1 5 10 15Asp Glu Phe Glu Glu Phe Pro Ala
Glu Asp Trp Ala Gly Leu Asp Glu 20 25 30Asp Glu Asp Ala His Val Trp
Glu Asp Asn Trp Asp Asp Asp Asn Val 35 40 45Glu Asp Asp Phe Ser Asn
Gln Leu Arg Ala Thr Val Leu Leu Met Ile 50 55 60Lys Val Tyr Glu Thr
Pro Tyr Gly Cys Tyr Ile Leu His Gln Lys Gly65 70 75 80Arg Met Cys
Ser Ala Phe Leu Cys Cys 8517363DNAHomo sapiensnucleic acid sequence
which expressing sDSS1 protein of Homo sapiens 17atgagccacc
accaccacca ccacgccggc gactacaagg acgacgacga caagggcagc 60gactacaagg
acgacgacga caagggcagc atgagcgaga agaagcagcc cgtggacctg
120ggcctgctgg aggaggacga cgagttcgag gagttccccg ccgaggactg
ggccggcctg 180gacgaggacg aggacgccca cgtgtgggag gacaactggg
acgacgacaa cgtggaggac 240gacttcagca accagctgcg cgccaccgtg
ctgctgatga tcctggtgtg cgagaccccc 300tacggctgct acgtgctgca
ccagaagggc cgcatgtgca gcgccttcct gtgctgctaa 360taa 363
* * * * *
References