CN108727484A - Human serum amyloid A 1 functional oligopeptides and its preparation method and application - Google Patents

Abstract

The invention discloses a kind of human serum amyloid A 1 functional oligopeptides and its preparation method and application.Specifically, the present invention provides a kind of human serum amyloid A 1 polypeptide fragment or its pharmaceutically acceptable salt, and the polypeptide fragment has following characteristics：(i) sequence of the polypeptide fragment derives from the amino acid sequence of human serum amyloid A 1；(ii) polypeptide fragment is M to N of human serum amyloid A 1 (SAA1) full length amino acid sequence (104 amino acid), and length is 42-67 amino acid, wherein M is any positive integer of 9-14, and N is any positive integer of 55-75.The human serum amyloid protein functional oligopeptides of the present invention have the ability for inhibiting proinflammatory cytokine expression, promoting anti-inflammatory cytokines expression, can be used for preparing the drug for the treatment of inflammation disease.

Description

Human serum amyloid A 1 functional oligopeptides and its preparation method and application

Technical field

The invention belongs to technical field of bioengineering, and in particular to a kind of human serum amyloid A 1 functional fragment and Preparation method and application.

Background technology

Serum amyloid A protein (Serum Amyloid A, SAA) is one kind that body is infected and is secreted when damaging Main acute phase protein.SAA is the general name of a polymorphism albumen, by the albumen (SAA1- of related but each tool separate gene SAA4 it) forms.Wherein, human serum amyloid A 1 (SAA1) is made of 104 amino acid, is mainly synthesized by liver cell, people SAA2 and SAA1 has the difference of 7 amino acid, they are acute phase protein.SAA3 is a pseudogene, and SAA4 is acute When phase reaction in change little, continue to generate on a small quantity in body.

Studies have shown that SAA contents in acute and chronic inflammation patients serum have a large amount of risings, therefore it is a reflection Infectious and noninfective inflammatory disease important biomolecule Testing index.SAA is not only Hospitalized Patients with Acute Exacerbations of Chronic Obstructive Pulmonary Disease Biomarker and acute myocardial infarction patient occur after receiving emergency treatment coronary intervention procedure the expection of cardiac rupture because Son, this provides important reference indicator for early detection cardiac rupture patient.Other numerous studies also indicate that, in pancreatitis, liver Inflammation, coronary heart disease, diabetes, chronic renal disease, rheumatoid arthritis, inflammatory enteritis, acute and chronic pneumonia and obesity In example, SAA is increased has very close relationship with disease activity and disease.Clinical research at present is also believed that all acute In phase reactive protein, SAA is most sensitive to acute inflammatory reaction, and appreciation amplitude is maximum.Research also shows SAA and alpha-fetoprotein simultaneously Equal tumor correlated albumens are similar, and the concentration in serum is proportionate with cancer.Therefore, clinically to SAA in disease The detection of content has additive diagnostic value in human serum.

Meanwhile SAA is not only a kind of simple marker of inflammation, also actively intervenes the correlated process of disease.In acute sense When dye or inflammation occur, the level of SAA can promote 1000 times in serum.High-caliber SAA will lead to amyloid lesion, and The risk of angiocardiopathy can be improved in the conjugate (LDL-SAA) of low-density lipoprotein and SAA；Meanwhile SAA is also with proinflammatory anti- The characteristic answered promotes the secretion of cytokine profiles, and such as IL-1 β, IL-6, MMPs, CCL20 etc., these cell factors are involved in The pathogenic process of disease caused by inflammation, e.g., rheumatic arthritis, muscular atrophy, gout, atherosclerosis etc..Due to SAA It is related to the generation of a variety of diseases, it has increasingly been taken seriously for the therapy of SAA.

At present it is known that SAA is combined with six kinds of receptors, Toll-like receptor (TLR) include TLR2 and TLR4, SRB1 (a class B scavenger receptor) and its g protein coupled receptor formyl peptide receptor 2 (FPR2), receptor for advanced glycation endproduct (RAGE), P2X7receptor).SAA with not With receptor combine played biological activity different, for example, SAA combined with FPR2 inducible synovia hyperplasia and its The proliferation of endothelial cell, so as to cause the generation of rheumatoid arthritis；SAA is combined the metabolism that can promote cholesterol with CD36； SAA is combined with SRB1 can generate inflammation-inducing factor, lead to the diseases such as atherosclerosis；SAA is combined with TLR4 can induce NO Generation.It can be seen that SAA1 plays various biological activity in body.

In order to more clearly carry out the research and analysis of structure and its biological activity to SAA1, this patent devises yeast Expression system expresses the recombined human SAA1 of no lipopolysaccharides interference and its polypeptide that structure is similar.This with yeast is host's Expression way has not only broken away from the problem of lipopolysaccharides puzzlement encountered in the research of SAA1 for many years, realize in vivo and in vitro not by Lipopolysaccharides interference SAA1 biological action, also using yeast expression system express the similar polypeptide of a variety of SAA1 structures into Row functional study, to find that SAA plays active structural domain and functional domain.Simultaneously also using detailed people SAA1 difference peptides The result of study for the biological activity that section is played, designs corresponding blocking agent, potential drug candidate is provided for clinical treatment.

However, this field, which still lacks, satisfactorily can effectively inhibit proinflammatory cytokine, so as to effectively treat inflammation The drug of disease or inflammation related disease.

Invention content

The purpose of the present invention is to provide one kind can effectively inhibiting proinflammatory cytokine, anti-inflammatory factors can be promoted to express, from And it can effectively treat the drug of inflammation or inflammation related disease.

The first aspect of the present invention, provides a kind of human serum amyloid A 1 polypeptide fragment or its is pharmaceutically acceptable Salt, the polypeptide fragment have following characteristics：

(i) sequence of the polypeptide fragment derives from the amino acid sequence of human serum amyloid A 1；With

(ii) polypeptide fragment is the of human serum amyloid A 1 (SAA1) full length amino acid sequence (104 amino acid) M to N, and length is 42-67 amino acid, wherein M is any positive integer of 9-14, and N is any just whole of 55-75 Number.

In another preferred example, the polypeptide fragment also has one or more (or whole) features selected from the group below：

(iii) polypeptide fragment have inhibit gramnegative bacterium endotoxin LPS inducing macrophages inflammatory cell because The ability that sublist reaches；

(iv) inflammatory cytokine is selected from：IL-6,IL-1β,TNF-α；

(v) polypeptide fragment has the ability for inhibiting gramnegative bacterium endotoxin LPS inductions septicemia lethal；

(vi) polypeptide fragment has the ability of inducing macrophage expression anti-inflammatory cytokines；

(vii) polypeptide fragment has the ability of p38MAPK phosphorylations in inducing macrophage.

In another preferred example, the anti-inflammatory cytokines are selected from：IL-10；The induction anti-inflammatory cytokines IL-10's Ability depends on Toll-like receptor 2 (TLR2).

In another preferred example, P_M-NIndicate that M to N are constituted from 1 amino acid sequence of serum amyloid A protein Polypeptide.

In another preferred example, 9,10 or 11 M.

In another preferred example, 58,59,60,61,62,63,64,65,66,67,68,69,70,71 or 72 N.

In another preferred example, the polypeptide is selected from the group：P_11-58、P_11-68、P_11-72、P_11-55、P_11-75、P_10-58、 P_10-68、P_10-72、P_10-55、P_10-75, or combinations thereof.

In another preferred example, the polypeptide has the 11- of wild-type human serum's amyloid A 1 amino acid sequence 58,11-68,11-72 or combinations thereof.

In another preferred example, the amino acid sequence of wild-type human serum's amyloid A 1 such as SEQ ID NO.:2 institutes Show.

In another preferred example, the mark of the N-terminal of the polypeptide fragment and/or C-terminal the purpose of also band is useful for purifies and separates Sign sequence (such as 6His or HIS6-SUMO-TEV segments).

In another preferred example, the polypeptide fragment be with yeast be host carry out expression acquisition polypeptide fragment.

In another preferred example, the amino acid sequence of the human serum amyloid A 1 is wild-type sequence.

In another preferred example, the amino acid sequence of the human serum amyloid A 1 such as SEQ ID NO.:Shown in 2.

In another preferred example, the proinflammatory cytokine is selected from the group：IL-6, IL-1 β, TNF-α, or combinations thereof.

In another preferred example, the polypeptide fragment has the ability for promoting anti-inflammatory cytokines expression.

In another preferred example, the anti-inflammatory factors are IL-10.

In another preferred example, the polypeptide fragment is recombination.

In another preferred example, the polypeptide is expressed in yeast.

In the second aspect of the present invention, a kind of fusion protein is provided, the fusion protein includes the first party of the present invention Human serum amyloid A 1 polypeptide fragment described in face and the sequence label together with the segment composition.

In another preferred example, the sequence label is blended in the N-terminal and/or C-terminal of the polypeptide fragment.

In another preferred example, the sequence label is selected from the group：His6, His6-SUMO, His6-SUMO-TEV piece Section.

In the third aspect of the present invention, a kind of polynucleotide sequence, the polynucleotide sequence coding present invention are provided First aspect described in human serum amyloid A 1 polypeptide fragment or the fusion protein described in the second aspect of the present invention.

In the fourth aspect of the present invention, a kind of Yeast expression carrier is provided, which contains the first of the coding present invention The polynucleotides of polypeptide fragment described in aspect.

In another preferred example, the carrier is the multinuclear containing the polypeptide fragment described in coding the first aspect of the present invention The Yeast expression carrier pPIC9K of thuja acid.

In the fifth aspect of the present invention, a kind of host cell is provided, the host cell is selected from the group：

(a) host cell containing the carrier described in the fourth aspect of the present invention；

(b) host that the polynucleotides of the polypeptide fragment described in coding the first aspect of the present invention are integrated in chromosome is thin Born of the same parents.

In another preferred example, the host cell is selected from the group：Yeast cells, the large intestine for expressing SAA1 albumen Bacillus.

In the sixth aspect of the present invention, a kind of side preparing the polypeptide fragment described in the first aspect of the present invention is provided Method, including step：

(a) under conditions suitable for the expression, the host cell described in the fifth aspect of the present invention is cultivated, to express this hair Polypeptide fragment described in bright first aspect；With

(b) polypeptide fragment described in the first aspect of the present invention is isolated from cultured products.

In the seventh aspect of the present invention, a kind of pharmaceutical composition is provided, it contains pharmaceutically acceptable carrier or tax Shape agent；And human serum amyloid A 1 polypeptide fragment described in the first aspect of the present invention is as active constituent.

In another preferred example, the dosage form of the pharmaceutical composition is ejection preparation, preparation capable of permeating skin, freeze dried powder.

In the eighth aspect of the present invention, it is more to provide human serum amyloid A 1 described in a kind of the first aspect of the present invention The purposes of peptide fragment or its pharmaceutically acceptable salt is used to prepare the drug for inhibiting inflammation and/or treating inflammation related disease.

In another preferred example, disease caused by the inflammation is selected from the group：Septicemia, is moved rheumatoid arthritis Pulse atherosclerosis, acute and chronic pneumonia, chronic enteritis, pancreatitis, hepatitis, diabetes, chronic renal disease, obesity, flesh Meat atrophy, gout.

In another preferred example, the inflammation is the inflammation caused by SAA1 is excessively high.

In the ninth aspect of the present invention, the human serum amyloid A 1 described in a kind of the first aspect of the present invention is provided The purposes of polypeptide fragment or its pharmaceutically acceptable salt, is used to prepare a composition or preparation, and the composition or preparation are used In stimulating expression of macrophage secretion anti-inflammatory factors IL-10.

In the tenth aspect of the present invention, a kind of method for treating disease caused by inflammation is provided, includes to pair of needs Human serum amyloid A 1 polypeptide fragment or its pharmaceutically acceptable salt or egg as described in application the first aspect of the present invention White complex.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Description of the drawings

Fig. 1 is SAA1 segments relative to the gel electrophoresis analysis figure after the position of overall length SAA1 and Yeast expression.

Fig. 2 shows SAA1 fragment blocks compared with overall length SAA1, inflammation inducing cell factor IL-6, IL-1 β and TNF-α table The ability comparison reached.

Fig. 3 shows the relationship of inflammatory cytokine IL-6, IL-1 β caused by SAA1 fragment blocks LPS and TNF-α expression Figure.

Fig. 4 is that 11-58 peptide fragments block LPS inflammation inducing cell factor IL-6, IL-1 β and the dosage of TNF-α expression to close System's figure.

Fig. 5 shows that 11-58 peptide fragments effectively inhibit dead mouse caused by LPS.

Fig. 6 shows that 11-58 peptide fragments itself are unable to the raising of inducing mouse lung myeloperoxidase (MPO), but can press down Raisings of the MPO of LPS inductions processed in lung；MPO is peculiar for neutrophil cell, increases reflection lung neutrophil cell Infiltration.

Fig. 7 show 11-58 peptide fragments inhibit LPS induction mouse lung tissue in inflammatory cytokine IL-6, IL-1 β and The expression of TNF-α.

Fig. 8 schemes for lung tissue section, and display receives the mouse of 11-58 peptide fragments, has one to the acute lung injury of LPS inductions Fixed resists, and keeps lung tissue's structural integrity and less inflammatory cell infiltration.

Fig. 9 shows that 11-58 peptide fragments induce the ability of map kinase activation and overall length SAA1 different by TLR2.

Figure 10 shows that 11-58 peptide fragments can regulate and control map kinase caused by LPS in macrophage and activate.

Figure 11 shows the expression of the mRNA and albumen of 11-58 peptide fragments induction anti-inflammatory cytokines IL-10.

Figure 12 is the comparison of induction anti-inflammatory cytokines IL-10 expression after 11-58 peptide fragments are used alone or share with LPS Figure.

Figure 13 shows the macrophage of the mouse from TLR2 and TLR4 gene delections, to 11-58 peptide fragments and overall length The IL-10 expression of SAA1 inductions has different reactions.

The RBL cell strains of Figure 14 display expression people FPR2 after by 11-58 peptide fragments and overall length SAA1 stimulations, generate different Migration reaction.

Figure 15 shows that 11-58 peptide fragments and overall length SAA1 have the ability that different inducing cells generates calcium current.

Specific implementation mode

The present inventor after extensive and in-depth study, by largely screening, it is new unexpectedly to develop a class formation for the first time The anti-inflammatory polypeptide of grain husk.The anti-inflammatory polypeptide of the present invention is the human serum amyloid A 1 functional oligopeptides with anti-inflammatory effect.This The small peptide (by taking small peptide 11-58 as an example) of invention, not only proinflammatory effect is remarkably decreased (or even loses or completely loses that cause is scorching to be made substantially With), and extremely can efficiently inhibit inflammatory reaction caused by LPS, and extremely can efficiently induce anti-inflammatory cytokines Expression.On this basis, the present invention is completed.

The research of the present inventor is also shown that SAA C-terminals are particularly significant to its proinflammatory effect, and C-terminal lacks more, leads Cause the proinflammatory effect of small peptide of the present invention weaker.

Specifically, the present inventor uses Yeast expression carrier pPIC9K, yeast strain GS115, expresses recombined human SAA1, and its segment derived from people SAA1.PPIC9K is the excretion vector of a high efficient expression exogenous proteins, is carried thereon SAA1 and its segment can be secreted into culture medium, facilitate subsequent purification work by the signal peptide α-factor.Utilize restriction enzyme site SAA1 is connected to α-factor downstream of carrier pPIC9K by XhoI and EcoRI, and centre is connected to Kex2 signal peptide cutting sequences, can be α-factor is cut off during secreting, expressing, discharges the N-terminal of SAA1.SAA1 segments after expression are in sds gel electrophoresis point It from rear, is dyed through Coomassie brilliant blue, shows estimated length (Fig. 1).

The structural domain and functional domain combined to study SAA1 with receptor, the present inventor devise the segment of a variety of SAA1 again Sequence, and analysis of biological activity has been carried out after expression.These SAA1 segments include but not limited to these following segment (numbers Represent beginning and end amino acid position)：11-58,11-68,11-72,27-72,27-90,29-104.These polypeptide fragments It is expressed using Yeast expression carrier pPIC9K and yeast strain GS115.These SAA1 segments are to bone marrow macrophage (BMDM) after being incubated, the expression that supernatant analyzes various cell factors is collected, it is found that SAA1C terminal deletions are more, The proinflammatory effect of its segment is weaker (Fig. 2).Above-mentioned segment lacks 10 to 28 amino acid in N-terminal.

Under same experiment condition, it is found that segment 11-58 has most apparent inhibiting effect to the LPS inflammatory reactions induced.Its In again with C-terminal removal amino acid at most, N-terminal lack the 11-58 segments of 10 amino acid, show strongest inhibitions LPS induction inflammation Property cell factor IL-6, TNF-α, the effect (Fig. 3) of IL-1 β expression.Using the 11-58 segments of various concentration, The segment is shown and concentration dependent inhibiting effect (Fig. 4).In addition, experiment is also shown that the SAA1 by yeast as host expresses Segment is more conducive to obtain the ability for inhibiting proinflammatory cytokine.

Then have detected anti-inflammatory effect of the 11-58 segments in Mice Body.In the detection of mouse lethal rate, selection C57BL/6 mouse are same strain with acute lung injury experiment.48 hours death rates are 90% under 20mg/kg LPS dosage. 11-58 segment dosage 5mg/kg are administered in abdominal cavity in advance, and LPS is administered after half an hour to be reduced to 40% by lethality, illustrate 11-58 Segment can be effectively reduced the death rate (Fig. 5) caused by LPS.

The LPS (15mg/kg) of relatively low-dose gives the injection of C57BL/6 mouse peritoneals, causes systemic inflammatory response, including Acute lung injury.11-58 segments are given as stated above, and it is horizontal can to effectively inhibit intrapulmonary MPO caused by LPS for as a result display Raising (Fig. 6).11-58 segments can also effectively inhibit the up-regulation of intrapulmonary inflammatory factor level caused by LPS, such as TNF-α, IL-6, IL-1 β (Fig. 7).It is observed according to lung sections hematoxylin eosin staining, it is found that 11-58 segments can effectively inhibit lung The infiltration (Fig. 8) of neutrophil cell.

Before the study found that overall length SAA1 can by TLR2 generate signal transduction, stimulate the work of intracellular map kinase Change, the phosphorylation level for being embodied in kinases rises.In order to compare 11-58 segments and overall length SAA1 on signal transduction functionality Difference, TLR2-HeLa stable cell strains give 0.5 μM of overall length SAA1 or 11-58 segment respectively.By the thorn of different time Swash, the phosphorylation level of three kinds of different map kinases is detected with the specific antibody of identification phosphorylating kinase.Fig. 9 shows 11-58 segments The phosphorylation of p38MAPK can be more stimulated compared with overall length SAA1, and loses the ability of stimulation JNK phosphorylations.

In order to further study signal transduction path and its effect of the inflammatory reaction caused by 11-58 segments inhibition LPS Mechanism.Utilize the 11-58 segments and LPS costimulation bone marrow macrophages of various concentration, wherein LPS adds prior to small peptide half an hour Enter into bone marrow macrophage, western-blot is then used to detect ERK, the phosphorylation level of JNK, p38MAPK are found 11-58 segments can inhibit ERK caused by LPS, the rising of JNK phosphorylation levels, and 11-58 segments itself have stimulation p38 phosphorus The horizontal ability risen of acidification, if shared with LPS, which further enhances the p38MAPK phosphorylations of LPS inductions (Figure 10).

IL-10 (IL-10) is a main anti-inflammatory factors.It was found that Bone Marrow Macrophage is pierced through 11-58 segments After swashing, there is a large amount of IL-10mRNA to express in 2-8 hours, and IL-10 protein levels also have raising, in 4-12 hours areas Interior particularly evident (Figure 11).In contrast, LPS induces seldom IL-10 to express, and when LPS and shared 11-58 segments, it is right There is no significant impact (Figure 12) for the function that its stimulation IL-10 is generated.

To detect generation of the 11-58 segments by which receptor for stimulating IL-10, from wild type C57BL/6 hour and together In the TLR2 knock out mice of sample genetic background, bone marrow macrophage is detached, uses overall length SAA1 and 11-58 segment to pierce respectively Swash, investigates the expression of IL-10.It was found that compared with overall length SAA1,11-58 segments can stimulate the mouse bone marrow cells of wild type huge Phagocyte generates the IL-10 of more (about 6 times).But in TLR2^-/-In mouse, overall length SAA1 and 11-58 segment stimulates IL-10 The ability of generation declines to a great extent (Figure 13).Thus it is speculated that 11-58 segments mainly generate IL-10 by TLR2 stimulating expression of macrophage.

Further compared 11-58 segments stimulates the difference of IL-10 with overall length SAA1 by TLR4, finds SAA1 and 11- Whether the generation of 58 segments stimulation IL-10 knocks out no dependence with TLR4 genes, regardless of being in mRNA or in albumen In level, 11-58 segments all induce more IL-10 to generate (Figure 13) compared with overall length SAA1.

Known overall length SAA1 can pass through FPR2 induced cell migrations.In contrast, 11-58 segments, which are unable to induced expression, has The Rat Granulocytes strain (FPR2-RBL) of FPR2 generates significant migration (Figure 14), illustrates that the removal of N-terminal and C-terminal leads to this SAA1 segments lose the function of induced cellular chemotaxis migration.

Known overall length SAA1 can stimulate the generation of intracellular calcium current by FPR2.This function is equally thin in FPR2-RBL It is detected on born of the same parents, as a result, it has been found that 11-58 segments lose the function (Figure 15) of eliciting calcium flux generation substantially.

Term

As used herein, term " polypeptide fragment of the present invention ", " polypeptide piece of human serum amyloid A 1 of the invention Section ", " segment of the present invention " or " small peptide of the present invention ", are used interchangeably, and refer to and derive from blood defined in first aspect present invention The polypeptide fragment or its pharmaceutically acceptable salt of clear amyloid A 1.Although furthermore, it is to be understood that preferred serum amyloid sample Albumin A 1 comes from people, but also may be from other mammals (such as other non-human primates).

The polypeptide fragment of human serum amyloid A 1

Invention broadly provides a kind of human serum amyloid A 1 polypeptide fragments, with following characteristics：

(i) sequence of the polypeptide fragment derives from the amino acid sequence of human serum amyloid A 1；With

(ii) polypeptide fragment is the of human serum amyloid A 1 (SAA1) full length amino acid sequence (104 amino acid) M to N, and length is 42-67 amino acid, wherein M is any positive integer of 9-14, and N is any just whole of 55-75 Number.

The representative polypeptide fragment of the present invention, including but not limited to P_11-58、P_11-68、P_11-72, or combinations thereof.It is wherein digital Represent beginning and end amino acid position in the amino acid sequence of human serum amyloid A 1.

As used herein, " separation " refers to that substance is separated from its primal environment (if it is crude, original Beginning environment is natural surroundings).As under the native state in active somatic cell polynucleotide and polypeptide be not isolate and purify , but same polynucleotide or polypeptide such as from native state with separated in other existing substances, then to isolate and purify 's.

As used herein, " polypeptide fragment of the present invention of separation " refer to polypeptide fragment of the present invention substantially free of naturally and its Relevant other albumen, lipid, carbohydrate or other materials.Those skilled in the art can use the purified technology of protein of standard pure Change polypeptide fragment of the present invention.Substantially pure polypeptide can generate single master tape in non-reducing polyacrylamide gel.This hair The purity of bright polypeptide fragment can use amino acid sequence analysis.

The polypeptide fragment of the present invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide, preferably recombinant polypeptide.The present invention Polypeptide fragment can be native purified product include via after protease hydrolytic product or chemically synthesized product, Or using recombinant technique from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell) It generates.According to the host used in recombinant production scheme, polypeptide of the invention can be glycosylated, or can be non-glycosylated 's.The polypeptide of the present invention may also include or not include the methionine residues of starting.

The invention also includes the segment of polypeptide fragment of the present invention, derivative and analogue.As used herein, term " piece Section ", " derivative " and " analog " refer to being kept substantially the identical biology work(of natural polypeptide fragment of the present invention of the invention Energy or active polypeptide.Polypeptide fragment, the derivative or the like of the present invention can be (i) there are one or multiple conservative or non-guarantors Keep acidic amino acid residue (preferably conservative amino acid) substituted polypeptide, and such substituted amino acid residue can be with It may not be by genetic code encoding, or (ii) more with substituent group in one or more amino acid residues Peptide, or (iii) mature polypeptide and another compound (for example extending the compound of polypeptide half-life period, such as polyethylene glycol) fusion Be formed by polypeptide, or (iv) additional amino acid sequence be fused to this polypeptide sequence and formed polypeptide (such as targeting sequencing or Secretion sequence or for purifying the sequence or proprotein sequence of this polypeptide, or the fusion protein with the formation of antigen I gG segments). According to the teaching of this article, these segments, derivative and analogue belong to scope known to those skilled in the art.

In the present invention, term " small peptide of the present invention ", which refers to, has the active SEQ ID NO of polypeptide fragment of the present invention:2 sequences Polypeptide.The term further includes having and polypeptide fragment identical function of the present invention, SEQ ID NO:The variant form of 2 sequences. These variant forms include (but being not limited to)：One or more (it is usually 1-50, preferably 1-30, more preferably 1-20 A, most preferably 1-10) amino acid missing, insertion and/or substitution, and add one or number in C-terminal and/or N-terminal A (being usually within 20, be more preferably within 5 within preferably 10) amino acid.For example, in the art, using When amino acid similar in performance is replaced, the function of protein is not usually changed.For another example, in C-terminal and/or N-terminal, which adds one or several amino acid generally also, will not change the function of protein.The term further includes polypeptide piece of the present invention The active fragment and reactive derivative of section.

Invention also provides the analog of polypeptide fragment of the present invention.The difference of these analogs and natural small peptide of the present invention can be with It is the difference on amino acid sequence, can also be the difference on the modified forms for do not influence sequence, or have both at the same time.These are more Peptide includes natural or induction genetic variant.Induction variant can be obtained by various technologies, such as by radiating or exposing Random mutagenesis is generated in mutagens, can also pass through the technology of site-directed mutagenesis or other known molecular biology.Analog is also Include the analog with the residue (such as D- amino acid) different from natural L-amino acids, and with non-naturally occurring or conjunction At amino acid (such as β, gamma-amino acid) analog.It should be understood that the polypeptide of the present invention is not limited to enumerated representativeness Polypeptide.

Modification (not changing primary structure usually) form include：The chemical derivative form of in vivo or in vitro polypeptide such as acetyl Change or carboxylated.Modification further includes glycosylation, is carried out in the synthesis and processing of polypeptide or in further processing step such as those Glycosylation modified and generation polypeptide.This modification can carry out glycosylated enzyme (such as mammal by the way that polypeptide to be exposed to Glycosylase or deglycosylation enzyme) and complete.Modified forms further include with phosphorylated amino acid residue (such as phosphoric acid junket ammonia Acid, phosphoserine, phosphothreonine) sequence.Further include being modified to improve its anti-proteolytic properties or optimization The polypeptide of solubility property.

In the present invention, " polypeptide fragment conservative variation's polypeptides of the present invention " refer to and SEQ ID NO:2 amino acid sequence It compares, there is at most 8, preferably at most 5, more preferably at most 3, most preferably at most 2 amino acid are by property is similar or phase Close amino acid is replaced and forms polypeptide.These conservative variation's polypeptides carry out amino acid substitution preferably based on table 1 and generate.

Table 1

The polynucleotides of the present invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people The DNA of work synthesis.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.Encoding mature polypeptide Coding region sequence can be with SEQ ID NO:Coding region sequence shown in 1 is identical or the variant of degeneracy.Such as this paper institutes With " variant of degeneracy ", which refers to coding in the present invention, has SEQ ID NO:The polypeptide piece of sequence shown in M-N in 2 Section, but with SEQ ID NO:The differentiated nucleic acid sequence of coding region sequence shown in 1.

Encode SEQ ID NO:The polynucleotides of 2 polypeptides shown in M-N include：The code sequence of encoding mature polypeptide Row；The coded sequence of mature polypeptide and various additional coding sequences；Coded sequence (and the optional additional code sequence of mature polypeptide Row) and non-coding sequence.

Polypeptide and polynucleotides in the present invention preferably provide in a separate form, are more preferably purified to homogeneous.

The nucleotide full length sequence of small peptide of the present invention or its segment can usually use PCR amplification method, recombination method or artificial conjunction At method obtain.It, can be according to related nucleotide sequence, especially open reading disclosed in this invention for PCR amplification method Frame sequence carrys out design primer, the commercially available libraries cDNA is used in combination or by the cDNA prepared by conventional method well known by persons skilled in the art Library expands as template and obtains related sequence.

Once obtaining related sequence, so that it may to obtain related sequence in large quantity with recombination method.This is typically will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.

In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, logical After first synthesizing multiple small fragments, it is then attached the very long segment of available sequence again.

At present, it is already possible to completely by chemical synthesis come obtain encoding albumen of the present invention (its segment or its derivative Object) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or such as carrier) and In cell.In addition, mutation can be also introduced into protein sequence of the present invention by chemical synthesis.

By the recombinant dna technology of routine (Science, 1984；224：1431), using the polynucleotide of the present invention Sequence can be used to express or produce the polypeptide fragment of the present invention of recombination.In general there are following steps：

(1) polynucleotides (or variant) of the coding small peptide of the present invention of the present invention, or with containing the polynucleotide Recombinant expression carrier converts or suitable host cell of transduceing；

(2) host cell that is cultivated in suitable culture medium；

(3) be separated from culture medium or cell, protein purification.

In the present invention, the polynucleotide sequence for encoding polypeptide of the present invention can be plugged into recombinant expression carrier.Term " recombination Expression vector " refers to bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus Such as adenovirus, retrovirus or other carriers.Applicable carrier includes but not limited in the present invention：It is expressed in bacterium Based on T7 expression vector (Rosenberg, et al.Gene, 1987,56：125)；It is expressed in mammalian cell PMSXND expression vectors (Lee and Nathans, J Bio Chem.263：3521,1988) it and in insect cell expresses From the carrier of baculoviral.As long as in short, can replicate and stablize in host, any plasmid and carrier can be used. One important feature of expression vector is to usually contain replication orgin, promoter, marker gene and translation control element.

The carrier for including above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence can be used for converting suitable When host cell, allow it to expression protein.

Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high Equal eukaryocytes, such as mammalian cell.Representative example has：Escherichia coli, streptomyces；The bacterium of salmonella typhimurium Cell；Fungal cell's such as yeast；Plant cell；The insect cell of drosophila S2 or Sf9；CHO, COS, 293 cells or Bowes are black The zooblast etc. of plain oncocyte.Preferred host cell is yeast cells.

It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original When core biology such as Escherichia coli, can absorb the competent cell of DNA can harvest after exponential phase of growth, use CaCl₂Method processing, institute With the step of it is generally well-known in the art.Another method is to use MgCl₂.If desired, conversion can also use the side of electroporation Method carries out.When host is eucaryote, following DNA transfection methods can be selected：Calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..

The transformant of acquisition can use conventional method culture, express the polypeptide of the coded by said gene of the present invention.According to used Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of suitable for host cell growth It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction) Cell is further cultured for a period of time by the promoter for inducing selection.

Recombinant polypeptide in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.Such as Fruit needs, its physics, chemical and other characteristics can be utilized to be separated by various separation methods and purify the albumen of recombination.This A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to：The renaturation process of routine is used Protein precipitant handles (salting-out method), centrifugation, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel filtration), inhales The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.

Purposes

(1) recombinant human serum amyloid A 1 polypeptide fragment of the invention, can be used to detect these products upon cell because The influence of sub (such as IL-6, TNF-α, IL-1 β, IL-10) secretion.

(2) recombination SAA1 polypeptide fragments of the invention, which have, inhibits proinflammatory cytokine expression and stimulation anti-inflammatory factors IL- The ability of 10 expression, can be used to prepare the drug for the treatment of inflammation disease；The inflammation disease is selected from the group：Rheumatism joint It is scorching, acute and chronic injury of lungs, chronic enteritis, atherosclerosis, angiocardiopathy, pancreatitis, hepatitis, diabetes, chronic Kidney trouble, obesity, muscular atrophy, gout.

(3) recombinant human serum amyloid A 1 polypeptide fragment of the invention has the characteristic for reducing LPS lethalities, energy Caused septicemia is accumulated for a large amount of endotoxin LPS after treating gram-negative bacterial infections.

Pharmaceutical composition and method of application

Recombinant human serum amyloid A 1 polypeptide fragment of the present invention can be formulated in nontoxic, inert and pharmaceutically may be used In the aqueous carrier medium of receiving, wherein pH ordinarily is about 5-8, and preferably pH is about 6-8, although pH value can be with being formulated substance Property and illness to be treated and be varied from.Prepared pharmaceutical composition can be administered by conventional route, Including (but being not limited to)：It is intramuscular, peritonaeum is interior, intravenous, subcutaneous, intradermal or local administration.

Recombinant human serum amyloid A 1 polypeptide fragment of the present invention can be directly used for the treatment of disease caused by inflammation, example Such as, it is used for the treatment of rheumatoid arthritis disease.When using recombinant human serum amyloid A 1 polypeptide fragment of the present invention, also It can be used simultaneously other therapeutic agents or be combined with other therapies, such as accumulated for endotoxin LPS after gram-negative bacterial infections Tire out the treatment of caused septicemia.

The present invention also provides a kind of pharmaceutical compositions, it contains the recombinant human serum amyloid of the present invention of safe and effective amount 1 polypeptide fragment of albumin A or combinations thereof；And pharmaceutically acceptable carrier or excipient.This kind of carrier includes (but and unlimited In)：Brine, buffer solution, glucose, water, glycerine, ethyl alcohol, and combinations thereof.Pharmaceutical preparation should match with administering mode.This hair Bright pharmaceutical composition can be made into injection form, such as with physiological saline or the aqueous solution containing glucose He other adjuvants It is prepared by conventional method.Pharmaceutical composition can be prepared by conventional method.Pharmaceutical composition for example injection, solution, Tablet and capsule preferably aseptically manufacture.The dosage of active constituent is therapeutically effective amount, such as about 1 microgram/thousand daily Gram about 5 mg/kg weight of weight-.In addition, recombinant human serum amyloid A 1 polypeptide fragment of the present invention can also be controlled with other Agent is treated to be used together.

It is by the recombinant human serum amyloid A 1 polypeptide fragment of the present invention of safe and effective amount when using pharmaceutical composition Or combinations thereof be applied to mammal, the wherein safe and effective amount typically at least about 10 micrograms/kg body weight, and most of In the case of no more than about 8 mg/kg weight, preferably the dosage is about 1 mg/kg body of about 10 micrograms/kg body weight- Weight.Certainly, specific dosage is also contemplated that the factors such as administration route, patient health situation, these are all skilled practitioners technical ability ranges Within.

Main advantages of the present invention are：

(1) heretofore described recombinant human serum amyloid A 1 polypeptide fragment, with full length recombinant human serum starch Sample albumin A 1 is compared, and has the activity for the induction proinflammatory cytokine expression being substantially reduced, notable raised induction anti-inflammatory cells The activity of factor IL-10 expression.

(2) inventors have surprisingly discovered that recombinant human serum amyloid A 1 polypeptide fragment LPS is induced it is more The expression of kind proinflammatory cytokine is inhibited.

(3) inventors have surprisingly discovered that recombinant human serum amyloid A 1 polypeptide fragment can protect mouse to exempt from Acute pulmonary damage caused by by LPS.

(4) present invention determines that these polypeptide fragments can reduce the mouse lethal rate of LPS by preliminary zoopery.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no Then percentage and number are calculated by weight.

Embodiment 1

The structure of the Yeast expression carrier of hSAA1 and its polypeptide fragment and its expression of target protein

The first step：Design primer clones hSAA1 genes and its polypeptide fragment (11-58,11-68,11-72,27-72,27- A variety of different peptide fragments such as 90,29-104), segment both ends carry restriction enzyme site XhoI/EcoRI.PCR obtains target fragment, XhoI/EcoRI double digestions, product recycling are connect with pPIC9k XhoI/EcoRI double digestion products, obtain expression vector.

Second step：Using electrotransformation method by expression vector transfecting yeast GS115, histidine auxotroph plate screening The monoclonal obtained is coated with G418 screening flat boards by yeast monoclonal, and G418 concentration is respectively 0.5mg/mL, 1mg/mL, 2mg/mL, 30 DEG C are cultivated 3-4 days, choose the Colony Culture grown on G418 concentration 2mg/mL tablets, methanol induction expression Target protein.Choose bulk fermentation and its purifying that the higher monoclonal of expression yield carries out next step.

Embodiment 2

The purifying of hSAA1 and its polypeptide fragment

Yeast fermentation broth supernatant is concentrated by ultrafiltration, is mixed 1-2 hours for 4 DEG C with Ni Sepharose HP, column is filled.20mM Sodium phosphate, 25mM imidazole, 100mM NaCl, pH 7.4 washs pillar, 20mM sodium Phosphate, 500mM imidazole, 100mM NaCl, pH 7.4 elutes target protein, SDS-PAGE detections.Then plus Enter the overnight digestion of 4 DEG C of His6-TEV enzymes, digestion products cross Ni again²⁺Affinity column, the His6-SUMO that digestion is got off and His6-TEV enzymes remove.The SAA1 or segment of purifying deposit in 20mM phosphate buffers (pH 7.4), can be placed on -80 DEG C long-term It preserves.Determine that SAA molecular weight and purity, BCA methods detect albumen concentration using SDS-PAGE, limulus reagent test detection endotoxin contains Amount.

Embodiment 3

The influence of hSAA1 and its polypeptide fragment to BMDM secrete cytokines

HSAA1 and its polypeptide fragment are added to 0.5 μM of final concentration in the BMDM cell culture fluids of free serum culture, Cell is collected after 5 hours, RNA, the table of reverse transcription cDNA, real-time PCR detection cell factors are extracted in TRIzol cracking Up to situation.For the experiment of LPS costimulations is added, small peptide is first added, after half an hour, 100ng/ml LPS are added.It is received after 5 hours Collect cell, the expression situation of real-time PCR detection cell factors.

Shown in Fig. 1 and Fig. 2, with the shortening of hSAA1 segments, proinflammatory effect gradually weakens, and is led along with inhibition LPS The inflammatory reaction effect of cause is reinforced.This explanation, SAA C-terminals play a significant role in inflammatory reaction, and C-terminal missing is got over More, proinflammatory effect is lost more, and wherein small peptide 11-58 has completely lost proinflammatory effect, and inhibits LPS to lead with efficient The inflammatory reaction of cause, the present inventor further investigate the scorching mechanism of action of its suppression.

Fig. 3 shows that different peptide fragments block the influence of inflammatory cytokine caused by LPS.The result shows that

Multiple polypeptide fragments (11-58,11-68,11-72,27-72,27-90,29-104,11-45) show centainly Inhibition, wherein the inhibition of peptide fragment 27-72,27-90,29-104,11-45 are relatively low.Make us it is surprising that Polypeptide fragment 11-58,11-68 and 11-72 show to exceed the high inhibition of meaning, for example, than peptide fragment 27-72 or 27-90 etc. The inhibitory activity of peptide fragment is higher by several times.Wherein, the inhibitory activity of the inhibitory activity of especially P11-58 and P11-68 and P27-72 It compares, improves about 5-10 times.

In numerous peptide fragments, P11-58 (i.e. segment 11-58) has most apparent inhibition to make the LPS inflammatory reactions induced With, the strongest inflammation inducing cell factor IL-6 of inhibition LPS of display, TNF-α, the effect of IL-1 β expression.

Embodiment 4

11-58 polypeptide fragments can inhibit the release of the pro-inflammatory cytokine of LPS inductions, and itself can induce higher level The secretion of IL-10

BMDM cells are detached, 11-58 small peptides are added to the BMDM cell culture of free serum culture with 0.5 μM of final concentration In liquid, after half an hour, 100ng/ml LPS are added, cell (or different time points collection cell), TRIzol are collected after 5 hours RNA, reverse transcription cDNA are extracted in cracking, and SYBR Green dyestuffs are added and carry out Real-time PCR detections, detection causes scorching thin Intracellular cytokine IL-6, IL-1 β, TNF-α, the mRNA expressions of suppression inflammatory cytokines IL-10.Ultimate analysis experiment gained Ct values, Specific gene transcriptional control is horizontal between calculating different samples.

As shown in figure 4, dose dependent is presented in IL-6, TNF α and IL-1 β that polypeptide fragment 11-58 inhibits LPS to generate.Such as Shown in Figure 11,12 and 13, as a result statistics is found, 11-58 polypeptide fragments individually stimulate bone marrow macrophage that can generate a large amount of IL- 10.When being used in combination in LPS, the ability that stimulation IL-10 is generated is not affected significantly.The overall length that compares SAA1,11-58 polypeptide Segment stimulation bone marrow macrophage can generate more IL-10 (Figure 13).

It is mainly the liter by inhibiting inflammatory factor caused by LPS that the above results, which show that 11-58 small peptides play anti-inflammatory effect, Height, while bone marrow macrophage being stimulated to generate anti-inflammatory factors IL-10 to play a role.

Embodiment 5

11-58 small peptides can inhibit ERK and JNK phosphorylation levels caused by LPS and raise

Separating mouse bone marrow macrophage is first added the 11-58 polypeptide fragments of various concentration, final concentration is added after one hour The LPS of 500ng/ml, mixing, 37 DEG C of cultures.After one hour, lytic cell, Western blot detect MAPK phosphorylation levels, The result is shown in Figure 10.LPS can induce the up-regulation of Bone Marrow Macrophage ERK, p38 and JNK phosphorylation level, and polypeptide fragment can be with ERK caused by LPS, the raising of the phosphorylation level of JNK are blocked, but itself can cause the raising of p38 phosphorylation levels.

Embodiment 6

It is TLR2 dependences that SAA, which induces the generation of IL-10,

The present inventor detaches the Bone Marrow Macrophage and TLR2 of wild type^-/-Bone Marrow Macrophage is used respectively ApoSAA and 11-58 polypeptide fragments stimulate, and investigate the expression of different time points IL-10, as a result see Fig. 6.11-58 polypeptide pieces Section can stimulate the Bone Marrow Macrophage of wild type to generate IL-10, but in TLR2^-/-In mouse, this ability is lost substantially It loses, therefore, the present inventor speculates, it is TLR2 dependences that 11-58 polypeptide fragment stimulating expression of macrophage, which generates IL-10,.

In the present embodiment, the signal transduction path that 11-58 small peptides activation TLR2 generates IL-10 is investigated.Cultivate TLR2-HeLa Stable cell strain, activation and its MAPK phosphorylation level of the detection 11-58 polypeptides to NF- κ B.As a result see Fig. 9.Overall length SAA1 is Positive control, in terms of activating MAPK phosphorylations, 11-58 polypeptides and overall length SAA1 are different, and SAA1 can activate ERK, P38, JNK phosphorylation.11-58 polypeptides cannot activate JNK phosphorylations, but can activate p38 phosphorylations and ERK phosphorylations；Compared to complete Long SAA1,11-58 polypeptide fragment can more effectively activate the phosphorylation of p38MAPK.And this activation is in TLR2^-/-Macrophage It is significantly reduced in cell, prompts TLR2 dependences.

Embodiment 7

11-58 peptide fragments can reduce lethality caused by LPS, and reduce acute lung injury caused by LPS

C57BL/6 mouse are used to detect SAA1 small peptides and inhibit mouse death rate caused by LPS, and specific experiment method is, Experiment is divided into four groups, every group of 10 mouse, small peptide group：The small peptide of 5mg/kg is injected intraperitoneally；PBS groups：Corresponding dosage is injected intraperitoneally PBS；LPS groups：The LPS of 20mg/kg is injected intraperitoneally；Small peptide+LPS groups：The small peptide of 5mg/kg is first injected intraperitoneally, after half an hour again The LPS of secondary intraperitoneal injection 20mg/kg.Mouse death rate is observed in 72 hours, draws survival rate curve.

In the detection of mouse lethal rate, 48 hours death rates are 90% under 20mg/kg LPS dosage.Abdominal cavity is administered in advance 11-58 polypeptide dosage 5mg/kg, LPS is administered after half an hour to be reduced to 40% by lethality, illustrate that 11-58 small peptides can be effective Reduce the death rate caused by LPS.As a result see Fig. 5.

Acute lung injury experiment mouse used is C57BL/6,6-8 week, male mouse, tests and is divided into four groups, every group of six mouse, Time point is 4 hours and 24 hours.Small peptide group：The small peptide of 5mg/kg is injected intraperitoneally；PBS groups：Corresponding dosage is injected intraperitoneally PBS；LPS groups：The LPS of 15mg/kg is injected intraperitoneally；Small peptide+LPS groups：The small peptide of 5mg/kg is first injected intraperitoneally, after half an hour again The LPS of 15mg/kg is injected intraperitoneally.After 4 hours and 24 hours, difference anesthetized mice, with not calcic, the PBS lavation lungs of magnesium are collected Lung-douching fluid calculates total number of cells in lung-douching fluid, then rejection tablet, hematoxylin eosin staining, is counted under microscope, in calculating The quantity of property granulocyte.It is fixed again by the maximum lung formalin lavation of a leaf for mouse, make paraffin section, hematoxylin-she Red colouring, the situation of observation lung infiltration.Remaining lung tissue is detected for real-time, neutrophil leucocyte detection in lung tissue (MPO Activity determinations).As shown in fig. 6,11-58 polypeptides can effectively inhibit the raising of intrapulmonary MPO levels caused by LPS.Such as Shown in Fig. 7,11-58 polypeptides can also effectively inhibit the up-regulation of intrapulmonary inflammatory factor level caused by LPS, such as TNF α, IL-6, IL-1β.It is observed according to lung sections hematoxylin eosin staining, as shown in figure 8, finding that 11-58 polypeptides can effectively inhibit intrapulmonary The infiltration of neutrophil leucocyte.

It discusses

The present invention develops a new class of polypeptide fragment, with SAA1 functional polypeptide 11-58 (P₁₁It -58), may for Working mechanism be by TLR2 stimulate IL-10 a large amount of generations, inhibit LPS induction pro-inflammatory cytokine generation, to play Inhibit the effect of inflammatory reaction.For LPS and overall length SAA1,11-58 polypeptide fragments itself not can induce cause it is scorching because A large amount of generations of son can not stimulate chemotactic and the infiltration of leucocyte, thus its anti-inflammatory effect becomes main biological activity. Having found that it is likely that for this 11-58 anti-inflammatory peptides brings new thinking for novel anti-inflammatory drug research and development.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Sequence table

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University of Macao

<120>Human serum amyloid A 1 functional oligopeptides and its preparation method and application

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Claims (10)

1. a kind of human serum amyloid A 1 polypeptide fragment or its pharmaceutically acceptable salt, which is characterized in that described is more Peptide fragment has following characteristics：

(i) sequence of the polypeptide fragment derives from the amino acid sequence of human serum amyloid A 1；With

(ii) polypeptide fragment is M to N of human serum amyloid A 1 full length amino acid sequence, and length is 42-67 amino acid, wherein M are any positive integer of 9-14, and N is any positive integer of 55-75.

2. a kind of fusion protein, which is characterized in that the fusion protein includes human serum amyloid protein described in claim 1 A1 polypeptide fragments and the sequence label together with the segment composition.

3. a kind of polynucleotide sequence, which is characterized in that the polynucleotide sequence coding human serum described in claim 1 forms sediment Fusion protein described in 1 polypeptide fragment of powder sample albumin A or claim 2.

4. a kind of Yeast expression carrier, which is characterized in that the carrier contains the multinuclear for encoding polypeptide fragment described in claim 1 Thuja acid.

5. a kind of host cell, which is characterized in that the host cell is selected from the group：

(a) host cell containing the carrier described in claim 4；

(b) host cell for the polynucleotides for encoding polypeptide fragment described in claim 1 is integrated in chromosome.

6. a kind of method preparing polypeptide fragment described in claim 1, which is characterized in that including step：

(a) under conditions suitable for the expression, the host cell described in claim 5 is cultivated, it is described in claim 1 to express Polypeptide fragment；With

(b) polypeptide fragment described in claim 1 is isolated from cultured products.

7. a kind of pharmaceutical composition, which is characterized in that it contains pharmaceutically acceptable carrier or excipient；And claim The 1 human serum amyloid A 1 polypeptide fragment is as active constituent.

8. the purposes of human serum amyloid A 1 polypeptide fragment or its pharmaceutically acceptable salt described in a kind of claim 1, It is characterized in that, being used to prepare the drug for inhibiting inflammation and/or treating inflammation related disease.

9. the use of a kind of human serum amyloid A 1 polypeptide fragment described in claim 1 or its pharmaceutically acceptable salt On the way, which is characterized in that be used to prepare a composition or preparation, the composition or preparation are secreted anti-inflammatory for stimulating expression of macrophage Factor IL-10.

10. a kind of method for treating disease caused by inflammation, which is characterized in that include applying claim 1 to the object of needs The human serum amyloid A 1 polypeptide fragment or its pharmaceutically acceptable salt or protein complexes.