CN102584976B - A kind of human serum amyloid A 1 and its preparation method and application - Google Patents
A kind of human serum amyloid A 1 and its preparation method and application Download PDFInfo
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- CN102584976B CN102584976B CN201210037294.6A CN201210037294A CN102584976B CN 102584976 B CN102584976 B CN 102584976B CN 201210037294 A CN201210037294 A CN 201210037294A CN 102584976 B CN102584976 B CN 102584976B
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Abstract
The invention discloses a kind of human serum amyloid A 1 and its preparation method and application.The invention also discloses the polymer of human serum amyloid A 1 and the polypeptide fragment of human serum amyloid A 1.Human serum amyloid A 1 of the present invention, polymer, polypeptide fragment all have the ability suppressing expression of inflammatory cytokines, can be used for inflammation-inhibiting and treatment inflammation related disease.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of human serum amyloid A 1 and its preparation method and application.
Background technology
Serum amyloid A protein (Serum Amyloid A, SAA) is a kind of main acute phase protein of body secretion when being infected and damaging.SAA is the general name of a polymorphism albumen, by be correlated with but the albumen (SAA1-SAA4) of each tool separate gene forms.Wherein, human serum amyloid A 1 (SAA1) is made up of 104 amino acid, and primarily of liver cell synthesis, people SAA2 and people SAA1 has 7 amino acid whose difference, and they are acute phase protein, acts on close in inflammatory reaction.People SAA3 is a pseudogene, and people SAA4 changes not quite in acute phase reaction, continues a small amount of generation in body.
Research shows, SAA1 all changes at acute and chronic inflammation patients serum intensive amount, is therefore the important biomolecule Testing index of a reflection infectivity and noninfective inflammatory disease.SAA1 is not only the biomarker of Hospitalized Patients with Acute Exacerbations of Chronic Obstructive Pulmonary Disease, and be also the expection factor that cardiac rupture occurs acute myocardial infarction patient after accepting emergency treatment coronary intervention procedure, this provides important references index for early discovery cardiac rupture patient.Other large quantity research also show, in pancreatitis, hepatitis, coronary heart disease, diabetes, chronic renal disease, rheumatoid arthritis and fat case, SAA1 raises has very close relationship with disease activity and disease.Current clinical study is also thought, in all acute phase reactive proteins, SAA1 is the most responsive to acute inflammatory reaction, and appreciation amplitude is maximum.Study simultaneously and also show that SAA1 is similar to tumor correlated albumens such as alpha-fetoprotein, its concentration in serum and cancer are proportionate.Therefore, to SAA1 detection of content in patients serum, there is additive diagnostic value clinically.
Meanwhile, SAA1 is not only a kind of simple marker of inflammation, also actively gets involved the correlated process of disease.When acute infection or inflammation occur, in serum, the level of SAA1 can promote 1000 times.High-caliber SAA1 will cause amyloid pathology, and the binding substances of low-density lipoprotein LDL and SAA1 (LDL-SAA) can improve the risk of cardiovascular disorder; Meanwhile, SAA1 also has the characteristic of proinflammatory reaction, promotes the secretion of cytokine profiles, as IL-1 β, IL-6, matrix metalloproteinase (matrix metalloproteinases, MMPs), CCL20 etc., these cytokines all participate in the pathogenic process of the disease that inflammation causes, as, rheumatic arthritis, amyotrophy, gout, atherosclerosis etc.Because SAA1 is relevant to the generation of various diseases, the methods for the treatment of for SAA1 more and more comes into one's own.
At present, known SAA1 and four kind of receptors bind, Toll-like receptor (Toll-like receptors, TLR) TLR2 and TLR4 is comprised, CD36 (category-B scavenger receptor, a class B scavenger receptor), and g protein coupled receptor formyl peptide receptor 2/ LXA4 acceptor (formyl peptide receptor2/lipoxin A4receptor, FPR2/LXA4R).The biologic activity that SAA1 plays from different receptors bind is different, and such as, SAA1 is combined with formyl peptide receptor 2 propagation can inducing synovia hyperplasia and endotheliocyte thereof, thus causes the generation of rheumatoid arthritis; SAA1 and CD36 combines the metabolism that can promote cholesterol; SAA1 and TLR2 combines can produce inflammation-inducing factor, causes the diseases such as atherosclerosis; SAA1 and TLR4 combines the generation can inducing NO.
As can be seen here, SAA1 plays various biological activity in body.At present, SAA1 all adopts escherichia coli expression to obtain, and combines the lipopolysaccharides on gram-negative bacteria cell wall and LPS, because SAA1 and lipopolysaccharides and lipid thereof have extremely strong keying action, is just difficult to be separated once combination, is difficult to obtain not containing the SAA1 of lipopolysaccharides.
In addition, this area also needs to develop the material that can suppress natural SAA activity, is used for the treatment of too high the caused inflammation of SAA and disease thereof.
Summary of the invention
The object of the invention is to, provide the preparation method of a kind of novel hSAA1, the hSAA1 of acquisition does not disturb by lipopolysaccharides, and studies its biological function.
A first aspect of the present invention, provides a kind of recombinant human serum amyloid A 1, and described recombinant human serum amyloid A 1 is the recombinant human serum amyloid A 1 with yeast expressed by host.
In another preference, described recombinant human serum amyloid A 1 has following one or more feature:
A () molecular weight is the monomer of 12 ~ 14KD;
B () suppresses the activity of natural human serum amyloid A protein;
C () has the ability suppressing expression of inflammatory cytokines.
In another preference, described inflammatory cytokine comprises IL-6, IL-1 β, TNF-α, IL-12, MMP9.
A second aspect of the present invention, provides a kind of polymer of human serum amyloid A 1, and described polymer is the polymer of the recombinant human serum amyloid A 1 described in first aspect.
In another preference, described polymer has following one or more feature:
A () described polymer is dimer, tripolymer, the tetramer or its combination;
B () suppresses the activity of natural human serum amyloid A protein;
C () has the ability suppressing expression of inflammatory cytokines.
In another preference, described dimeric molecular weight is about 25 ~ 27KD; Described trimerical molecular weight is about 38 ~ 40KD; Described tetrameric molecular weight is about 41 ~ 43KD.
In another preference, described inflammatory cytokine comprises IL-6, IL-1 β, TNF-α, IL-12 and MMP9.
In another preference, described polymer is the polymer of the human serum amyloid A 1 with yeast expressed by host.
A third aspect of the present invention, provide a kind of human serum amyloid A 1 polypeptide fragment, described polypeptide fragment has following characteristics simultaneously:
I the sequence of () this polypeptide fragment derives from the aminoacid sequence of human serum amyloid A 1;
(ii) length of this polypeptide fragment is 20-100 amino acid;
(iii) this polypeptide fragment suppresses the activity of natural human serum amyloid A protein;
(iv) this polypeptide fragment has the ability suppressing expression of inflammatory cytokines.
In another preference, described polypeptide fragment is the polypeptide fragment with yeast expressed by host.
In another preference, described polypeptide has the aminoacid sequence of the 7-30 position of wild-type human serum's amyloid A 1 aminoacid sequence, 11-45 position, 27-72 position, 27-90 position, 29-104 position, 27-104 position, 9-72 position or 72-104 position.
In another preference, the aminoacid sequence of wild-type human serum's amyloid A 1 is as shown in SEQ ID NO.:2.
In another preference, the N end of described polypeptide fragment and/or C end are also with the sequence label (as 6His or HIS6-SUMO-TEV fragment) for objects such as purifies and separates.
A fourth aspect of the present invention, provides a kind of Yeast expression carrier, the gene containing coding human serum amyloid A 1; Or
This carrier contains the polynucleotide of the polypeptide fragment described in the third aspect.
In another preference, described carrier is containing coding human serum amyloid A 1 gene or the Yeast expression carrier pPIC9K of polynucleotide containing the polypeptide fragment described in the coding third aspect, and coding human serum amyloid A 1 gene is on pPIC9K carrier between XhoI and EcoRI restriction enzyme site.
A fifth aspect of the present invention, provides a kind of yeast host cell, and described host cell is selected from lower group:
(a) host cell containing the carrier described in fourth aspect;
The host cell of the gene of coding hSAA1 albumen is integrated with in (b) karyomit(e);
The host cell of the polynucleotide of the polypeptide fragment of coding described in the third aspect is integrated with in (c) karyomit(e).
A sixth aspect of the present invention, provides the preparation method of a kind of recombinant human serum amyloid A 1 or recombinant human serum amyloid A 1 polymer or human serum amyloid A 1 polypeptide fragment, comprises,
I () under conditions suitable for the expression, cultivates host cell described in the 5th aspect, thus express recombinant human serum amyloid A 1 of the present invention or polymer of the present invention or polypeptide fragment of the present invention; With
(ii) from cultured products, isolate described recombinant human serum amyloid A 1 or its polymer or its polypeptide fragment.
A seventh aspect of the present invention, provides a kind of pharmaceutical composition, and it contains pharmaceutically acceptable carrier or vehicle; And be selected from the activeconstituents of lower group:
(a1) recombinant human serum amyloid A 1 of the present invention;
(a2) polymer of human serum amyloid A 1 of the present invention;
(a3) human serum amyloid A 1 polypeptide fragment of the present invention.
In another preference, the formulation of described pharmaceutical composition is injection formulations, preparation capable of permeating skin, lyophilisate.
A eighth aspect of the present invention, a kind of recombinant human serum amyloid A 1 is provided, or recombinant human serum amyloid A 1 polymer, or the purposes of human serum amyloid A 1 polypeptide fragment, for the preparation of inflammation-inhibiting and/or the medicine for the treatment of inflammation related disease.
In another preference, described inflammation related disease is selected from lower group: rheumatic arthritis, atherosclerosis, cardiovascular disorder, pancreatitis, hepatitis, diabetes, chronic renal disease, obesity, amyotrophy and gout.
In another preference, described inflammation is too high the caused inflammation of SAA.
A ninth aspect of the present invention, a kind of human serum amyloid A 1 of restructuring or its polypeptide fragment or its polymer are provided, described recombinant human serum amyloid A 1 or its polypeptide fragment or its polymer are expressed in yeast, and suppress the activity of natural human serum amyloid A protein.
In another preference, described suppression is competitive inhibition.
In another preference, described recombinant human serum amyloid A 1 or its polypeptide fragment or its polymer have the ability suppressing expression of inflammatory cytokines, and have one of following characteristics:
A the molecular weight of () human serum amyloid A 1 monomer is 12 ~ 14KD;
B the polymer described in () is dimer, tripolymer, the tetramer or its combination;
C the sequence of () this polypeptide fragment derives from the aminoacid sequence of human serum amyloid A 1, and the length of this polypeptide fragment is 20-100 amino acid.
In another preference, described inflammatory cytokine comprises IL-6, IL-1 β, TNF-α, IL-12 and MMP9.
In another preference, described dimeric molecular weight is about 25 ~ 27KD; Described trimerical molecular weight is about 38 ~ 40KD; Described tetrameric molecular weight is about 41 ~ 43KD.
A tenth aspect of the present invention, provides a kind of method of inflammation-inhibiting or treatment inflammation related disease, comprises the step using the component being selected from lower group to the object of needs:
(a) recombinant human serum amyloid A 1 of the present invention;
The polymer of (b) human serum amyloid A 1 of the present invention;
(c) human serum amyloid A 1 polypeptide fragment of the present invention.
In another preference, described object is Mammals.More preferably, described object is behaved.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 is the expression level figure that dot-blotting detects the hSAA1 of yeast expression.
Fig. 2 is SDS-PAGE and the Western-blotting detection figure of the hSAA1 purified product of yeast expression.
Fig. 3 is SDS-PAGE figure after the hSAA1 polypeptide fragment purifying of yeast expression.
Fig. 4 is that the hSAA1 of yeast expression stimulates THP-1 cell, the detection figure of IL-6 cytokine secretion.
Fig. 5 is that the hSAA1 of yeast expression stimulates THP-1 cell, the detection figure of TNF α cytokine secretion.
Fig. 6 is that the hSAA1 monomer of yeast expression stimulates THP-1 cell, the detection figure of IL-1 β cytokine secretion.
Fig. 7 is that the hSAA1 polypeptide fragment of yeast expression stimulates THP-1 cell, the detection figure of IL-6 cytokine secretion.
Fig. 8 is that the hSAA1 polypeptide fragment of yeast expression stimulates THP-1 cell, the detection figure of IL-1 β cytokine secretion.
Fig. 9 is that the hSAA1 polymer of yeast expression stimulates THP-1 cell, the detection figure of IL-1 β cytokine secretion.
Embodiment
Present inventor is through extensively and in depth studying, unexpected discovery, when hSAA1 albumen or its fragment are expressed by Yeast system, not only can obtain glycosylated restructuring hSAA1 or its fragment, and this specific hSAA1 or its fragment are the inhibitor of natural hSAA unexpectedly, when share with natural hSAA, significantly can reduce the activity of (or suppression) natural hSAA.On this basis, the present invention is completed.
Particularly, the present invention adopts the yeast cell of the gene yeast expression vector transfection containing coding hSAA1 albumen, can obtain hSAA1 and polymer thereof, do not have lipopolysaccharides to disturb through cultivation, screening, secretion inducing, purifying.Testing surface, by yeast as the hSAA1 of host expresses or polymer, has the ability suppressing inflammatory cytokine.
Further, in order to study structural domain and the functional domain of hSAA1 and receptors bind, contriver has synthesized the aminoacid sequence of the polypeptide of Multi-encoding hSAA1, use yeast to obtain the polypeptide fragment of hSAA1 as host expresses, result shows also have as the hSAA1 polypeptide fragment of host expresses the ability suppressing inflammatory cytokine by yeast.
Term
As used herein, term " inflammation related disease " refers to be attended by inflammation or by the too high disease caused of expression of inflammatory cytokines, include but not limited to, rheumatic arthritis, atherosclerosis, cardiovascular disorder, pancreatitis, hepatitis, diabetes, chronic renal disease, obesity, amyotrophy and gout.
Recombinant human serum amyloid A 1 and polymer thereof
Recombinant human serum amyloid A 1 of the present invention (restructuring hSAA1) is the recombinant human serum amyloid A 1 with yeast expressed by host.
In another preference, described recombinant human serum amyloid A 1 has following one or more feature:
A () molecular weight is the monomer of 12 ~ 14KD;
B () suppresses the activity of natural human serum amyloid A protein;
C () has the ability suppressing expression of inflammatory cytokines.
In another preference, described inflammatory cytokine comprises IL-6, IL-1 β, TNF-α, IL-12, MMP9.
Recombinant human serum amyloid A 1 of the present invention, not in conjunction with lipopolysaccharides.
In another preference, described suppression is competitive inhibition.
The nucleotide sequence of encoding recombinant human's serum amyloid A protein 1 is as shown in SEQ ID NO.:1.
The aminoacid sequence of recombinant human serum amyloid A 1 is as shown in SEQ ID NO.:2.
The polymer of human serum amyloid A 1 provided by the invention is the polymer of recombinant human serum amyloid A 1 of the present invention.
In another preference, described polymer has following one or more feature:
A () described polymer is dimer, tripolymer, the tetramer or its combination;
B () suppresses the activity of natural human serum amyloid A protein;
C () has the ability suppressing expression of inflammatory cytokines.
In another preference, described dimeric molecular weight is about 25 ~ 27KD; Described trimerical molecular weight is about 38 ~ 40KD; Described tetrameric molecular weight is about 41 ~ 43KD.
In another preference, described inflammatory cytokine comprises IL-6, IL-1 β, TNF-α, IL-12, MMP9.
In another preference, described polymer is the polymer of the human serum amyloid A 1 with yeast expressed by host.
In another preference, described suppression is competitive inhibition.
Recombinant human serum amyloid A 1 of the present invention and polymeric preparation method thereof, comprise step:
A () provides the Yeast expression carrier of the gene containing coding human serum amyloid A 1;
B () provides the yeast host cell containing described Yeast expression carrier;
(c) under conditions suitable for the expression, the yeast host cell described in cultivation, thus express recombinant human serum amyloid A 1 of the present invention or polymer of the present invention; With
D () isolates described recombinant human serum amyloid A 1 or its polymer from cultured products.
In another preference, the gene of coding human serum amyloid A 1 is connected to commercial vector (as pPIC9K) after amplification, double digestion, obtains the Yeast expression carrier described in step (a).PPIC9K is the excretion vector of a high expression exogenous protein, with signal peptide α-factor on it, hSAA1 can be secreted in substratum.
In another preference, XhoI and EcoRI double digestion is utilized hSAA1 gene to be connected to α-factor downstream through XhoI and EcoRI double digestion carrier pPIC9K, centre is connected to Kex2signal cleavage (carrier is self-contained), in the process of secreting, expressing, α-factor can be excised, the N-terminal of release hSAA1.
In another preference, in step (b), adopt Yeast expression carrier transfecting yeast GS115.
In another preference, electric method for transformation in step (b), is adopted to carry out transfection.
In another preference, in step (c), adopt histidine auxotroph slat chain conveyor yeast, screening yeast mono-clonal.
In another preference, described step (d) adopts G418 to screen.
In another preference, described step (e) adopts methyl alcohol to carry out abduction delivering, obtains described recombinant human serum amyloid A 1 and polymer thereof.
In another preference, described method also comprises the step that the mono-clonal higher to expression amount carries out bulk fermentation and purifying.
In another preference, described purifying refers to and utilizes affinity chromatography to carry out purifying.
The polypeptide fragment of human serum amyloid A 1
A kind of human serum amyloid A 1 polypeptide fragment, has following characteristics:
I the sequence of () this polypeptide fragment derives from the aminoacid sequence of human serum amyloid A 1;
(ii) length of this polypeptide fragment is 20-100 amino acid;
(iii) this polypeptide fragment suppresses the activity of natural human serum amyloid A protein;
(iv) this polypeptide fragment has the ability suppressing expression of inflammatory cytokines.
In another preference, described polypeptide fragment is the polypeptide fragment with yeast expressed by host.
Polypeptide fragment of the present invention, includes but not limited to P7-30, P11-45, P27-72, P27-90, P29-104, P27-104, P9-72, P72-104 or its combination.Wherein digitized representation top and end amino acid position in the aminoacid sequence of human serum amyloid A 1.Namely described polypeptide has the aminoacid sequence of the 7-30 position of wild-type human serum's amyloid A 1 aminoacid sequence, 11-45 position, 27-72 position, 27-90 position, 29-104 position, 27-104 position, 9-72 position or 72-104 position.
In another preference, the aminoacid sequence of wild-type human serum's amyloid A 1 is as shown in SEQ ID NO.:2.
Polypeptide of the present invention can be recombinant polypeptide, improvement on synthesis, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (such as, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to recombinant production scheme, polypeptide of the present invention can be glycosylated, can be maybe nonglycosylated.
In another preference, polypeptide fragment of the present invention is recombinant polypeptide, for yeast is that host carries out expressing the polypeptide fragment obtained.
The preparation method of the polypeptide fragment of human serum amyloid A 1 of the present invention, comprises step:
A () provides the Yeast expression carrier of the polynucleotide of the polypeptide fragment containing coding human serum amyloid A 1;
B () provides the yeast host cell containing described Yeast expression carrier;
(c) under conditions suitable for the expression, culturing yeast host cell, thus express polypeptide fragment of the present invention; With
(ii) from cultured products, described polypeptide fragment is isolated.
In another preference, the polynucleotide of the polypeptide fragment of coding human serum amyloid A 1 are connected to pPIC9K after amplification, double digestion, obtain the Yeast expression carrier described in step (a).
In another preference, XhoI and EcoRI double digestion is utilized to be connected on XhoI and EcoRI double digestion carrier pPIC9K by the polynucleotide of the polypeptide fragment of coding human serum amyloid A 1.
In another preference, in step (b), adopt Yeast expression carrier transfecting yeast GS115.
In another preference, electric method for transformation in step (b), is adopted to carry out transfection.
In another preference, in step (c), adopt histidine auxotroph slat chain conveyor yeast, screening yeast mono-clonal.
In another preference, described step (d) adopts G418 to screen.
In another preference, described step (e) adopts methyl alcohol to carry out abduction delivering, obtains described recombinant human serum amyloid A 1 and polymer thereof.
In another preference, described method also comprises the step that the mono-clonal higher to expression amount carries out bulk fermentation and purifying.
In another preference, the N end band of described polypeptide fragment has HIS6-SUMO-TEV fragment, both can strengthen the expression output of polypeptide, also can facilitate purifying, and purified product can cut release polypeptide fragment with TEV enzyme.
In another preference, yeast expression system of the present invention, expresses the polypeptide of recombinant human SAA1 and the similar thereof not having lipopolysaccharides to disturb.This with yeast for the phraseology of host not only to have broken away from the problem of the lipopolysaccharides puzzlement encountered in SAA1 research for many years, realize in vivo and in vitro not by the biological action of the SAA1 of lipopolysaccharides interference, the polypeptide that yeast expression system also can be utilized to express multiple SAA1 similar carries out functional study, thus finds that SAA plays active structural domain and functional domain.The result of study of the biologic activity that the different peptide section of detailed people SAA1 simultaneously also can be utilized to play, designs corresponding blocker, for clinical treatment provides potential drug candidate.
Restructuring hSAA1 also using conventional procedures expressed above, add HIS6 affinity tag at C-terminal, expressed albumen and polypeptide thereof, after SDS-PAGE, Western-blotting confirm as target protein matter, utilize affinity chromatography to carry out purifying.
Purposes
Restructuring hSAA1 after this patent purifying and polypeptide thereof stimulate THP-1 cell strain, detect the impact that these products upon cell factors (as IL6, TNF α, IL-1 β, IL12P40, MMP9 etc.) are secreted.Result shows, restructuring hSAA1 of the present invention, polymer, polypeptide fragment have the ability suppressing expression of inflammatory cytokines, the medicine of the disease that can cause for the preparation for the treatment of inflammation.
Further, Activity determination is carried out to purified product and commercial SAA (can the generation of the incite inflammation factor, bring out inflammation) co-stimulatory cell, show that the hSAA1 monomer of yeast expression and polymer significantly reduce the proinflammatory effect of commercial SAA.Because the hSAA1 monomer of yeast expression and polymer and the expression of polypeptide to cytokine profiles thereof have remarkable restraining effect, therefore may be used for treating the disease that in inflammatory reaction process, SAA excessive secretion causes.Meanwhile, the hSAA1 monomer of yeast expression, polymer and polypeptide can suppress LPS irritation cell to secrete some to cause scorching cytokine (as IL-1 β), also may be used for treating the disease caused by inflammatory reaction.
The disease that described inflammation causes is selected from lower group: rheumatic arthritis, atherosclerosis, cardiovascular disorder, pancreatitis, hepatitis, diabetes, chronic renal disease, obesity, amyotrophy, gout.
Pharmaceutical composition and method of application
Recombinant human serum amyloid A 1 of the present invention, polymer, polypeptide fragment, can be formulated in nontoxic, inertia with in pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, preferably pH is about 6-8, although pH value can with being formulated the character of material and illness to be treated and changing to some extent.The pharmaceutical composition prepared can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
Recombinant human serum amyloid A 1 of the present invention, polymer, polypeptide fragment can be directly used in the treatment of the disease that inflammation causes, such as, for the treatment of rheumatoid arthritis disease.Use recombinant human serum amyloid A 1 of the present invention, polymer, polypeptide fragment time, also can use simultaneously other treatment agent or with other therapy couplings.
Present invention also offers a kind of pharmaceutical composition, it contains the recombinant human serum amyloid A 1 of the present invention of safe and effective amount, polymer, polypeptide fragment or its combination; And pharmaceutically acceptable carrier or vehicle.This kind of carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should match with administering mode.Pharmaceutical composition of the present invention can be made into injection form, such as, be prepared by ordinary method with physiological saline or the aqueous solution containing glucose and other assistant agents.Pharmaceutical composition is prepared by ordinary method.Pharmaceutical composition such as injection, solution, Tablet and Capsula should aseptically manufacture.The dosage of activeconstituents is treatment significant quantity, such as every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, recombinant human serum amyloid A 1 of the present invention, polymer, polypeptide fragment also can use together with other treatment agent.
When making pharmaceutical composition, in Mammals by the recombinant human serum amyloid A 1 of the present invention of safe and effective amount, polymer, polypeptide fragment or its combined administration, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and be in most of the cases no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should consider the factor such as route of administration, patient health situation, and these are all within skilled practitioners skill.
Major advantage of the present invention is:
(1). the present inventor finds the expression inhibiting effect to multiple inflammatory cytokine of recombinant human serum amyloid A 1, polymer, polypeptide fragment unexpectedly.
(2). experimentation on animals is determined, recombinant human serum amyloid A 1 of the present invention, polymer, polypeptide fragment can reduce the mouse lethal rate of LPS.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number be weight percentage and parts by weight.
Embodiment 1
The preparation of restructuring hSAA1
Synthesize following primer:
Upstream primer (SEQ ID NO:3):
5’GTCG CTC GAG AAA AGA GAG GCT CGAAGCTTCTTTTCGTTC3’；
Downstream primer (SEQ ID NO:4):
5’GTCG GAA TTC TCA GTG GTG GTG GTG GTG GTG GTATTTCTCAGGCAG3’；
With ordinary method extracting people mRNA, the cDNA obtained after reverse transcription, as template.Use above-mentioned primer, by conventional PCR method, amplification obtains people hSAA1 full-length gene.To pcr amplification product, after XhoI/EcoRI double digestion, be inserted into the α-factor signal peptide downstream of the carrier pPIC9k (purchased from Invitrogen company) cut through same enzyme, obtain carrier pPIC9k-hSAA1.When hSAA1 secreting, expressing, α-factor signal peptide can be cut, thus the N-terminal of release hSAA1.
Utilize electric method for transformation, above-mentioned recombinant expression vector pPIC9k-hSAA1 proceeded to conventional Pichia pastoris GS115, and carry out the recon selecting conversion as follows:
Adopt the yeast of histidine auxotroph slat chain conveyor transfection, screening yeast mono-clonal, by dull and stereotyped for obtained mono-clonal coating G418 screening, G418 concentration is respectively 0.5mg/mL, 1mg/mL, 2mg/mL, cultivate 3-4 days for 30 DEG C, choose the Colony Culture that G418 concentration 2mg/mL flat board grows, methanol induction expresses recombinant human hSAA1 protein, and its C-terminal is with the HIS6 affinity tag (being introduced by primer) being convenient to affinity purification.
DOT-BLOTTING is adopted to detect the expression output of target protein matter in supernatant liquor.Fig. 1 is the DOT-BLOTTING detected result that 6 yeast mono-clonals express hSAA1, chooses and expresses bulk fermentation and the purifying thereof that the higher mono-clonal of output carries out next step.
Embodiment 2
The preparation of hSAA1 polypeptide fragment
Adopt ordinary method gene chemical synthesis his6-sumo-tev sequence, as shown in SEQ ID NO:5, utilize restriction enzyme site XhoI to access the α-factor signal peptide downstream of carrier pPIC9k (Invitrogen company), obtain the pPIC9k carrier pPIC9k-his6-sumo-tev with his6-sumo-tev.
In employing table 1, segment name is the primer of P27-72, and by conventional PCR method, amplification obtains the people hSAA1 gene fragment of encode polypeptide fragments P27-72 respectively.To pcr amplification product, after XhoI/EcoRI double digestion, be inserted into the his6-sumo-tev downstream in the carrier pPIC9k-his6-sumo-tev cut through same enzyme, obtain carrier pPIC9k-his6-sumo-tev-hSAA1P27-72.
Utilize electric method for transformation, above-mentioned recombinant expression vector pPIC9k-his6-sumo-tev-hSAA1P27-72 proceeded to conventional Pichia pastoris GS115, and carry out the recon selecting conversion as follows:
Adopt the yeast of histidine auxotroph slat chain conveyor transfection, screening yeast mono-clonal, by dull and stereotyped for obtained mono-clonal coating G418 screening, G418 concentration is respectively 0.5mg/mL, 1mg/mL, 2mg/mL, cultivate 3-4 days for 30 DEG C, choose the Colony Culture that G418 concentration 2mg/mL flat board grows, methanol induction expresses recombinant human hSAA1 polypeptide fragment P27-72 (corresponding to 27-72 position in SEQ ID NO.:2).
Adopt aforesaid method to prepare recombinant human hSAA1 polypeptide fragment P27-90, recombinant human hSAA1 polypeptide fragment P29-104, difference is primer, as shown in table 1.
Table 1 primer sequence table
Embodiment 3
The purifying of restructuring hSAA1 and polypeptide fragment thereof
Yeast fermentation broth supernatant embodiment 1 and embodiment 2 obtained, through ultrafiltration and concentration, mixes 1-2 hour with Ni Sepharose HP4 DEG C, dress post.Adopt lavation buffer solution (20mM sodium phosphate (sodium phosphate), 25mM imidazoles (imidazole), 500mM NaCl, pH7.4) pillar is washed, elution buffer (20mM sodium phosphate sodium phosphate, 500mM imidazoles imidazole, 500mM NaCl, pH7.4) wash-out target protein matter, adopts SDS-PAGE and Western-blotting to detect, and result respectively as shown in Figures 2 and 3.
Current existing escherichia expression system, only can obtain monomer.Surprisingly, adopt yeast expression system, hSAA1 that is new, multimeric forms can be obtained, i.e. the polymer of hSAA1.
As shown in Figure 2, for SDS-PAGE and Western-blotting of the hSAA1 purified product of yeast expression detects figure, wherein, swimming lane 1 is hSAA1 monomer (the ≈ 13KD of yeast expression, molecular weight is greater than the molecular weight 12KDa calculated based on aminoacid sequence), swimming lane 2 is hSAA1 polymers (dimer ≈ 26KD, tripolymer ≈ 39KD, tetramer ≈ 52KD) of yeast expression.
As shown in Figure 3, for SDS-PAGE figure after the hSAA1 polypeptide fragment purifying of yeast expression, wherein, swimming lane 3 is that the SUMO-hSAA1 (P27-72) of yeast expression (is connected with the hSAA1 polypeptide fragment P27-72 of SUMO, ≈ 21KD), swimming lane 2 is that the SUMO-hSAA1 (P27-90) of yeast expression (is connected with the hSAA1 polypeptide fragment P27-90 of SUMO, ≈ 23KD), swimming lane 1 is the SUMO-hSAA1 (P27-104) (being connected with the hSAA1 polypeptide fragment P27-104 of SUMO, ≈ 24KD) of yeast expression
Embodiment 4
HSAA1 and polypeptide fragment thereof are on the impact of THP-1 cell strain cytokine secretion
Commercial SAA, hSAA1, hSAA1 polymer, polypeptide fragment (tev enzyme cuts after product) are joined Human THP-1 cells (the ATCC TIB-202 of the routine of serum-free culture with the final concentration of 0.5 μM
tMpeople's Acute monocytic strain leukemia cell line) in nutrient solution (106 cells/ml), 5 h before harvest cells, TRIzol cracking, extracts RNA, and reverse transcription is the expression level that cDNA, real-time PCR detects cytokine.
Shown in Fig. 4, use the hSAA1 monomer of yeast expression and polymer to stimulate THP-1 cell after 5 hours respectively, detect the expression level of IL-6RNA in cell.Result shows, after commercial SAA (0.5 μM) stimulates THP-1 cell, the level that IL-6 raises is higher than the hSAA1 monomer of yeast expression and hSAA1 polymer; Irritation cell after the hSAA1 monomer (0.5 μM) of commercial SAA (0.5 μM) and yeast expression and polymer (0.5 μM) are mixed respectively, compared with stimulating separately THP-1 cell with commercial SAA, IL-6 expression level declines.Show, the hSAA1 monomer of yeast expression and polymer suppress the proinflammatory effect of commercial SAA.
In addition, after adopting micro-LPS (5ng/mL) to stimulate THP-1, IL-6 expression level improves, and after mixing with the hSAA1 monomer (0.5 μM) of yeast expression and stimulating, and IL-6 improves the standard and to stimulate separately higher than both.Prompting, LPS can act synergistically with the hSAA1 of yeast expression, causes THP-1 emiocytosis IL-6 level to improve.
Fig. 5 is the detection of TNF alpha levels after stimulation THP-1 cell, and result is consistent with IL-6, and the hSAA1 monomer of yeast expression and polymer have the effect of the activity suppressing commercial SAA.But after micro-LPS (5ng/mL) Co stituation THP-1, TNF alpha levels declines, prompting, the hSAA1 monomer of yeast expression has the effect that competitive inhibition LPS stimulates THP-1 emiocytosis TNF α.
Fig. 6 is the detection of IL-1 β level after stimulation THP-1 cell, and the hSAA1 monomer of yeast expression has the effect suppressing commercial SAA, meanwhile, also inhibited to LPS irritation cell secretion IL-1 β.
After Fig. 7 and Fig. 8 is respectively polypeptide fragment stimulation THP-1 cell, the expression level of IL-6 and IL-1 β detects, find that hSAA1 (27-72) induces IL-6 and IL-1 β level to be minimum, and the effect of commercialization SAA can be suppressed, also there are some with LPS simultaneously and act synergistically.The corresponding raising of hSAA1 (27-90), hSAA1 (29-104) induced levels, but also there is the effect with the activity suppressing commercialization SAA.
For Fig. 8 and hSAA1 (27-72), time alone commercial SAA (0.5 μM), the expression level of IL-1 β is 75 times, and time alone hSAA1 (P27-72 polypeptide) (0.5 μM), the expression level of IL-1 β is 40 times.Surprisingly, when SAA (0.5 μM) and P27-72 polypeptide (0.5 μM) being share, the expression level of IL-1 β can be dropped to 37 times from 75 times.This shows that P27-72 polypeptide can reduce the rising (IL-1 β raises the generation that can cause inflammation disease) of the IL-1 β that SAA causes.
Fig. 6 is the detection of IL-1 β level after stimulation THP-1 cell, and the hSAA1 polymer of yeast expression has the effect suppressing commercial SAA, meanwhile, also inhibited to LPS irritation cell secretion IL-1 β.
Because the hSAA1 monomer of yeast expression and polymer and the expression of polypeptide to cytokine profiles thereof have restraining effect, therefore as potential medicine, the disease that in inflammatory reaction process, SAA excessive secretion causes can be used for the treatment of.Meanwhile, the hSAA1 monomer of yeast expression, polymer and polypeptide can suppress LPS irritation cell to secrete some to cause scorching cytokine (as IL-1 β), also as potential medicine, can be used for the treatment of the disease caused by inflammatory reaction.
Comparative example 1
By ordinary method, adopt coli expression system, the hSAA1 of Preparation of amino acid sequence as shown in SEQ ID NO.:2.For the nonglycosylated hSAA1 of this prokaryotic expression, measure it to the impact on THP-1 cell strain cytokine secretion by the method that embodiment 4 is identical.
Result shows, be used alone the hSAA1 (0.5 μM) of escherichia coli expression, or use after mixing with commercial SAA (0.5 μM), can promote the expression of inflammatory factor IL-6, TNF α, IL-1 β, the hSAA1 of escherichia coli expression is not inhibited to the activity of commercial SAA.
Embodiment 5
HSAA1 and polypeptide fragment thereof and LPS and SAA abdominal injection BALB/c mouse cause mouse inflammatory reaction
The present embodiment devises the hSAA1 of yeast expression, polymer and polypeptide in Mice Body to the inhibiting experiment of the inflammatory reaction that LPS causes.Select the male BALB/c mouse in 5-7 week, often organize 3, five groups altogether, be respectively LPS group (LPS15mg/Kg), LPS/hSAA1 low dose group (LPS15mg/Kg, hSAA115mg/Kg), dosage group (LPS15mg/Kg in LPS/hSAA1, hSAA130mg/Kg), LPS/hSAA1 high dose group (LPS15mg/Kg, hSAA1100mg/Kg), control group (PBS), abdominal injection, the survival time of record mouse.
The table 2 mouse survival time
As shown in table 2, find according to mouse death rate test experience, the hSAA1 monomer of yeast expression and polymer thereof can alleviate the dead mouse that LPS causes, extend the mouse survival time, wherein hSAA1 (27-72) fragment is best to suppression LPS mouse lethal rate activity, can suppress the death of mouse completely.
Embodiment 6
Set up arthritis mouse model, joint cavity injection hSAA1 and polypeptide fragment thereof, detect arthritic development
By ordinary method, with collagen protein inducing mouse arthritis model.Then, at intraarticular injection hSAA1 and polypeptide fragment thereof, the effect (administration group and non-administered group, n=6) that observation hSAA1 and polypeptide fragment thereof develop mouse arthritis.
Use is quantized classified estimation by arthritic clinical table disease severity.Based on swelling, erythema and distortion are divided into 0-4:0, without swelling; 1, in a toes swelling or Mild edema; 2, moderate swelling affects some toes; 3, some swelling affect most of toes; 4, the most serious swelling and/or tetanic.
Result shows, compared with arthritis mouse model group (non-administered group), hSAA1 test group and hSAA1 polypeptide fragment group (P27-72 polypeptide) arthritic symptom have obvious alleviation (score value is significantly lower than the control group of non-administration).
Embodiment 7
Pharmaceutical composition
Prepare a pharmaceutical composition, described composition comprises:
(a) P27-72 polypeptide (or P27-90 polypeptide); With
(b) physiological saline.
Embodiment 8
Pharmaceutical composition
Prepare a pharmaceutical composition, described composition comprises:
HSAA1 albumen prepared by (a) embodiment 1; With
(b) physiological saline.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (13)
1. a recombinant human serum amyloid A 1, is characterized in that, described recombinant human serum amyloid A 1 for the recombinant human serum amyloid A 1 with yeast expressed by host, described yeast be Pichia pastoris GS115.
2. a polymer for human serum amyloid A 1, is characterized in that, described polymer is the polymer of recombinant human serum amyloid A 1 according to claim 1, and described polymer is dimer, tripolymer or the tetramer.
3. a human serum amyloid A 1 polypeptide fragment, is characterized in that, described polypeptide fragment is the recombinant polypeptide fragment with Pichia pastoris GS115 expressed by host, and described polypeptide fragment has following characteristics simultaneously:
I the sequence of () this polypeptide fragment derives from the aminoacid sequence of human serum amyloid A 1;
(ii) length of this polypeptide fragment is 20-100 amino acid;
(iii) this polypeptide fragment suppresses the activity of natural human serum amyloid A protein;
(iv) this polypeptide fragment has the ability suppressing expression of inflammatory cytokines,
Wherein, described inflammatory cytokine is selected from: IL-6, IL-1 β, TNF-α;
And the aminoacid sequence of described polypeptide fragment is the aminoacid sequence of the 27-72 position of wild-type human serum's amyloid A 1 aminoacid sequence, 27-90 position or 29-104 position.
4. a Yeast expression carrier, is characterized in that, this carrier contains the polynucleotide of polypeptide fragment according to claim 3 of encoding.
5. a yeast host cell, is characterized in that, described host cell is selected from lower group:
(a) host cell containing carrier according to claim 4;
The host cell Pichia pastoris GS115 of the gene of coding human serum amyloid A 1 albumen is integrated with in (b) karyomit(e);
The host cell of the polynucleotide of polypeptide fragment according to claim 3 of encoding is integrated with in (c) karyomit(e).
6. a preparation method for recombinant human serum amyloid A 1 according to claim 1 or polymer according to claim 2 or polypeptide fragment according to claim 3, is characterized in that, comprise step:
I () under conditions suitable for the expression, cultivates host cell according to claim 5, thus express recombinant human serum amyloid A 1 according to claim 1 or polymer according to claim 2 or polypeptide fragment according to claim 3; With
(ii) from cultured products, isolate described recombinant human serum amyloid A 1 or its polymer or its polypeptide fragment.
7. a pharmaceutical composition, is characterized in that, it contains pharmaceutically acceptable carrier or vehicle; And be selected from the activeconstituents of lower group:
(a1) recombinant human serum amyloid A 1 according to claim 1;
(a2) polymer of human serum amyloid A 1 according to claim 2;
(a3) human serum amyloid A 1 polypeptide fragment described in claim 3.
8. a purposes for recombinant human serum amyloid A 1 according to claim 1, is characterized in that, for the preparation of inflammation-inhibiting reaction and/or the medicine for the treatment of inflammation related disease.
9. purposes as claimed in claim 8, it is characterized in that, described inflammation related disease is sacroiliitis.
10. a polymeric purposes for human serum amyloid A 1 according to claim 2, is characterized in that, for the preparation of inflammation-inhibiting reaction and/or the medicine for the treatment of inflammation related disease.
11. purposes as claimed in claim 10, it is characterized in that, described inflammation related disease is sacroiliitis.
The purposes of 12. 1 kinds of human serum amyloid A 1 polypeptide fragments according to claim 3, is characterized in that, for the preparation of inflammation-inhibiting reaction and/or the medicine for the treatment of inflammation related disease.
13. purposes as claimed in claim 12, it is characterized in that, described inflammation related disease is sacroiliitis.
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