CN102584976A - Human serum amyloid A1 and preparation method and application thereof - Google Patents
Human serum amyloid A1 and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a human serum amyloid A1 and a preparation method and an application thereof. The invention further discloses a polymer of the human serum amyloid A1 and a polypeptide fragment of the human serum amyloid A1. The human serum amyloid A1, the polymer and the polypeptide fragment disclosed by the invention have the capabilities of inhibiting the expression of inflammatory cytokines, and can be used for inhibiting inflammations and treatment inflammation-relevant diseases.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of human serum amyloid A 1.
Background technology
(Serum Amyloid A is that body is infected and a kind of main acute phase protein of excretory when damaging SAA) to serum amyloid A protein.SAA is a proteic general name of polymorphum, is made up of the relevant but albumen (SAA1-SAA4) of each tool separate gene.Wherein, human serum amyloid A 1 (SAA1) is made up of 104 amino acid, and mainly synthetic by liver cell, people SAA2 and people SAA1 have 7 amino acid whose difference, and they are acute phase protein, and effect is close in inflammatory reaction.People SAA3 is a pseudogene, and people SAA4 changes in acute phase reaction not quite, in body, continues a small amount of the generation.
Research shows that SAA1 all changes at acute and chronic inflammation patients serum intensive amount, therefore is that a reflection important biomolecule infectious and non-infectious inflammatory disease detects index.SAA1 is not only the biomarker of chronic obstructive pulmonary disease acute exacerbation phase, also is the expection factor that cardiac rupture takes place after accepting emergency treatment coronary artery intervene operation the acute myocardial infarction patient, and this provides the important references index for early discovery cardiac rupture patient.Other big quantity research show that also in pancreatitis, hepatitis, coronary heart disease, mellitus, chronic renal disease, rheumatoid arthritis and fat case, SAA1 rising and disease activity and course of disease development have very confidential relation.Clinical study at present thinks that also in all acute phase reactive proteins, SAA1 is the most responsive to acute inflammatory reaction, and the appreciation amplitude is maximum.Research simultaneously shows that also tumor correlated albumens such as SAA1 and ALPHA-FP are similar, and its concentration and cancer in serum is proportionate.Therefore, the detection of content in the patients serum has auxiliary diagnosis value to SAA1 clinically.
Simultaneously, 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 generation, the level of SAA1 can promote 1000 times in the serum.High-caliber SAA1 will cause the amyloid pathology, and the binding substances of low-density lipoprotein LDL and SAA1 (LDL-SAA) can improve cardiovascular disease risk; Simultaneously, SAA1 also has short scorching reaction properties, promotes the secretion of the various kinds of cell factor, like IL-1 β; IL-6, and matrix metalloproteinase (matrix metalloproteinases, MMPs), CCL20 etc.; These cytokines are all participated in the pathogenic process of the disease that inflammation causes, as, rheumatic arthritis; Amyotrophy, gout, atherosclerosis etc.Because SAA1 is relevant with the generation of multiple disease, more and more come into one's own to the treat-ment of SAA1.
At present; Known SAA1 and four kinds of receptors bind, (Toll-like receptors TLR) comprises TLR2 and TLR4 to Toll appearance acceptor; CD36 (category-B scavenger receptor; A class B scavenger receptor), and g protein coupled receptor formyl peptide receptor 2/ lipoxin A 4 acceptors (formyl peptide receptor 2/lipoxin A4 receptor, FPR2/LXA4R).SAA1 is different with the BA that different receptors bind is brought into play, and for example, SAA1 combines to induce the propagation of synovia hyperplasia and endotheliocyte thereof with formyl peptide receptor 2, thereby causes the generation of rheumatoid arthritis; SAA1 combines to promote the metabolism of SUV with CD36; SAA1 combines with TLR2 to produce the inflammation incitant, causes diseases such as atherosclerosis; SAA1 combines to induce the generation of NO with TLR4.
This shows that SAA1 brings into play various biological in body active.At present, SAA1 all adopts escherichia coli expression to obtain, in conjunction with the LPS on the gram-negative bacteria cell wall be LPS because SAA1 has extremely strong keying action with LPS and lipid thereof,, be difficult to obtain not contain the SAA1 of LPS in case combine just to be difficult to separation.
In addition, this area also need be developed and can suppress the active material of natural SAA, is used to treat too high inflammation that causes of SAA and disease thereof.
Summary of the invention
The objective of the invention is to, the preparation method of a kind of novel hSAA1 is provided, the hSAA1 of acquisition is not disturbed by LPS, and its biological function is studied.
First aspect of the present invention provides a kind of recombinant human serum amyloid A 1, and said recombinant human serum amyloid A 1 is for being the expressed recombinant human serum amyloid A 1 of host with the yeast.
In another preference, said recombinant human serum amyloid A 1 has following one or more characteristic:
(a) molecular weight is the monomer of 12～14KD;
(b) activity of inhibition natural human serum amyloid A protein;
(c) has the ability that inflammatory cytokine is expressed that suppresses.
In another preference, said inflammatory cytokine comprises IL-6, IL-1 β, TNF-α, IL-12, MMP9.
Second aspect of the present invention provides a kind of polymer of human serum amyloid A 1, and said polymer is the polymer of the described recombinant human serum amyloid A 1 of first aspect.
In another preference, said polymer has following one or more characteristic:
(a) said polymer is a dimer, tripolymer, the tetramer or its combination;
(b) activity of inhibition natural human serum amyloid A protein;
(c) has the ability that inflammatory cytokine is expressed that suppresses.
In another preference, said dimeric molecular weight is about 25～27KD; Said trimerical molecular weight is about 38～40KD; Said tetrameric molecular weight is about 41～43KD.
In another preference, said inflammatory cytokine comprises IL-6, IL-1 β, TNF-α, IL-12 and MMP9.
In another preference, said polymer is for being the polymer of the expressed human serum amyloid A 1 of host with the yeast.
The third aspect of the invention provides a kind of human serum amyloid A 1 polypeptide fragment, and described polypeptide fragment has following characteristic simultaneously:
(i) sequence of this polypeptide fragment derives from the aminoacid sequence of human serum amyloid A 1;
(ii) the 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 that inflammatory cytokine is expressed that suppresses.
In another preference, said polypeptide fragment is for being the expressed polypeptide fragment of host with the yeast.
In another preference, said polypeptide has the aminoacid sequence of 7-30 position, 11-45 position, 27-72 position, 27-90 position, 29-104 position, 27-104 position, 9-72 position or the 72-104 position of wild-type human serum amyloid A 1 aminoacid sequence.
In another preference, the aminoacid sequence of wild-type human serum amyloid A 1 is shown in SEQ ID NO.:2.
In another preference, the N of said polypeptide fragment end and/or C end also have the sequence label (like 6His or HIS6-SUMO-TEV fragment) that is used for purposes such as purifies and separates.
Fourth aspect of the present invention provides a kind of Yeast expression carrier, contains the gene of the human serum amyloid A 1 of encoding; Or
This carrier contains the polynucleotide of the described polypeptide fragment of the third aspect.
In another preference; Said carrier is the Yeast expression carrier pPIC9K that contains coding human serum amyloid A 1 gene or contain the polynucleotide of the described polypeptide fragment of coding claim 3, and coding human serum amyloid A 1 gene is between XhoI on the pPIC9K carrier and EcoRI restriction enzyme site.
The 5th aspect of the present invention provides a kind of yeast host cell, and said host cell is selected from down group:
(a) contain the host cell of the described carrier of claim 5;
(b) be integrated with the host cell of coding hSAA1 proteic gene in the karyomit(e);
(c) be integrated with the host cell of the polynucleotide of coding claim 3 described polypeptide fragment in the karyomit(e).
The 6th 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 the condition that is fit to expression, cultivate the described host cell in the 5th aspect, thereby 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.
The 7th aspect of the present invention provides a kind of pharmaceutical composition, and it contains pharmaceutically acceptable carrier or vehicle; And the activeconstituents that is selected from down 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.
Eight aspect of the present invention; A kind of recombinant human serum amyloid A 1 is provided; Perhaps recombinant human serum amyloid A 1 polymer, the perhaps purposes of human serum amyloid A 1 polypeptide fragment is used to prepare the medicine of inflammation-inhibiting and/or treatment inflammation related disease.
In another preference, described inflammation related disease is selected from down group: rheumatic arthritis, atherosclerosis, cardiovascular disorder, pancreatitis, hepatitis, mellitus, chronic renal disease, obesity, amyotrophy and gout.
In another preference, said inflammation is the too high inflammation that causes of SAA.
The 9th aspect of the present invention; A kind of human serum amyloid A 1 or its polypeptide fragment or its polymer of reorganization are provided; Said 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 inhibition is a competitive inhibition.
In another preference, said recombinant human serum amyloid A 1 or its polypeptide fragment or its polymer have the ability that suppresses the inflammatory cytokine expression, and have one of following characteristic:
(a) the monomeric molecular weight of human serum amyloid A 1 is 12～14KD;
(b) described polymer is a dimer, tripolymer, the tetramer or its combination;
(c) 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, said inflammatory cytokine comprises IL-6, IL-1 β, TNF-α, IL-12 and MMP9.
In another preference, said dimeric molecular weight is about 25～27KD; Said trimerical molecular weight is about 38～40KD; Said tetrameric molecular weight is about 41～43KD.
The tenth aspect of the present invention provides the method for a kind of inflammation-inhibiting or treatment inflammation related disease, comprises to the object of needs and uses the step that is selected from down the component of organizing:
(a) recombinant human serum amyloid A 1 of the present invention;
(b) polymer of 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 a Mammals.More preferably, said object is behaved.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and hereinafter can mutual combination between specifically described each technical characterictic in (like embodiment), thus constitute new or optimized technical scheme.As space is limited, this tired no longer one by one stating.
Description of drawings
Fig. 1 detects the expression level figure of the hSAA1 of yeast expression for dot-blotting.
Fig. 2 is that the SDS-PAGE and the Western-blotting thereof of the hSAA1 purified product of yeast expression detects figure.
Fig. 3 is SDS-PAGE figure behind 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
The present inventor is through extensively and in depth research; The unexpected discovery; When hSAA1 albumen or its fragment are expressed through Yeast system, not only can obtain glycosylated reorganization hSAA1 or its fragment, and this specific hSAA1 or its fragment are the suppressor factor of natural hSAA unexpectedly; When share, can significantly reduce the activity of (or inhibition) natural hSAA with natural hSAA.On this basis, accomplished the present invention.
Particularly, the present invention adopts the yeast cell that contains the transfection of the proteic gene yeast expression vector of coding hSAA1, can obtain hSAA1 and polymer thereof through cultivation, screening, secretion inducing, purifying, does not have LPS to disturb.Testing surface by hSAA1 or the polymer of yeast as host expresses, has the ability that suppresses inflammatory cytokine.
Further; For structural domain and the domain of studying hSAA1 and receptors bind; The contriver has synthesized the amino acid sequence of polypeptide of multiple coding hSAA1; Use the polypeptide fragment of yeast as host expresses acquisition hSAA1, the result shows the ability that is also had the inhibition inflammatory cytokine by yeast as the hSAA1 polypeptide fragment of host expresses.
Term
As used herein; Term " inflammation related disease " refers to be attended by inflammation or expresses the too high disease that causes by inflammatory cytokine; Include but not limited to rheumatic arthritis, atherosclerosis, cardiovascular disorder, pancreatitis, hepatitis, mellitus, 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 (reorganization hSAA1) is to be the expressed recombinant human serum amyloid A 1 of host with the yeast.
In another preference, said recombinant human serum amyloid A 1 has following one or more characteristic:
(a) molecular weight is the monomer of 12～14KD;
(b) activity of inhibition natural human serum amyloid A protein;
(c) has the ability that inflammatory cytokine is expressed that suppresses.
In another preference, said inflammatory cytokine comprises IL-6, IL-1 β, TNF-α, IL-12, MMP9.
Recombinant human serum amyloid A 1 of the present invention does not combine LPS.
In another preference, said inhibition is a competitive inhibition.
The nucleotide sequence of coding recombinant human serum amyloid A 1 is shown in SEQ ID NO.:1.
The aminoacid sequence of recombinant human serum amyloid A 1 is 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, said polymer has following one or more characteristic:
(a) said polymer is a dimer, tripolymer, the tetramer or its combination;
(b) activity of inhibition natural human serum amyloid A protein;
(c) has the ability that inflammatory cytokine is expressed that suppresses.
In another preference, said dimeric molecular weight is about 25～27KD; Said trimerical molecular weight is about 38～40KD; Said tetrameric molecular weight is about 41～43KD.
In another preference, said inflammatory cytokine comprises IL-6, IL-1 β, TNF-α, IL-12, MMP9.
In another preference, said polymer is for being the polymer of the expressed human serum amyloid A 1 of host with the yeast.
In another preference, said inhibition is a competitive inhibition.
Recombinant human serum amyloid A 1 of the present invention and polymeric preparation method thereof comprise step:
(a) Yeast expression carrier of the gene that contains the human serum amyloid A 1 of encoding is provided;
(b) yeast host cell that contains said Yeast expression carrier is provided;
(c) under the condition that is fit to expression, cultivate described yeast host cell, thereby express recombinant human serum amyloid A 1 of the present invention or polymer of the present invention; With
(d) from cultured products, isolate described recombinant human serum amyloid A 1 or its polymer.
In another preference, the gene of coding human serum amyloid A 1 through amplification, be connected to commercially available carrier (like pPIC9K) behind the double digestion, is obtained the described Yeast expression carrier of step (a).PPIC9K is an excretion vector that efficiently expresses exogenous protein, has signal peptide α-factor on it, can hSAA1 be secreted in the substratum.
In another preference; Utilize XhoI and EcoRI double digestion that the hSAA1 gene is connected to the α-factor downstream through XhoI and EcoRI double digestion carrier pPIC9K; The centre is connected to Kex2 signal cleavage (carrier is self-contained); Can discharge the N-terminal of hSAA1 with α-factor excision in the process of secreting, expressing.
In another preference, adopt Yeast expression carrier transfection yeast GS115 in the step (b).
In another preference, adopt electric method for transformation to carry out transfection in the step (b).
In another preference, adopt the dull and stereotyped culturing yeast of histidine auxotroph in the step (c), screening yeast mono-clonal.
In another preference, said step (d) adopts G418 to screen.
In another preference, said step (e) adopts methyl alcohol to carry out abduction delivering, obtains said recombinant human serum amyloid A 1 and polymer thereof.
In another preference, said method also comprises the step of the higher mono-clonal of expression amount being carried out bulk fermentation and purifying.
In another preference, said purifying is meant 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 characteristic:
(i) sequence of this polypeptide fragment derives from the aminoacid sequence of human serum amyloid A 1;
(ii) the 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 that inflammatory cytokine is expressed that suppresses.
In another preference, said polypeptide fragment is for being the expressed polypeptide fragment of host with the yeast.
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.Digitized representation top and end amino acid position in the aminoacid sequence of human serum amyloid A 1 wherein.It is the aminoacid sequence that said polypeptide has 7-30 position, 11-45 position, 27-72 position, 27-90 position, 29-104 position, 27-104 position, 9-72 position or the 72-104 position of wild-type human serum amyloid A 1 aminoacid sequence.
In another preference, the aminoacid sequence of wild-type human serum amyloid A 1 is shown in SEQ ID NO.:2.
Polypeptide of the present invention can be recombinant polypeptide, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of chemosynthesis, or uses recombinant technology from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell), to produce.The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.
In another preference, polypeptide fragment of the present invention is a recombinant polypeptide, expresses the polypeptide fragment of acquisition for the host for yeast.
The preparation method of the polypeptide fragment of human serum amyloid A 1 of the present invention comprises step:
(a) Yeast expression carrier of the polynucleotide of the polypeptide fragment that contains the human serum amyloid A 1 of encoding is provided;
(b) yeast host cell that contains said Yeast expression carrier is provided;
(c) be fit under the condition of expressing, the culturing yeast host cell, thus express polypeptide fragment of the present invention; With
(ii) from cultured products, isolate described polypeptide fragment.
In another preference, the polynucleotide of the polypeptide fragment of coding human serum amyloid A 1 through amplification, be connected to pPIC9K behind the double digestion, are obtained the described Yeast expression carrier of step (a).
In another preference, utilize will the encode polynucleotide of polypeptide fragment of human serum amyloid A 1 of XhoI and EcoRI double digestion to be connected on XhoI and EcoRI double digestion carrier pPIC9K.
In another preference, adopt Yeast expression carrier transfection yeast GS115 in the step (b).
In another preference, adopt electric method for transformation to carry out transfection in the step (b).
In another preference, adopt the dull and stereotyped culturing yeast of histidine auxotroph in the step (c), screening yeast mono-clonal.
In another preference, said step (d) adopts G418 to screen.
In another preference, said step (e) adopts methyl alcohol to carry out abduction delivering, obtains said recombinant human serum amyloid A 1 and polymer thereof.
In another preference, said method also comprises the step of the higher mono-clonal of expression amount being carried out bulk fermentation and purifying.
In another preference, the N of said polypeptide fragment end has the HIS6-SUMO-TEV fragment, both can strengthen polypeptide expression output, also can make things convenient for purifying, and purified product can be cut the release polypeptide fragment with the TEV enzyme.
In another preference, yeast expression system of the present invention is expressed the polypeptide that does not have LPS interferential recombinant human SAA1 and similar thereof.This is the problem that host's phraseology has not only been broken away from the LPS puzzlement of being run in the SAA1 research for many years with the yeast; Realize that inside and outside research does not receive the biological action of LPS interferential SAA1; The polypeptide that yeast expression system also capable of using is expressed multiple SAA1 similar carries out functional study, thereby finds SAA performance active structures territory and domain.The result of study of the BA that the detailed different peptide sections of people SAA1 also capable of using are simultaneously brought into play designs corresponding blocker, for clinical treatment provides the potential drug candidate.
More than the expressed also available ordinary method of reorganization hSAA1, add the HIS6 affinity tag at C-terminal, expressed albumen and polypeptide thereof after Western-blotting confirms as target protein matter, utilize affinity chromatography to carry out purifying through SDS-PAGE.
Purposes
Reorganization hSAA1 and the polypeptide thereof of this patent after with purifying stimulates the THP-1 cell strain, detects the influence of these product pair cell factors (like IL6, TNF α, IL-1 β, IL12 P40, MMP9 etc.) excretory.The result shows, reorganization hSAA1 of the present invention, polymer, polypeptide fragment have and suppress the ability that inflammatory cytokine is expressed, and can be used to prepare the medicine of the disease that the treatment inflammation causes.
Further, purified product and the common irritation cell of commercial SAA (can induce the generation of inflammatory factor, bring out inflammation) are carried out the activity detection, show the hSAA1 monomer of yeast expression and the proinflammatory effect that polymer significantly reduces commercial SAA.Because the hSAA1 monomer of yeast expression and polymer and polypeptide thereof have remarkable restraining effect to the expression of the various kinds of cell factor, therefore can be used for treating the disease that inflammatory reaction process SAA excessive secretion is caused.Simultaneously, hSAA1 monomer, polymer and the polypeptide of yeast expression can suppress the secretion of LPS irritation cell, and some cause scorching cytokine (like IL-1 β), also can be used to treat the caused disease of inflammatory reaction.
The disease that described inflammation causes is selected from down group: rheumatic arthritis, atherosclerosis, cardiovascular disorder, pancreatitis, hepatitis, mellitus, 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, inert and the pharmaceutically acceptable aqueous carrier medium; Wherein pH is about 5-8 usually; Preferably pH is about 6-8, although the pH value can change with being prepared Substance Properties and illness to be treated to some extent.The pharmaceutical composition for preparing can carry out administration through 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 directly be used for the treatment of diseases that inflammation causes, and for example, are used for the rheumatoid arthritis treatment of diseases.When using recombinant human serum amyloid A 1 of the present invention, polymer, polypeptide fragment, also can use simultaneously the other treatment agent or with other therapy couplings.
The present invention also provides a kind of pharmaceutical composition, and it contains recombinant human serum amyloid A 1 of the present invention, polymer, polypeptide fragment or its combination of safe and effective amount; And pharmaceutically acceptable carrier or vehicle.This type carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical prepn should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example prepares through ordinary method with the saline water or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition can prepare through ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example 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 with the other treatment agent.
When making pharmaceutical composition; Be that recombinant human serum amyloid A 1 of the present invention, polymer, polypeptide fragment or its combined administration with safe and effective amount is in Mammals; Wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight; And in most of the cases be 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 is factor such as considered route of administration, patient health situation also, and these all are within the skilled practitioners skill.
Major advantage of the present invention is:
(1). the 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 confirms that recombinant human serum amyloid A 1 of the present invention, polymer, polypeptide fragment can reduce the mouse lethality rate of LPS.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; People such as Sambrook for example; Molecular cloning: the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber be weight percentage and parts by weight.
The preparation of reorganization hSAA1
Synthetic following primer:
Upstream primer (SEQ ID NO:3):
5’GTCG?CTC?GAG?AAA?AGA?GAG?GCT?CGAAGCTTCTTTTCGTTC?3’；
Downstream primer (SEQ ID NO:4):
5’GTCG?GAA?TTC?TCA?GTG?GTG?GTG?GTG?GTG?GTG?GTATTTCTCAGGCAG?3’；
With ordinary method extracting people mRNA, the cDNA that after reverse transcription, obtains is as template.Use above-mentioned primer, through conventional PCR method, amplification obtains people hSAA1 full-length gene.To pcr amplification product, behind the XhoI/EcoRI double digestion, be inserted into α-factor signal peptide downstream of the carrier pPIC9k (available from Invitrogen company) that cuts through same enzyme, obtain carrier pPIC9k-hSAA1.When the hSAA1 secreting, expressing, α-factor signal peptide can be excised, thereby discharges the N-terminal of hSAA1.
Utilize electric method for transformation, change above-mentioned recombinant expression vector pPIC9k-hSAA1 over to conventional pichia spp GS115, and select the recon that transforms as follows:
Adopt the dull and stereotyped yeast of cultivating transfection of histidine auxotroph, screening yeast mono-clonal, the mono-clonal coating G418 screening that is obtained is dull and stereotyped; G418 concentration is respectively 0.5mg/mL, 1mg/mL, 2mg/mL; Cultivated 3-4 days for 30 ℃; Choose the mono-clonal of being grown on the G418 concentration 2mg/mL flat board and cultivate, methanol induction express recombinant people hSAA1 protein, its C-terminal have the HIS6 affinity tag (introducing through primer) of being convenient to affinity purification.
Adopt DOT-BLOTTING to detect the expression output of target protein matter in the supernatant.Fig. 1 is the DOT-BLOTTING detected result that 6 yeast mono-clonals are expressed hSAA1, chooses the higher mono-clonal of expression output and carries out next step bulk fermentation and purifying thereof.
The preparation of hSAA1 polypeptide fragment
Adopt the synthetic his6-sumo-tev sequence of ordinary method gene; Shown in SEQ ID NO:5; Utilize restriction enzyme site XhoI to insert α-factor signal peptide downstream of carrier pPIC9k (Invitrogen company), acquisition has the pPIC9k carrier pPIC9k-his6-sumo-tev of his6-sumo-tev.
The sheet name section is the primer of P27-72 in the employing table 1, and through conventional PCR method, amplification obtains the people hSAA1 gene fragment of coded polypeptide fragment P27-72 respectively.To pcr amplification product, behind the XhoI/EcoRI double digestion, be inserted into the his6-sumo-tev downstream among the carrier pPIC9k-his6-sumo-tev that cuts through same enzyme, obtain carrier pPIC9k-his6-sumo-tev-hSAA1P27-72.
Utilize electric method for transformation, change above-mentioned recombinant expression vector pPIC9k-his6-sumo-tev-hSAA1P27-72 over to conventional pichia spp GS115, and select the recon that transforms as follows:
Adopt the dull and stereotyped yeast of cultivating transfection of histidine auxotroph, screening yeast mono-clonal, the mono-clonal coating G418 screening that is obtained is dull and stereotyped; G418 concentration is respectively 0.5mg/mL; 1mg/mL, 2mg/mL cultivated 3-4 days for 30 ℃; Choose the mono-clonal of being grown on the G418 concentration 2mg/mL flat board and cultivate methanol induction express recombinant people hSAA1 polypeptide fragment P27-72 (corresponding to 27-72 position among the SEQ ID NO.:2).
Adopt method for preparing recombinant human hSAA1 polypeptide fragment P27-90, recombinant human hSAA1 polypeptide fragment P29-104, difference is primer, and is as shown in table 1.
Table 1 primer sequence table
The purifying of reorganization hSAA1 and polypeptide fragment thereof
The yeast fermentation broth supernatant that embodiment 1 and embodiment 2 are obtained mixed 1-2 hour the dress post through ultrafiltration and concentration with 4 ℃ of Ni Sepharose HP.Adopt lavation buffer solution (20mM sodium phosphate (sodium phosphate), 25mM imidazoles (imidazole), 500mM NaCl; PH 7.4) the washing pillar, elution buffer (20mM sodium phosphate sodium phosphate, 500mM imidazoles imidazole; 500mM NaCl; PH 7.4) wash-out target protein matter, adopt SDS-PAGE and Western-blotting thereof to detect, the result is respectively as shown in Figures 2 and 3.
Present existing escherichia expression system only can obtain monomer.Unexpected is that the employing yeast expression system can obtain hSAA1 new, multimeric forms, i.e. the polymer of hSAA1.
As shown in Figure 2; For the SDS-PAGE and the Western-blotting thereof 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 that calculates based on aminoacid sequence); Swimming lane 2 is hSAA1 polymers (dimer ≈ 26KD, tripolymer ≈ 39KD, tetramer ≈ 52KD) of yeast expression.
As shown in Figure 3, be SDS-PAGE figure behind the hSAA1 polypeptide fragment purifying of yeast expression, wherein; Swimming lane 3 (is connected with the hSAA1 polypeptide fragment P27-72 of SUMO for the SUMO-hSAA1 of yeast expression (P27-72); ≈ 21KD), swimming lane 2 is the SUMO-hSAA1 of yeast expression (P27-90) (being connected with the hSAA1 polypeptide fragment P27-90 of SUMO, ≈ 23KD); Swimming lane 1 is the SUMO-hSAA1 of yeast expression (P27-104) (being connected with the hSAA1 polypeptide fragment P27-104 of SUMO, ≈ 24KD)
Embodiment 4
HSAA1 and polypeptide fragment thereof are to the influence of THP-1 cell strain cytokine secretion
Commercial SAA, hSAA1, hSAA1 polymer, polypeptide fragment (the tev enzyme is cut after product) are joined people THP-1 cell (the ATCC TIB-202 of the routine of serum-free culture with the final concentration of 0.5 μ M
TMThe acute monocyte strain property of people leukemia cell line) nutrient solution (10
6Cells/ml) in, collecting cell after 5 hours, RNA is extracted in the TRIzol cracking, and reverse transcription is cDNA, and real-time PCR detects the cytokine expression level.
Shown in Figure 4, hSAA1 monomer and the polymer with yeast expression stimulates the THP-1 cell after 5 hours respectively, detects the expression level of IL-6RNA in the cell.The result shows that after commercial SAA (0.5 μ M) stimulated the THP-1 cell, the level that IL-6 raises was higher than the hSAA1 monomer and the hSAA1 polymer of yeast expression; Commercial SAA (0.5 μ M) is mixed the back irritation cell respectively with the hSAA1 monomer (0.5 μ M) and the polymer (0.5 μ M) of yeast expression, stimulate the THP-1 cell to compare separately with commercial SAA, the IL-6 expression level descends.Show the proinflammatory effect that the hSAA1 monomer of yeast expression and polymer suppress commercial SAA.
In addition, adopt micro-LPS (5ng/mL) to stimulate THP-1 after, the IL-6 expression level improves, and after mixing stimulation with the hSAA1 monomer (0.5 μ M) of yeast expression, IL-6 improves the standard and is higher than both and stimulates separately.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 behind the stimulation THP-1 cell, and the result is consistent with IL-6, and the hSAA1 monomer of yeast expression and polymer have the active effect that suppresses commercial SAA.But, stimulate THP-1 jointly with micro-LPS (5ng/mL) after, the TNF alpha levels descends, 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 behind the stimulation THP-1 cell, and the hSAA1 monomer of yeast expression has the effect that suppresses commercial SAA, and is simultaneously, also inhibited to LPS irritation cell secretion IL-1 β.
Fig. 7 and Fig. 8 are respectively polypeptide fragment stimulates the expression level of IL-6 behind the THP-1 cell and IL-1 β thereof to detect; Find that it is minimum that hSAA1 (27-72) induces IL-6 and IL-1 β level thereof; And can suppress the effect of commercialization SAA, also have some synergies with LPS simultaneously.HSAA1 (27-90), hSAA1 (29-104) induces the corresponding raising of level, but also exists and the active effect that suppresses commercialization SAA.
With Fig. 8 and hSAA1 (27-72) is example, and during singly with commercial SAA (0.5 μ M), the expression level of IL-1 β is 75 times, and during singly with hSAA1 (P27-72 polypeptide) (0.5 μ M), the expression level of IL-1 β is 40 times.Unexpected is, SAA (0.5 μ M) and P27-72 polypeptide (0.5 μ M) when share, can be dropped to 37 times from 75 times with the expression level of IL-1 β.This shows that the P27-72 polypeptide can reduce the rising of the IL-1 β that SAA causes (IL-1 β raises and can cause the generation of multiple diseases associated with inflammation).
Fig. 6 is the detection of IL-1 β level behind the stimulation THP-1 cell, and the hSAA1 polymer of yeast expression has the effect that suppresses commercial SAA, and is simultaneously, also inhibited to LPS irritation cell secretion IL-1 β.
Because the hSAA1 monomer of yeast expression and polymer and polypeptide thereof have restraining effect to the expression of the various kinds of cell factor, therefore can be used as the potential medicine, be used for treating the disease that inflammatory reaction process SAA excessive secretion is caused.Simultaneously, hSAA1 monomer, polymer and the polypeptide of yeast expression can suppress the secretion of LPS irritation cell, and some cause scorching cytokine (like IL-1 β), also can be used as the potential medicine, are used to treat the caused disease of inflammatory reaction.
Comparative Examples 1
Use ordinary method, adopt coli expression system, the hSAA1 of preparation aminoacid sequence shown in SEQ ID NO.:2.For the nonglycosylated hSAA1 of this prokaryotic expression, measure it to influence to THP-1 cell strain cytokine secretion with embodiment 4 identical methods.
The result shows; Use the hSAA1 (0.5 μ M) of escherichia coli expression separately; Perhaps mix the back with commercial SAA (0.5 μ M) and use, can both 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.
HSAA1 and polypeptide fragment thereof and LPS and SAA abdominal injection BALB/c mouse thereof cause the mouse inflammatory reaction
HSAA1, polymer and the polypeptide that present embodiment has designed yeast expression in the mouse body to the inhibiting experiment of the inflammatory reaction that LPS caused.Select the 5-7 male BALB/c mouse in week, 3 every group, five groups altogether, be respectively LPS group (LPS 15mg/Kg); The LPS/hSAA1 low dose group (LPS 15mg/Kg, hSAA115mg/Kg), (the LPS 15mg/Kg of dose groups among the LPS/hSAA1; HSAA1 30mg/Kg), LPS/hSAA1 high dose group (LPS 15mg/Kg, hSAA1 100mg/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 the mouse death rate test experience; The hSAA1 monomer and the polymer thereof of yeast expression can be alleviated the dead mouse that LPS causes; Prolong the mouse survival time, wherein hSAA1 (27-72) fragment is best to suppressing LPS mouse lethality rate activity, can suppress the death of mouse fully.
Embodiment 6
Set up the mouse arthritis model, joint cavity injection hSAA1 and polypeptide fragment thereof detect arthritic development
Through ordinary method, with collagen protein inducing mouse arthritis model.Then, at intraarticular injection hSAA1 and polypeptide fragment thereof, the effect that observation hSAA1 and polypeptide fragment thereof develop mouse arthritis (administration group and not administration group, n=6).
Arthritic clinical table disease severity is divided the level assessment with usage quantity.Based on swelling, erythema and distortion are divided into 0-4:0, no swelling; 1, in toes swelling or Mild edema; 2, moderate swelling influences some toes; 3, some swelling influence most of toes; 4, the most serious swelling and/or tetanic.
The result shows, compares with mouse arthritis model group (not administration group), and hSAA1 test group and hSAA1 polypeptide fragment group (P27-72 polypeptide) arthritic symptom have obvious alleviation (score value significantly is lower than the control group of not administration).
Embodiment 7
Pharmaceutical composition
Prepare a pharmaceutical composition, said compsn comprises:
(a) P27-72 polypeptide (or P27-90 polypeptide); With
(b) saline water.
Embodiment 8
Pharmaceutical composition
Prepare a pharmaceutical composition, said compsn comprises:
(a) the hSAA1 albumen of embodiment 1 preparation; With
(b) saline water.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. a recombinant human serum amyloid A 1 is characterized in that, said recombinant human serum amyloid A 1 is for being the expressed recombinant human serum amyloid A 1 of host with the yeast.
2. the polymer of a human serum amyloid A 1 is characterized in that, said polymer is the polymer of the described recombinant human serum amyloid A 1 of claim 1.
3. a human serum amyloid A 1 polypeptide fragment is characterized in that, described polypeptide fragment has following characteristic simultaneously:
(i) sequence of this polypeptide fragment derives from the aminoacid sequence of human serum amyloid A 1;
(ii) the 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 that inflammatory cytokine is expressed that suppresses.
4. polypeptide fragment as claimed in claim 3; It is characterized in that said polypeptide has the aminoacid sequence of 7-30 position, 11-45 position, 27-72 position, 27-90 position, 29-104 position, 27-104 position, 9-72 position or the 72-104 position of wild-type human serum amyloid A 1 aminoacid sequence.
5. a Yeast expression carrier is characterized in that, this carrier contains the gene of the human serum amyloid A 1 of encoding; Or
This carrier contains the polynucleotide of the described polypeptide fragment of coding claim 3.
6. a yeast host cell is characterized in that, said host cell is selected from down group:
(a) contain the host cell of the described carrier of claim 5;
(b) be integrated with the host cell of coding hSAA1 proteic gene in the karyomit(e);
(c) be integrated with the host cell of the polynucleotide of coding claim 3 described polypeptide fragment in the karyomit(e).
7. the preparation method of the described recombinant human serum amyloid A 1 of claim 1 or described polymer of claim 2 or the described polypeptide fragment of claim 3 is characterized in that, comprises step:
(i) be fit to cultivate the described host cell of claim 6 under the condition of expressing, thereby expressing the described recombinant human serum amyloid A 1 of claim 1 or described polymer of claim 2 or the described polypeptide fragment of claim 3; With
(ii) from cultured products, isolate described recombinant human serum amyloid A 1 or its polymer or its polypeptide fragment.
8. a pharmaceutical composition is characterized in that, it contains pharmaceutically acceptable carrier or vehicle; And the activeconstituents that is selected from down group:
(a1) the described recombinant human serum amyloid A 1 of claim 1;
(a2) polymer of the described human serum amyloid A 1 of claim 2;
(a3) the said human serum amyloid A 1 polypeptide fragment of claim 3.
9. described recombinant human serum amyloid A 1 of claim 1; The perhaps polymer of the described human serum amyloid A 1 of claim 2; The perhaps purposes of the said human serum amyloid A 1 polypeptide fragment of claim 3; It is characterized in that, be used to prepare the medicine of inflammation-inhibiting and/or treatment inflammation related disease.
10. the human serum amyloid A 1 of a reorganization or its polypeptide fragment or its polymer; It is characterized in that; Said 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.
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