CN1304568C - Novel human complement C1r-like serine proteinase analogue, and its encoding sequence and use - Google Patents
Novel human complement C1r-like serine proteinase analogue, and its encoding sequence and use Download PDFInfo
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- CN1304568C CN1304568C CNB2004100257426A CN200410025742A CN1304568C CN 1304568 C CN1304568 C CN 1304568C CN B2004100257426 A CNB2004100257426 A CN B2004100257426A CN 200410025742 A CN200410025742 A CN 200410025742A CN 1304568 C CN1304568 C CN 1304568C
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Abstract
The present invention provides a novel human complement C1r-like serine proteinase analogue (C1r-like serine proteinase analogue, CLSPa protein) which is polynucleotide of the CLSPa coding protein and a method for producing the CLSPa protein by the recombinant technology. The present invention also discloses the polynucleotide use for coding the CLSPa protein, and the CLSPa protein has the function of protease activity resistance.
Description
Technical field
The invention belongs to biotechnology and medical field, specifically, the present invention relates to the polynucleotide of new coding people complement C1r sample serine protease analog molecules (C1r-like serine protease analog abbreviates CLSPa albumen as), and the polypeptide of this polynucleotide encoding.The invention still further relates to the purposes and the preparation of these polynucleotide and polypeptide.Specifically, polypeptide of the present invention is a kind of new complement C1r sample serine protease analog molecules.
Background technology
Complement (Complement) provides the approach of removing invading micro-organism fast and efficiently as the important and conservative system of a class in the natural immunity (Innate immunity) for body.Complement system is except the direct killing mechanism of complement, the multiple small pieces molecule that discharges in the complement activation process has biological effect widely, comprises chemotactic neutrophil leucocyte and lymphocyte, opsonophagocytosis, participation adjusting cell and humoral immunoresponse(HI) etc.The basis that these functions are achieved be exactly in the complement system each composition have serine protease, can activate the downstream substrate, excited the complement cascade reaction.C1 is the start-up portion in the classical activated pathway of complement system, is made up of C1q, C1r and three subunits of C1s, and C1q is the subunit with recognition reaction, and C1r and C1s are the subunit with katalysis.C1r is a single chain polypeptide, is made up of about 700 amino-acid residues, and molecular weight is about 83kDa, at the have an appointment serine protease structural domain of 250 amino-acid residues of C end, and with non-complement sex pilus propylhomoserin proteolytic enzyme (as trypsinase and Chymotrypsin) height homology; Holding 450 amino acid of having an appointment at N, contain two CUB and an EGF structural domain, is a kind of serine stretch protein proenzyme.C1r extensively is present in blood plasma and the body fluid, and in solution, C1r is a binary, and the assignment of genes gene mapping of coding C1r is in people's No. 12 the short arm of a chromosome 12p13.The CUB-EGF structural domain of C1r N end is at Ca
2+Existence under the C1s-C1r-C1r-C1s tetramer constituted played main effect, and then form stable C1 complex body.And CCP1, CCP2, the SP structural domain of its C end are its active parts with katalysis, can cracking C1s, make its activation, and further activate C3 convertase, thereby activate whole classical pathway.In the ordinary course of things, C1r is combining with C1INH (C1inhibitor), and in case when having IC to be attached to C1q to go up, the restraining effect of C1INH removes at once, the conformation of C1r changes, and has enzymic activity.
Serine protease is one of most important enzyme molecule of body, account for 1/3rd of protein in body enzyme, its physiological characteristic has possessed His-Asp-Ser catalysis ring exactly, has found also that in recent years other catalysis ring is as Ser-His-Glu, Ser-Lys/His, His-Ser-His and N-terminal Ser.The serine protease of knowing comprises C1r/C1s, seminose in conjunction with serine protease, ovochymase, spermadhesin and II type transmembrane serine protease etc., and they have participated in multinomial physiological functions such as complement activation, ovulation, insemination, tissue reconstruction, cell migration, tumor-infiltrated and transfer, digestion, fetal development, organ formation.Every vital movement of known road body is all being carried out under the network regulation of precision, and its mechanism has feedback regulation, neutralization to suppress, competition suppresses etc., and serine protease effect system is also being kept this running balance.Serine protease and their inhibitor are prevalent in nature animal and plant and the microorganism, and in many important physical systems, played critical regulating and controlling effect, joined the release of order reaction and polypeptide or proteohormone etc. as blood coagulation, fibrinolytic, complement.At present, nearly more than 20 serpin families are proved, and its basic structural feature has alpha-helix, beta sheet, alpha-helix/beta sheet and many disulfide linkage folding.These inhibitor structurally effect substrate with serine protease are very close, and avtive spot that can desmoenzyme plays sealing or the effect of degraded destructive.
Because complement pathway is relevant with numerous physiological processs, therefore, this area presses for the exploitation various materials relevant with complement pathway.
Summary of the invention
The purpose of this invention is to provide a kind of new people's complement C1r sample serine protease analog molecules CLSPa albumen with and fragment, analogue and derivative.
Another object of the present invention provides the polynucleotide of these polypeptide of coding.
Another object of the present invention provides the method for these polypeptide of production and the purposes of this polypeptide and encoding sequence.
In a first aspect of the present invention, novel isolated CLSPa polypeptide is provided, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQIDNO:2 aminoacid sequence.
Preferably, this polypeptide is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the arrestin enzymic activity function by (a) polypeptides derived;
(c) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the complement-mediated killing effect function by (a) polypeptides derived.
More preferably, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) polynucleotide of the above-mentioned people CLSPa polypeptide of coding; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence (ORF sequence) that (a) has 18-1478 position among the SEQ ID NO:1; (b) has the sequence (full length sequence) of 1-3345 position among the SEQ ID NO:1; Or (c) has a sequence (encoding sequence of mature polypeptide) of 126-1478 position among the SEQ ID NO:1.
In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.
In a fourth aspect of the present invention, provide preparation to have the method for the polypeptide of people CLSPa protein-active, this method comprises: (a) under the proteic condition of suitable expressing human CLSPa, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate polypeptide with people CLSPa protein-active.
In a fifth aspect of the present invention, provide and above-mentioned people CLSPa polypeptid specificity bonded antibody.
In a sixth aspect of the present invention, the compound of simulation, promotion, antagonism people CLSPa polypeptide active is provided, and the compound that suppresses people CLSPa polypeptide expression.The method of screening and/or prepare these compounds also is provided.Preferably, this compound is encoding sequence or its segmental antisense sequences of people CLSPa polypeptide.
In a seventh aspect of the present invention, provide and whether had the proteic method of CLSPa in the test sample, it comprises: sample is contacted with the proteic specific antibody of CLSPa, observe whether form antibody complex, formed antibody complex and just represented to exist in the sample CLSPa albumen.
In a eighth aspect of the present invention, a kind of disease relevant with people CLSPa polypeptide unconventionality expression or method of disease susceptibility of detecting is provided, this method comprises: whether have sudden change in the nucleotide sequence of detection coding said polypeptide.
In a ninth aspect of the present invention, provide the purposes of polypeptide of the present invention and encoding sequence.Polypeptide for example of the present invention can be used to screen the agonist that promotes people CLSPa polypeptide active, and perhaps screening suppresses the antagonist of people CLSPa polypeptide active or is used to the peptide finger print identification.The proteic encoding sequence of people CLSPa of the present invention or its fragment can be used as primer and be used for pcr amplification reaction, perhaps are used for hybridization as probe, perhaps are used to make gene chip or microarray.A kind of preferred purposes is with the inhibitor of CLSPa polypeptide as the arrestin enzymic activity.
In a tenth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains people CLSPa polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier of safe and effective amount.Non-medicinal composition also is provided, and said composition contains CLSPa polypeptide and the acceptable carrier (as solvent) of 0.001-99.99wt%.These compositions can be used for the activity (activity that more preferably suppresses serine protease) of arrestin enzyme, or are used to suppress the complement-mediated killing effect.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Description of drawings
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Fig. 1 is the cDNA sequence of people CLSPa of the present invention, and wherein non-coding sequence is represented with lowercase, and encoding sequence is with capitalizing conventional letter representation.
Fig. 2 is the full length amino acid sequence of people CLSPa of the present invention.
Fig. 3 is the RT-PCR expression analysis of people CLSPa.Figure A is the expression in human tumor cell line of people CLSPa; Figure B is that the health adult tissue of people CLSPa distributes.
Fig. 4 is that the Western that the carrier for expression of eukaryon of people CLSPa is expressed in eukaryotic cell analyzes.
Fig. 5 is the people CLSPa of the present invention hemolytic restraining effect of complement-mediated is detected.*P＜0.01
Fig. 6 is that the proteic protease activity of people CLSPa of the present invention detects.
To be that people CLSPa of the present invention is proteic detect the protease activity restraining effect Fig. 7.Wherein Fig. 7 A reality the CLSPa albumen of purifying to the restraining effect of tryptic protease activity; Fig. 7 B has shown the restraining effect of CLSPa albumen to the endogenous protein enzymic activity.* P＜0.05 is compared with the control vector group; * P＜0.01 is compared with trypsinase.
Embodiment
Extensive studies is separated to a new complement C1r sample serine protease analog molecules CLSPa to the inventor first through going deep into.CLSPa and known serine protease---complement C1r/C1s has higher homology, and has the effect that suppresses complement-mediated haemolysis effect and other serine proteases.Therefore this shows that the CLSPa molecule is a kind of novel, natural serine protease analogue, but the activity of competitive inhibition serine protease, thereby the inflammatory reaction of complement-mediated plays regulating and controlling effect, thereby may be worth having important development and application aspect the immunodiagnosis in a plurality of fields such as antitumor, resisting transplant rejection, anti-inflammatory response and the immunotherapy.Finished the present invention on this basis.
In the present invention, term " CLSPa albumen ", " CLSPa polypeptide ", " CLSPa protein polypeptide " or " complement C1r sample serine protease analog molecules CLSPa " are used interchangeably, and all refer to contain the albumen or the polypeptide of people's complement C1r sample serine protease analog molecules CLSPa aminoacid sequence (SEQ ID NO:2).They comprise the complement C1r sample serine protease analog molecules CLSPa that contains or do not contain initial methionine.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating CLSPa albumen or polypeptide " is meant that the CLSPa polypeptide is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying CLSPa albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.The purity of CLSPa polypeptide can be used amino acid sequence analysis.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises the proteic fragment of people CLSPa, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of natural human CLSPa albumen of the present invention or active polypeptide basically.Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
In the present invention, term " people CLSPa polypeptide " refers to have the SEQ ID NO:2 polypeptide of sequence of people CLSPa protein-active.This term also comprises having and variant form people CLSPa albumen identical function, SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of people CLSPa and reactive derivative.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people CLSPa DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people CLSPa polypeptide to obtain.The present invention also provides other polypeptide, as comprises people CLSPa polypeptide or its segmental fusion rotein (as the fusion rotein that forms with GST etc.).Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people CLSPa polypeptide.Usually, this fragment have people CLSPa peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analogue of people CLSPa albumen or polypeptide.The difference of these analogues and natural human CLSPa polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, " people CLSPa albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding CLSPa.
Polypeptide among the present invention and polynucleotide preferably provide with isolating form, more preferably are purified to homogeneous.
People CLSPa Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition gene of the present invention.When particularly being difficult to obtain the cDNA of total length from the library, can preferably use RACE method (the terminal rapid amplifying method of RACE-cDNA), the primer that is used for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment by gel electrophoresis separation and purifying amplification.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or CLSPa albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the CLSPa polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention people CLSPa polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, people CLSPa polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125); The pMSXND expression vector of in mammalian cell, expressing (Lee and Nathans, J Bio Chem.263:3521,1988) and at the carrier that derives from baculovirus of expressed in insect cells.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains people CLSPa DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage PL promotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser of 100 to 270 base pairs of replication origin side in late period one, at the polyoma enhanser of replication origin side in late period one and adenovirus enhanser etc.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The people CLSPa albumen or the polypeptide of reorganization are of use in many ways.These purposes include, but is not limited to: the direct disease due to the low or forfeiture and be used to screen and promote or antibody, polypeptide or other part of antagonism CLSPa protein function as pharmacological agent CLSPa protein function.The peptide molecule that can suppress or stimulate people CLSPa protein function that can be used for seeking therapeutic value with the recombinant human CLSPa protein screening peptide library of expressing.
On the other hand, the present invention also comprises people CLSPa DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people CLSPa gene product or fragment.Preferably, refer to that those can combine with people CLSPa gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people CLSPa, comprise that also those do not influence the antibody of people CLSPa protein function.The present invention also comprise those can with modify or without the people CLSPa gene product bonded antibody of modified forms.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people CLSPa gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human CLSPa albumen or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler,
Eur.J.Immunol.6:511,1976; People such as Kohler,
Eur.J.Immunol.6:292,1976; People such as Hammerling,
In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people CLSPa protein function and the antibody that does not influence people CLSPa protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people CLSPa gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people CLSPa gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).
The proteic antibody of anti-people CLSPa can be used in the immunohistochemistry technology, detects the people CLSPa albumen in the biopsy specimen.
Antibody among the present invention can be used for treating or prevention and the relevant disease of people CLSPa albumen.The antibody that gives suitable dosage can stimulate or block proteic generation of people CLSPa or activity.
Antibody also can be used for being designed to the immunotoxin at a certain privileged sites in the body.As the monoclonal antibody of people CLSPa albumen high-affinity can with bacterium or plant poison (as diphtheria toxin, ricin, abrine etc.) covalent attachment.A kind of usual method is with sulfydryl linking agent such as SPDP, attacks the amino of antibody, by the exchange of disulfide linkage, toxin is incorporated on the antibody, and this hybrid antibody can be used for killing the cell of people CLSPa protein positive.
The production of polyclonal antibody can choose CLSPa albumen or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.
Utilize albumen of the present invention,, can filter out with CLSPa albumen interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.
Albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist or acceptor etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
Polypeptide of the present invention can be directly used in disease treatment, for example, is used for the treatment of antitumor (as pernicious neoplastic hematologic disorder), resisting transplant rejection, anti-inflammatory response (as the muscle dysfunction disease) aspect.When using CLSPa albumen of the present invention, also can use the other treatment agent simultaneously, as TNF α etc.
The present invention also provides a kind of pharmaceutical composition, and it contains CLSPa polypeptide of the present invention or its agonist, antagonist and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by 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, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that the CLSPa albumen of safe and effective amount or its antagonist, agonist are applied to 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 also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
The proteic polynucleotide of people CLSPa also can be used for multiple therapeutic purpose.Gene therapy technology can be used for treating because cell proliferation, growth or the metabolic disturbance due to the proteic expression of CLSPa of the proteic nothing expression of CLSPa or unusual/non-activity.The CLSPa albumen that the gene therapy vector (as virus vector) of reorganization can be designed to express variation is to suppress endogenic CLSPa protein-active.Deriving from viral expression vector such as retrovirus, adenovirus, adeno-associated virus (AAV), hsv, parvovirus etc. can be used for the CLSPa transgenosis to cell.The method that structure carries the recombinant viral vector of CLSPa gene is found in existing document (Sambrook, et al.).Recombinant human CLSPa gene can be packaged in the liposome in addition, and then is transferred in the cell.
Suppress the oligonucleotide (comprising sense-rna and DNA) of people CLSPa mRNA and ribozyme also within the scope of the invention.Ribozyme is the enzyme sample RNA molecule that a kind of energy specificity is decomposed specific RNA, and its mechanism of action is to carry out the endonuclease effect after ribozyme molecule and the hybridization of complementary target RNA-specific.The RNA of antisense and DNA and ribozyme can obtain with existing any RNA or DNA synthetic technology, as the technology widespread use of solid phase phosphoamide chemical synthesis synthetic oligonucleotide.Antisense rna molecule can be transcribed acquisition by the dna sequence dna of this RNA that encodes in external or body.This dna sequence dna has been incorporated into the downstream of rna polymerase promoter of carrier.In order to increase the stability of nucleic acid molecule, available several different methods is modified it, and as increasing the sequence length of both sides, the connection between the ribonucleoside is used phosphoric acid thioester bond or peptide bond but not phosphodiester bond.
Polynucleotide imports tissue or intracellular method comprises: directly be injected into polynucleotide in the in-vivo tissue; Or external by carrier (as virus, phage or plasmid etc.) earlier with the polynucleotide transfered cell in, again cell is transplanted in the body etc.
Can be incorporated into the rondom polypeptide storehouse that solid formation forms by the various amino acid that may make up by screening with the protein bound peptide molecule of people CLSPa obtains.During screening, must carry out mark to people CLSPa protein molecular.
The invention still further relates to the diagnostic testing process of quantitative and detection and localization people CLSPa protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The people CLSPa protein level that is detected in the test can be with laying down a definition the importance of people CLSPa albumen in various diseases and be used to the disease of diagnosing CLSPa albumen to work.
Whether having the proteic method of CLSPa in a kind of detection test sample is to utilize the proteic specific antibody of CLSPa to detect, and it comprises: sample is contacted with the CLSPa protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample CLSPa albumen.
The proteic polynucleotide of CLSPa can be used for the diagnosis and the treatment of CLSPa protein related diseases.Aspect diagnosis, the proteic polynucleotide of CLSPa can be used for detecting the proteic expression of CLSPa CLSPa abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of CLSPa as the CLSPa dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of CLSPa albumen and also can detect the proteic transcription product of CLSPa.
The sudden change that detects the CLSPa gene also can be used for the disease of diagnosing CLSPa albumen relevant.The form of CLSPa protein mutation comprises that the point mutation compared with normal wild type CLSPa dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.
Sequence of the present invention identifies it also is valuable to karyomit(e).In brief, the proteic cDNA of CLSPa prepares PCR primer (preferred 15-35bp) according to the present invention, sequence can be positioned on the karyomit(e).Then, these primers are used for the somatocyte hybrid cell that the PCR screening contains each bar human chromosome.Have only those hybrid cells that contain corresponding to the people's gene of primer can produce the fragment of amplification.
In case sequence is positioned to chromosome position accurately, the physical location of this sequence on karyomit(e) just can be associated with the gene map data.These data for example are found in, V.Mckusick, Mendelian Inheritancein Man (can by with the online acquisition of Johns Hopkins University Welch Medical Library).Can pass through linkage analysis then, determine gene and navigated to relation between the disease on the chromosomal region already.
The present invention also provides non-medicinal composition, and said composition contains CLSPa polypeptide and the acceptable carrier (as solvent) of 0.001-99.99wt% (more preferably 0.01-99.9wt%).These compositions can be used for the activity (activity that more preferably suppresses serine protease) of arrestin enzyme, or are used to suppress the complement-mediated killing effect.
In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.Polynucleotide of the present invention are isolated from human dendritic cell cDNA library.Its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 3345 bases, and its open reading frame is positioned at the 18-1478 position, and the coding total length is 487 amino acid whose people CLSPa albumen (SEQ ID NO:2).But this CLSPa albumen arrestin enzymic activity, and can suppress the complement-mediated killing effect, therefore can be diseases such as treatment is antitumor, resisting transplant rejection, anti-inflammatory response new treatment approach is provided, thereby have great application prospect.
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 and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the clone of people CLSPa cDNA
Extract the total RNA of human dendritic cell with Trizol reagent (Life Technologies).Then, from total RNA, separate poly (A) mRNA.Poly (A) mRNA after reverse transcription forms cDNA, is cloned test kit (Life Technologies) with SuperScriptII cDNA fragment orientation is inserted on the multiple clone site of carrier, transform DH5 α bacterium and form the cDNA plasmid library.Measure the sequence of random choose clone's 5 ' end with dideoxy method.CDNA sequence and the existing public dna sequence data storehouse measured are compared, found that a cDNA clone's dna sequence dna is new full-length cDNA.By synthetic a series of primers the contained dna sequence dna of new clone is carried out two-way mensuration.Computer Analysis shows that cloning contained full-length cDNA is a new cDNA sequence (as SEQ ID NO:1 and shown in Figure 1), the new protein (as SEQ ID NO:2 and shown in Figure 2) of encoding.This protein is named as people's complement C1r sample serine protease analog molecules (CLSPa), and its encoding gene name is people's complement C1r sample serine protease analog molecules gene.
Sequence SEQ ID NO:1 total length is 3345bp, comprises 3 ' end non-coding region of 5 of 17bp ' end non-coding region and 1864bp, and coding contains 487 amino acid whose polypeptide.Calculate in theory not that the molecular weight of glycosylated ripe molecule is about 53.484 dalton, calculating iso-electric point is 6.75.Structural analysis shows that the proteic 1-36 of CLSPa position comprises a secreting signal peptide, the 39-164 position comprise a CUB structural domain (
C1r/C1s, an embryonicsea urchin protein
UEgf, and
BOne morphogenetic protein I domain), comprise a serine protease structural domain (serine protease domain) in the 244-483 position, this shows that CLSPa belongs to serine protease sample molecule.
They are different with known for the BLAST analysis revealed, and C1r has certain homology with people's complement component, and similarity is 40%, and consistence is 52%.It is 48% and 58% that the similarity of the two corresponding C UB structural domain and serine protease structural domain is respectively.
Embodiment 2: carry out the cell expressing analysis of people CLSPa with the RT-PCR method
Extract the total RNA of dendritic cell that is in the corresponding clone of logarithmic phase, human peripheral blood mononuclear cell and stimulates the human peripheral blood mononuclear cell source of different time with Trizol reagent, get 5 μ g cell total rnas and 1 μ g Oligo-dT through LPS
12-18Mix, carry out reverse transcription.The reverse transcription system is 20 μ l, and reaction adds 80 μ lddH after finishing
2O dilutes.Pcr amplification CLSPa primer is as follows: adopted primer 5 '-ATGCCTGGACCCAGAGTGTGGG-3 ' (SEQ ID NO:3) is arranged, antisense primer 5 '-TCAATTCTTGCCATTCATCACTCCCTTGATCC-3 ' (SEQ ID NO:4), simultaneously with β-actin as positive control.The PCR reaction volume is 50 μ l, wherein contain reverse transcription template 10 μ l, 0.5mM primer, 0.2mM dNTP and 1U rTaq archaeal dna polymerase (Takara), the amplification parameter be 95 ℃ 15 seconds, 57 ℃ 30 seconds, 72 ℃ 90 seconds, capable 1.5% agarose gel electrophoresis of PCR product is tentatively confirmed after 32 circulations.The dna sequence analysis result shows that the DNA sequences encoding of this PCR product and the 18-1481 shown in the SEQ ID NO:1 are identical.
RT-PCR result shows (Fig. 3), and in all solid tumor cells that detect and blood tumor cell system, CLSPa mRNA is expressed in human prostata cancer PC-3 and the ovarian cancer SK-OV-3 cell, and other clone there is no expression.In health adult tissue, people CLSPa mRNA wide expression is in adult's healthy tissues, expression amount is higher in placenta, liver, kidney, pancreas, there is moderate to express at lung, spleen, prostate gland, ovary, colon, peripheral blood tissue, and expression is lower in heart, skeletal muscle, thymus gland, testis, small intestine, does not reach but see Table in brain.
In this embodiment, conventional extractive dendritic cell mRNA, and reverse transcription becomes cDNA as template increases with the PCR Oligonucleolide primers of 5 ' and 3 ' following end of sequence, obtains people CLSPa DNA as inserting fragment.
5 ' the end Oligonucleolide primers sequence of using in the PCR reaction is:
5’-CAGGATCCATGCCTGGACCCAGAGTG-3’(SEQ ID NO：5)
This primer contains the restriction enzyme site of BamHI restriction enzyme, is the part encoding sequence of translation initiation and people CLSPa after this restriction enzyme site;
3 ' end primer sequence is:
5’-GTGAATTCTCAATTCTTGCCATTCATCACT-3’(SEQ ID NO：6)
This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people CLSPa of EcoRI restriction enzyme.
With the PCR product purification that obtains after BamHI-EcoR I enzyme cut and recombinate according to a conventional method with plasmid pGEM-3ZF again and be converted into the competence e. coli bl21, the picking positive colony is identified back purifying and order-checking (377 type sequenators of ABI company, BigDye Terminator test kit, PE company).The people CLSPa cDNABamHI-EcoR I endonuclease bamhi of correct sequence is cloned into expression vector pGEX-2T (Pharmacia), forms carrier pGEX-2T-CLSPa, then transformed into escherichia coli BL21.Positive colony is cut evaluation with BamHI-EcoR I enzyme, the capable 0.8% agarose gel electrophoresis analysis of product.Confirm through order-checking, inserted designed CLSPa encoding sequence.
Choosing the positive BL21 clone who expresses CLSPa is inoculated in 100ml 2 * YTA substratum, 37 ℃ of 300rpm shaking culture 12-15hr, 2 * YTA the substratum that is diluted in preheating at 1: 10 continues shaking culture 1.5hr, add behind the 100mMIPTG to 0.1mM 30 ℃ and induce 2-6hr, 5,4 ℃ of centrifugal 10min of 000g remove supernatant, put and use 50ml 1 * PBS (0.14M NaCl on ice, 2.7mM KCl, 10.1mM.2HPO4,1.8mM KH2PO4, pH7.3) resuspended, add 20%Triton X-100 to 1% jog 30min again after ultrasonic (B.Braun Labsonic U) fragmentation, then 12,4 ℃ of centrifugal 10min of 000g, supernatant with 0.8 μ m membrane filtration after, cross 1ml 50% glutathione S epharose 4B chromatography column, behind 1 * PBS thorough washing, add 500ul gsh elution buffer (10mM gsh, 50mM Tris-HCl, pH 8.0) room temperature leaves standstill after 30 minutes and collects elutriant, repeats wash-out 2-3 time, obtains people CLSPa-GST fusion rotein.
Electrophoresis detection shows that the molecular weight of fusion rotein conforms to predictor.
Embodiment 4: anti-people CLSPa production of antibodies
The recombinant human CLSPa fusion rotein that obtains among the embodiment 3 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people CLSPa gene translation product with it.
Found that antibody can combine with the recombinant human CLSPa fusion rotein of embodiment 3 preparations specifically.
Embodiment 5: people CLSPa Construction of eukaryotic and gene transfection
In this embodiment, conventional extractive dendritic cell mRNA, and reverse transcription becomes cDNA as template increases with the PCR Oligonucleolide primers of 5 ' and 3 ' following end of sequence, obtains people CLSPa full length coding region DNA as inserting fragment.The PCR reaction parameter be 95 ℃ 15 seconds, 57 ℃ 30 seconds, 72 ℃ 90 seconds, 25 circulations were extended 10 minutes for back 72 ℃,
Upstream primer is that (5 ' end contains EcoR I site to 5 '-GGAATTCATGCCTGGACCCAGAGTGTGG-3 ', and the initial code of people CLSPa coding region) (SEQ ID NO:7), downstream primer are 5 '-CTAAGCTTCAATTCTTGCCATTCATCACTCC-3 ' (5 ' end contains Hind III site) (SEQ ID NO:8).
Behind the PCR product purification directly and pGEM-T carrier (Promega company) recombinate according to a conventional method and be converted into competence bacterium DH5 α.Picking white clone identifies, also order-checking (sequencing primer is T7 and SP6) of purifying.With the purified rear clone of EcoR I-Hind III endonuclease bamhi of correct sequence to expression vector pcDNA3.1/Myc-His (-) B (Invitrogen company).After enzyme was cut the evaluation positive colony, recon was designated as pCLSPa.Confirm through order-checking, inserted designed people CLSPa encoding sequence.
People CLSPa eukaryotic expression plasmid DNA and control plasmid pcDNA3.1/Myc-His (-) B with the commercially available 293T HEKC of LipofectAMINE reagent (Invitrogen company) cotransfection, were detected after the transfection in 48 hours.
Embodiment 6: the Western of people CLSPa eukaryotic expression product detects
After the transfection 48 hours, collecting cell (5 * 10
6Cell/sample), wash one time with PBS after, extract reagent (Pierce company) with the T-PER tissue protein and extract whole protein, concrete operations are undertaken by the test kit explanation.Use BCA method (Pierce company) to measure protein concentration afterwards, after being adjusted to unanimity, the per 20 μ l of sample and 6 times of sample-loading buffer (100mmol/L Tris-HCL of 4 μ l, 200mmol/L DTT, 4%SDS, 0.2% tetrabromophenol sulfonphthalein, 20% glycerine, PH6.8) mixing, after 100 ℃ of water-baths were boiled 5 minutes, row 12%SDS-PAGE electrophoresis moved on to the protein transduction in the polyacrylamide on the nitrocellulose filter in 4 ℃ with the 100V constant voltage subsequently, ponceau dyeing and label orientation mark the Marker position.4 hours (the TBST solution of 10% skim-milk) of room temperature blocking-up, the anti-His tag antibody in mouse source was diluted in the skim-milk solution by 1: 1000, after 2 hours, wash 3 times each 10 minutes in room temperature reaction with TBST (the TBS solution of 0.05%Tween20) with cellulose nitrate film.In addition corresponding two of horseradish peroxidase (HRP) mark anti-(dilutions in 1: 2000) then, incubated at room 60 minutes, TBST gives a baby a bath on the third day after its birth inferior, each 10 minutes, A liquid in the Western trace luciferase assay reagent (Cell Signaling company) and B liquid is mixed with equal-volume, and incubated at room was after 1 minute, dry, press mold, exposure is developed.
The result shows, can detect the people CLSPa Recombinant Protein Expression of band His label in the 293T HEKC of people CLSPa carrier for expression of eukaryon transfection, and molecular weight is about 70kD, is the single specificity band, and is bigger slightly than CLSPa expection molecular weight.Because CLSPa albumen comprises the N glycosylation site, may be therefore to the proteic glycosylation modified increase that causes its apparent molecular weight.And in control plasmid pcDNA3.1/Myc-His (-) B cells transfected, do not detect expression (Fig. 4).
Embodiment 7: the hemolysis inhibition test of complement-mediated
The 293T cell of pCLSPa of having collected transfection washes one time resuspended (4 * 10 with PBS
5Cell/ml) in 100 μ l GVB
2+Damping fluid (Sigma company), with it multigelation, thus lysing cell, 15, centrifugal 10 minutes of 4 ℃ of 000g, collecting cell cracking supernatant.In 96 orifice plates, per 300 μ l GVB
2+Contain 10% people's complement serum (Sigma company) in the damping fluid, sheep red blood cell of 10% antibody sensitized (Sigma company) and the different dilution CLSPa cracking supernatant liquors that contain, mixing is put 37 ℃ and was hatched 30 minutes.Enzyme connection instrument adopts the 415nm wavelength to detect each sample OD value, and cracking per-cent calculates by following formula: cracking degree (%)=100 * (experimental group OD value-spontaneous release group OD value)/(maximum release group OD value-spontaneous release group OD value).Maximum release group and spontaneous release group are used distilled water and GVB respectively
2+Damping fluid substitutes and contains CLSPa cracking supernatant liquor.
The result shows, two kinds of concentration (1: 3 and 1: 10) contain the cytotoxicity that CLSPa lysis supernatant liquor can significantly reduce complement-mediated, and transfection the not effect of lysis supernatant liquor of control plasmid, prompting CLSPa has played modulability restraining effect (Fig. 5) in the complement activation system.
The protease activity of embodiment 8:CLSPa detects
The 293T cell of pCLSPa of having collected transfection utilizes the crosslinked CLSPa albumen that has the myc label at anti-myc antibody (Invitrogen company) purifying of immobilization protein G (Pierce company) after the cracking.Use BCA method (Pierce company) to measure protein concentration afterwards, be adjusted to unanimity.Protease substrate mixed solution (the Bz-Arg-pNA that the CLSPa albumen 400ng of purifying and pNA are crosslinked, MeOSuc-Ala-Ala-Pro-Val-pNA, Boc-Gly-Gly-Leu-pNA, Suc-Ala-Ala-Pro-Phe-pNA) (Calbiochem company) is at the 0.025M of 0.2ml MES, 125mM NaCl, hatched altogether 2 hours for 37 ℃ in 2mM EGTA (pH 7.2) solution, simultaneously with trypsin trypsin) (0.25%) as positive control.Under the 405nm wavelength, detect the amount of the product that adds lustre to, with the reflection protease activities.
The result shows that compare with positive control, the CLSPa albumen of purifying does not have tangible protease activity (Fig. 6).
Embodiment 9:CLSPa detects the restraining effect of protease activity
Utilize the protease activity detection architecture of embodiment 8 to detect the restraining effect of CLSPa to tryptic protease activity.Method is as follows: at the 0.025M of 0.2ml MES, 125mM NaCl was hatched 2 hours for 37 ℃ in 2mM EGTA (pH 7.2) solution altogether, detected under the 405nm wavelength then with CLSPa albumen, trypsinase trypsin and the protease substrate mixed solution of purifying.
Simultaneously, utilize the protease activity detection architecture of embodiment 8 to detect the restraining effect of CLSPa to the endogenous protein enzymic activity.With the pCLSPa of equivalent or control vector transfection 293T lysis supernatant according to different enzymes: substrate ratio and protease substrate mixed solution were hatched 2 hours for 37 ℃ altogether, and the result detects under the 405nm wavelength.
The result shows that the CLSPa albumen of purifying can suppress the protease activity (Fig. 7 A) of trypsinase trypsin.Simultaneously, the containing CLSPa lysis supernatant liquor and can significantly reduce the endogenous protein enzymic activity of two kinds of ratios (1: 3 and 1: 10), and transfection the not effect (Fig. 7 B) of lysis supernatant liquor of control plasmid.This shows that CLSPa not only can suppress the enzymic activity of serine protease, and can suppress the enzymic activity of other proteolytic enzyme, and has played the modulability restraining effect in the complement activation system.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Second Military Medical University, PLA
＜120〉Novel Human complement C1r sample serine protease analogue, its encoding sequence and purposes
<130>044558
<160>8
<170>PatentIn version 3.1
<210>1
<211>3345
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>1
cagatgtcca gttccagatg cctggaccca gagtgtgggg gaaatatctc tggagaagcc 60
ctcactccaa aggctgtcca ggcgcaatgt ggtggctgct tctctgggga gtcctccagg 120
cttgcccaac ccggggctcc gtcctcttgg cccaagagct accccagcag ctgacatccc 180
ccgggtaccc agagccgtat ggcaaaggcc aagagagcag cacggacatc aaggctccag 240
agggctttgc tgtgaggctc gtcttccagg acttcgacct ggagccgtcc caggactgtg 300
caggggactc tgtcacaatc tcattcgtcg gttcggatcc aagccagttc tgtggtcagc 360
aaggctcccc tctgggcagg ccccctggtc agagggagtt tgtatcctca gggaggagtt 420
tgcggctgac cttccgcaca cagccttcct cggagaacaa gactgcccac cttcacaagg 480
gctttctggc cctctaccaa accgtggctg tgaactatag tcagcccatc agcgaggcca 540
gcaggggctc tgaggccatc aacgcacctg gagacaaccc tgccaaggtc cagaaccact 600
gccaggagcc ctattatcag gccgcggcag caggggcact cacctgtgca accccaggga 660
cctggaaaga cagacaggat ggggaggagg ttcttcagtg tatgcctgtc tgcggacggc 720
cagtcacccc cattgcccag aatcagacga ccctcggttc ttccagagcc aagctgggca 780
acttcccctg gcaagccttc accagtatcc acggccgtgg gggcggggcc ctgctggggg 840
acagatggat cctcactgct gcccacaccg tctaccccaa ggacagtgtt tctctcagga 900
agaaccagag tgtgaatgtg ttcttgggcc acacagccat agatgagatg ctgaaactgg 960
ggaaccaccc tgtccaccgt gtcgttgtgc accccgacta ccgtcagaat gagtcccata 1020
actttagcgg ggacatcgcc ctcctggagc tgcagcacag catccccctg ggccccaacg 1080
tcctcccggt ctgtctgccc gataatgaga ccctctaccg cagcggcttg ttgggctacg 1140
tcagtgggtt tggcatggag atgggctggc taactactga gctgaagtac tcgaggctgc 1200
ctgtagctcc cagggaggcc tgcaacgcct ggctccaaaa gagacagaga cccgaggtgt 1260
tttctgacaa tatgttctgt gttggggatg agacgcaaag gcacagtgtc tgccaggggg 1320
acagtggcag cgtctatgtg gtatgggaca atcatgccca tcactgggtg gccacgggca 1380
ttgtgtcctg gggcataggg tgtggcgaag ggtatgactt ctacaccaag gtgctcagct 1440
atgtggactg gatcaaggga gtgatgaatg gcaagaattg accctggggg cttgaacagg 1500
gactgaccag cacagtggag gccccaggca acagagggcc tggagtgagg actgaacact 1560
ggggtagggg ttgggggtgg ggggttgggg gaggcaggga aatcctattc acatcactgt 1620
tgcaccaagc cactgcaaga gaaaccccca cccggcaagc ccgccccatc ccagacagga 1680
agcagagtcc cacagaccgc tcctcctcac cctctacctc cctgtgctca tgcactaggc 1740
cccgggaagc ctgtacatct caacaacttt cgccttgaat gtccttagaa ccaccttccc 1800
ctacttcatc tgttgacaca gcttttatac tcacctgtgg aagagtcagc tactcacccg 1860
ctattagagt atggaggaag gggttttcat tgcattgcat ttctgaaaca ttcctaagac 1920
cctttagttg accttcaaat attcaagcta ttctgcagct ccaagatgca attatagaaa 1980
cagctccttt tttattttat gtcctctata tgccaggtgc ttcacctgtt atttcactta 2040
atcctcatac catatttgca aaggatgtgt tattatctat gtgtgacaaa tgaggaaact 2100
gaggctcagg ggataaaggg acttgcccaa gtcccacagc tggtgtgtga ctgcagagac 2160
tgtgctcttc ccagtgtgct gcaatacttc tcaaccctcc tctaacctgc tgtgtcaccc 2220
gctttccctc ccagccccca catccttacc attttccctc cctgggaatt cctgcttctg 2280
cgaaaatggt atcctctagc tcacactttc ctaatggccc catctcctgc agaagccagg 2340
tgagcccagc actggactga agttcttgca gacaccccac ctgtgcccct atcatcaggg 2400
gaactgctcc acctgagagg accaactctt taatttttag taaaacctga aggtgatggg 2460
ccgggcgcag tggctcacgc ctgtaatccc aacaccttag gagtccgagg tgggtggatc 2520
acgaggtcag gagatccagc ccatcctggc caacatggtg aaaccccatc tctactaaaa 2580
atacaaaaat tagccgggcg tggtgacacg tgcctgtagt cccagctact cgggaggctg 2640
aggcaggaga atcacttgaa cctgggaggc ggaggttgca gtgagctaag atcacgccac 2700
tgcactccag cctgcggaca gaccaagact tcatcccccc caaaaaaaaa agattggagg 2760
tgatttacag tgaaagacac aaataaaata caactgttca atggaaatag aaaataaaca 2820
ccataaaaga gagaagagag gtaatttgtt agcatcaaga gtcaagttgc tatatggtca 2880
aaggttaaat ttatctctaa aaaatggcag gattcaaagt tgtacataca tgtgattact 2940
tctgtttttt acacccacat acagtacaaa agattattaa aaatattccc aaaaggcagg 3000
tgcaatgatg cacacttata cccccagcca ctcaggaggc tgatgcaaga ggatcgcttg 3060
agcccaggag ttgaagtcca gcctaagcaa catagtgaaa ccccatctcc aaaaatataa 3120
taataattct ctcaaaatac taaacagagg tggttttatt gataagattt tggctgtttg 3180
gttttccact attctctatt ggctaaaatt tgtttaatga gcatgaaatg tttttatttt 3240
attttgctta tttttatgat tgcaaaaaat gatatgagtt tctccctgcc aaggcaaaaa 3300
atatatatat atacctataa aaaaaaaaaa aaaaaaaaaa aaaaa 3345
<210>2
<211>487
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>2
Met Pro Gly Pro Arg Val Trp Gly Lys Tyr Leu Trp Arg Ser Pro His
1 5 10 15
Ser Lys Gly Cys Pro Gly Ala Met Trp Trp Leu Leu Leu Trp Gly Val
20 25 30
Leu Gln Ala Cys Pro Thr Arg Gly Ser Val Leu Leu Ala Gln Glu Leu
35 40 45
Pro Gln Gln Leu Thr Ser Pro Gly Tyr Pro Glu Pro Tyr Gly Lys Gly
50 55 60
Gln Glu Ser Ser Thr Asp Ile Lys Ala Pro Glu Gly Phe Ala Val Arg
65 70 75 80
Leu Val Phe Gln Asp Phe Asp Leu Glu Pro Ser Gln Asp Cys Ala Gly
85 90 95
Asp Ser Val Thr Ile Ser Phe Val Gly Ser Asp Pro Ser Gln Phe Cys
100 105 110
Gly Gln Gln Gly Ser Pro Leu Gly Arg Pro Pro Gly Gln Arg Glu Phe
115 120 125
Val Ser Ser Gly Arg Ser Leu Arg Leu Thr Phe Arg Thr Gln Pro Ser
130 135 140
Ser Glu Asn Lys Thr Ala His Leu His Lys Gly Phe Leu Ala Leu Tyr
145 150 155 160
Gln Thr Val Ala Val Asn Tyr Ser Gln Pro Ile Ser Glu Ala Ser Arg
165 170 175
Gly Ser Glu Ala Ile Asn Ala Pro Gly Asp Asn Pro Ala Lys Val Gln
180 185 190
Asn His Cys Gln Glu Pro Tyr Tyr Gln Ala Ala Ala Ala Gly Ala Leu
195 200 205
Thr Cys Ala Thr Pro Gly Thr Trp Lys Asp Arg Gln Asp Gly Glu Glu
210 215 220
Val Leu Gln Cys Met Pro Val Cys Gly Arg Pro Val Thr Pro Ile Ala
225 230 235 240
Gln Asn Gln Thr Thr Leu Gly Ser Ser Arg Ala Lys Leu Gly Asn Phe
245 250 255
Pro Trp Gln Ala Phe Thr Ser Ile His Gly Arg Gly Gly Gly Ala Leu
260 265 270
Leu Gly Asp Arg Trp Ile Leu Thr Ala Ala His Thr Val Tyr Pro Lys
275 280 285
Asp Ser Val Ser Leu Arg Lys Asn Gln Ser Val Asn Val Phe Leu Gly
290 295 300
His Thr Ala Ile Asp Glu Met Leu Lys Leu Gly Asn His Pro Val His
305 310 315 320
Arg Val Val Val His Pro Asp Tyr Arg Gln Asn Glu Ser His Asn Phe
325 330 335
Ser Gly Asp Ile Ala Leu Leu Glu Leu Gln His Ser Ile Pro Leu Gly
340 345 350
Pro Asn Val Leu Pro Val Cys Leu Pro Asp Asn Glu Thr Leu Tyr Arg
355 360 365
Ser Gly Leu Leu Gly Tyr Val Ser Gly Phe Gly Met Glu Met Gly Trp
370 375 380
Leu Thr Thr Glu Leu Lys Tyr Ser Arg Leu Pro Val Ala Pro Arg Glu
385 390 395 400
Ala Cys Asn Ala Trp Leu Gln Lys Arg Gln Arg Pro Glu Val Phe Ser
405 410 415
Asp Asn Met Phe Cys Val Gly Asp Glu Thr Gln Arg His Ser Val Cys
420 425 430
Gln Gly Asp Ser Gly Ser Val Tyr Val Val Trp Asp Ash His Ala His
435 440 445
His Trp Val Ala Thr Gly Ile Val Ser Trp Gly Ile Gly Cys Gly Glu
450 455 460
Gly Tyr Asp Phe Tyr Thr Lys Val Leu Ser Tyr Val Asp Trp Ile Lys
465 470 475 480
Gly Val Met Asn Gly Lys Asn
485
<210>3
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>3
atgcctggac ccagagtgtg gg 22
<210>4
<211>32
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>4
tcaattcttg ccattcatca ctcccttgat cc 32
<210>5
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>5
caggatccat gcctggaccc agagtgtg 28
<210>6
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>6
gtgaattctc aattcttgcc attcatcact 30
<210>7
<211>28
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>7
ggaattcatg cctggaccca gagtgtgg 28
<210>8
<211>31
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>8
ctaagcttca attcttgcca ttcatcactc c 31
Claims (10)
1. isolating CLSPa polypeptide is characterized in that this polypeptide is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the arrestin enzymic activity function by (a) polypeptides derived;
(c) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the complement-mediated killing effect function by (a) polypeptides derived.
2. polypeptide as claimed in claim 1 is characterized in that, this polypeptide is the polypeptide with SEQ ID NO:2 aminoacid sequence.
3. isolating polynucleotide is characterized in that, it is selected from down group:
(a) polynucleotide of polypeptide according to claim 1 of encoding;
(b) with polynucleotide (a) complementary polynucleotide.
4. polynucleotide as claimed in claim 3 is characterized in that, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.
5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:
(a) has the sequence of 18-1478 position among the SEQ ID NO:1;
(b) has the sequence of 1-3345 position among the SEQ ID NO:1;
(c) has the sequence of 126-1478 position among the SEQ ID NO:1.
6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
7. a genetically engineered host cell is characterized in that, it contains the described carrier of claim 6.
8. the preparation method of a peptide species is characterized in that, this method comprises:
(a) under conditions suitable for the expression, cultivate the described host cell of claim 7;
(b) from culture, isolate described CLSPa polypeptide.
9. energy and the described CLSPa polypeptid specificity of claim 1 bonded antibody.
10. the purposes of the described CLSPa polypeptide of claim 1 is characterized in that, it is used as the inhibitor of arrestin enzymic activity, or is used to prepare the medicine that suppresses the complement-mediated killing effect.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100257426A CN1304568C (en) | 2004-07-05 | 2004-07-05 | Novel human complement C1r-like serine proteinase analogue, and its encoding sequence and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100257426A CN1304568C (en) | 2004-07-05 | 2004-07-05 | Novel human complement C1r-like serine proteinase analogue, and its encoding sequence and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1721528A CN1721528A (en) | 2006-01-18 |
| CN1304568C true CN1304568C (en) | 2007-03-14 |
Family
ID=35912136
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100257426A Active CN1304568C (en) | 2004-07-05 | 2004-07-05 | Novel human complement C1r-like serine proteinase analogue, and its encoding sequence and use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1304568C (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1351609A (en) * | 1999-04-09 | 2002-05-29 | 巴斯福股份公司 | Low-molecular inhibitors of complement proteases |
| CN1364802A (en) * | 2001-01-10 | 2002-08-21 | 上海博德基因开发有限公司 | New polypeptide-complement Clr 78.76 and polynucleotide for encoding such polypeptide |
| CN1433466A (en) * | 1999-12-02 | 2003-07-30 | 詹斯·C·詹斯尼厄斯 | MASP-3, complement-fixing enzyme, and uses for it |
-
2004
- 2004-07-05 CN CNB2004100257426A patent/CN1304568C/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1351609A (en) * | 1999-04-09 | 2002-05-29 | 巴斯福股份公司 | Low-molecular inhibitors of complement proteases |
| CN1433466A (en) * | 1999-12-02 | 2003-07-30 | 詹斯·C·詹斯尼厄斯 | MASP-3, complement-fixing enzyme, and uses for it |
| CN1364802A (en) * | 2001-01-10 | 2002-08-21 | 上海博德基因开发有限公司 | New polypeptide-complement Clr 78.76 and polynucleotide for encoding such polypeptide |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1721528A (en) | 2006-01-18 |
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