CN1304410C - Tumor necrosis factor-Gamma - Google Patents

Tumor necrosis factor-Gamma Download PDF

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CN1304410C
CN1304410C CNB941952118A CN94195211A CN1304410C CN 1304410 C CN1304410 C CN 1304410C CN B941952118 A CNB941952118 A CN B941952118A CN 94195211 A CN94195211 A CN 94195211A CN 1304410 C CN1304410 C CN 1304410C
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tnf
polypeptide
sequence
cell
dna
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CN1174558A (en
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余国良
倪健
克雷格·A·罗森
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Human Genome Sciences Inc
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Abstract

The present invention discloses human TNF-gamma polypeptide, DNA (RNA) for coding the polypeptide, and a method for preparing the polypeptide by recombination technology. The present invention also discloses a method for inhibiting the cell growth of tumors or cancers by using polypeptide, quickening the healing of wound, providing resistance against infection, inducing inflammatory activity and stimulating the growth of certain cell types to treat diseases, for example restenosis. The present invention also discloses a diagnostic method for detecting mutation in a TNF-gamma nucleic acid sequence or an overexpression of the TNF-gamma polypeptide. The present invention also discloses an antagonist against the polypeptide and an application for treating cachexia, septic shock, cerebral malaria, inflammation, arthritis and graft rejection.

Description

Tumor necrosis factor-Gamma
The present invention relates to new identified polynucleotides, the polypeptide of described polynucleotide encoding, the production of the application of described polynucleotide and polypeptide and described polynucleotide and polypeptide.In more detail, identified that polypeptide of the present invention is a newcomer of tnf family cytokines and is called " TNF-γ " hereinafter.The invention still further relates to the effect that suppresses described polypeptide.
Huamn tumor necrosis factory alpha (TNF-α) and β (TNF-β or lymphotoxin) are meant the member of the polypeptide amboceptor of plurality of classes, comprising disturbing rope, interleukin and somatomedin, be generically and collectively referred to as cytokine (Beutler, B. and Cerami, A, Annu.Rev.Immunol., 7:625-625 (1989)).
Initial tumour necrosis factor (TNF-α and TNF-β) is owing to its anti-tumor activity is found.Yet it is counted as a pleiotropy cytokine that plays an important role in immunomodulatory and inflammation now.So far, the relevant cytokine family member of 8 kinds of known TNF is arranged, TNF-α, TNF-β (lymphotoxin α), LT-β and F as part, CD30, CD27, CD40 and 4-1BB acceptor.These protein have conservative C-end sequence and variable N-end sequence, and except TNF-β, they are through being often used as membrane anchor.When they and TNF receptors bind, TNF-α and TNF-β work as the homotype triplet.
TNF can comprise monocyte by many cell types, inoblast, and the T cell, NK cell (NK) cell is also mainly produced by the activated scavenger cell.The someone reports the quick necrosis of TNF-α in tumour, immunostimulation, and autoimmune disease, transplant rejection, the opposing parasite produces antiviral response, the sepsis shock, growth regulating works in blood vessel endothelium effect and the metabolic effect.TNF-α also excites endotheliocyte to secrete the various factors, comprises PAI-1, IL-1, and GM-CSF and IL-6 promote cell proliferation.In addition, TNF-α is just regulating various cell adhesion molecule and is resembling the E-selection factor, ICAM-1 and VCAM-1.The someone proves that TNF-α and Fas part can inducing apoptosis again.
Inducing the first step of the various cell responses that mediated by TNF or LT is that they combine with the specific cell surface receptor.Identified the TNF acceptor (Hohman of two uniquenesses that are approximately 55KDa (TNF-R1) and 75KDa (TNF-R2), H.P.etal, J.Biol.Chem.264:14927-14934 (1989), and separated corresponding to the people and the mouse cDNA of two kinds of acceptor types and carried out qualitative (Loetscher, H.etal., Cell, 61:351 (1990)).Two TNF-Rs share typical cell surface receptor structure and comprise the extracellular, stride film, the intracellular region territory.
According to the similarity of structural amino acid sequence homology and function, identified that polypeptide of the present invention is a newcomer of TNF family, for example, TNF-γ is a proinflammatory protein.
According to an aspect of the present invention, provide a kind of new mature polypeptide, it is TNF-γ, with and biologic activity go up useful fragment, analogue and derivative with diagnostics or treatment.Polypeptide of the present invention is the people source.
According to a further aspect in the invention, provide coding people TNF-the isolated nucleic acid molecule of γ, comprised mRNA, DNA, cDNA, genomic dna and analogue thereof and bioactive and diagnosis is gone up or useful fragment and derivative thereof gone up in treatment.
According to a further aspect in the invention, the method of producing described polypeptide by recombinant technology is provided, described method is included under the condition that promotes said protein expression, cultivates the reorganization protokaryon and/or the eukaryotic host cell that contain people TNF-γ nucleotide sequence, reclaims described protein subsequently.
According to a further aspect in the invention, the method of the polynucleotide that utilize the described polypeptide or the described polypeptide of encoding is provided, to be used to screen agonist and antagonist and to be used for the treatment of purpose, for example wound healing suppresses tumor regrowth, and the opposing parasite is provided, the resistance of bacterium and virus, induce inflammation, inducing endothelial cell and some hemapoietic propagation, treatment restenosis and the method that prevents some autoimmune disorders.
According to a further aspect in the invention, provide and comprise its length and be enough to nucleic acid probe with the nucleic acid molecule of people TNF-γ sequence-specific hybridization.
According to a further aspect in the invention, provide TNF-γ stimulant, it is simulated TNF-γ and is incorporated into TNF-γ acceptor and replys to cause TNF-γ type.
According to a further aspect in the invention, provide the antagonist of described polypeptide, they can be used for suppressing the effect of described polypeptide, for example, prevent the sepsis shock, inflammation, cerebral malaria, the activation of HIV virus, transplant rejection, bone resorption and emaciation.
According to a further aspect in the invention, provide the diagnostic assay method that is used to detect not enough with the expression of the nucleotide sequence of this polypeptide of coding overexpression diseases associated in TNF-γ polypeptide.
These and other aspects of the present invention should be conspicuous for those skilled in the art according to the instruction of this paper.
Below accompanying drawing show embodiment of the present invention, do not mean the scope of the present invention that restriction comprises as claim.
Fig. 1 has illustrated the cDNA sequence of polypeptide of the present invention and the aminoacid sequence of the deduction of correspondence.Initial 25 amino acid (underscore) are the leader sequences of inferring.Used amino acid whose standard single-letter abbreviation.
Fig. 2 shows the aminoacid sequence contrast between TNF-γ and other TNF family member.TNF-γ contains the conservative amino acid residues just like the TNF family shown in the shadow zone.
Fig. 3 A is a rna blot analysis that shows people's tissue of expressing TNF-γ.Survey RNA with the TNF-γ cDNA of mark from pointed tissue.Because Fig. 3 A has shown the bands of a spectrum of a uniqueness, so TNF-γ mRNA mainly is present in the kidney.As if other swimming marks have shown strong hybridization, but in fact, have non-specific stain.
Fig. 3 B shows that TNF-γ mainly expresses the rna blot analysis of (swimming lane 9) at HUVEC cell (Human umbilical vein endothelial cells).The 6th swimming lane and the 8th swimming lane are non-specific stains.Survey RNA with the TNF-γ cDNA of mark from pointed clone.Swimming lane 1 is CAMA1 (mammary cancer); Swimming lane 2 is AN3CA (uterus carcinoma); Swimming lane 3 is SK.UT.1 (uterus carcinoma); Swimming lane 4 is MG63 (osteoblastomas); Swimming lane 5 is HOS (osteoblastomas); Swimming lane 6, MCF7 (mammary cancer); Swimming lane 7, OVCAR-3 (ovarian cancer); Swimming lane 8, CAOV-3 (ovarian cancer); Swimming lane 9, HUVEC; Swimming lane 10, AOSMIC (unstriated muscle); Swimming lane 11, the foreskin inoblast.
Fig. 4 is the gel photograph after the TNF-γ electrophoresis that will be produced by bacterial expression and purifying.
Fig. 5 is the gel photograph by the TNF-γ of baculovirus expression.
Fig. 6 A is the photo of WEHI 164 cells, and wherein said cell comprises and be untreated in (upper left corner) and be exposed to TNF-α, after TNF-β and the TNF-γ.Cell with non-circular form of elongation is a dissolved.The TNF that adds is approximately 0.5mg/ml.Photo is shooting in 72 hours after adding TNF.
Fig. 6 B shows with TNF-α and TNF-β and compares, and TNF-γ suppresses the ability of WE HI 164 cells growth.
Fig. 7 shows reorganization TNF-γ, the ability of beta induced WE HI 164 necrocytosiss of TNF-α and TNF-.
Fig. 8 shows reorganization TNF-α, and TNF-β, TNF-γ induce the ability of morphological change in the L929 cell.Morphological change is by the indication of black circle cell.The reorganization TNF that produces with the E.coli that is approximately 0.5mg/ml handles cell.Photo is shooting in 72 hours after adding TNF.Morphological change points out that cell is killed.
Fig. 9 is TNF-γ, and TNF-α and TNF-β are to the graphic extension of the influence of venous endothelial cell.Utilize MT S measuring method that quantitative assay is carried out in TNF-α and the cell proliferation of TNF-β after the TNF-γ that reaches intestinal bacteria production handles venous endothelial cell with commercial acquisition.
Figure 10 is the photo of HL 60 cells, and contrast has shown HL 60 cells that separate; In the lower right corner, cytoadherence phenomenon has together been illustrated TNF-α and TNF-γ inducing cell adhesion and cell-cells contacting.
Figure 11 illustrates that TNF-γ is not that sTNF RI (p55) and sTNF RII (p75) combine significantly with two kinds of known soluble TNF acceptors.
According to an aspect of the present invention, a kind of nucleic acid (polynucleotides) of separation is provided, its coding have Fig. 1 deduction amino acid sequence mature polypeptide or by the mature polypeptide of on October 26th, 1994 with the clone's of ATCC preserving number 75927 preservations cDNA coding.
Can be from the polynucleotides of people's kidney and umbilical venous endothelial cell acquisition coding polypeptide of the present invention. Polynucleotides of the present invention are to find in originating from a cDNA library of Human umbilical vein endothelial cells. There are structural dependence in it and T NF family. It contains the ORF that a coding has the protein of 174 amino acid residues, and wherein 25 amino acid residues that approximately begin of this protein are the targeting sequencing of inferring, so mature protein contains 149 amino acid. Protein and rabbit TNF-α have the homology of top at the C-end, have 38% homogeneity and 58% similitude in 111 amino acid spans. There is (referring to Fig. 2) too in sequence conservative in T NF family member in TNF-γ. Bold-faced letter represents the amino acid residue guarded. Show in the rna blot analysis as Fig. 3 B that TNF-γ mR NA expresses specifically in Human umbilical vein endothelial cells.
Polynucleotides of the present invention can be rna form or dna form, and wherein DNA comprises cD NA, genomic DNA, and synthetic DNA. DNA can be two strands or strand, and if strand can be coding strand or non-coding (antisense) chain. The coded sequence of encoding mature polypeptide can be same as coded sequence that Fig. 1 shows or preservation clone's coded sequence or can be a different coded sequence, wherein because the result of the rich remaining or degeneracy of genetic code, this different coded sequence coding is same as the DNA of Fig. 1 or the mature polypeptide of preservation cDNA coding.
The polynucleotides of the mature polypeptide of the mature polypeptide of code pattern 1 or preservation cDNA coding include, but are not limited to: the coded sequence that only has mature polypeptide; The coded sequence of mature polypeptide and additional code sequence such as leading or secretion sequence or proteinogen sequence; The coded sequence of mature polypeptide (with randomly comprising the additional code sequence) and non-coding sequence are such as 5 ' or 3 ' non-coding sequence of the coded sequence of introne or mature polypeptide.
So wood language " polynucleotides of coded polypeptide " comprises the polynucleotides that only comprise polypeptid coding sequence and comprises additional code and/or the polynucleotides of non-coding sequence.
The invention further relates to above the variant of the polynucleotides of narration, its coding has fragment, analog and the derivative of polypeptide of the polypeptide of the amino acid sequence that Fig. 1 infers or preservation clone's cDNA coding. The variant of polynucleotides can be the variant that the non-natural of naturally occurring equipotential polynucleotides variant or polynucleotides exists.
So, the present invention includes coding and being same as the polynucleotides of identical mature polypeptide of the mature polypeptide shown in Fig. 1 or preservation clone cDNA coding and the variant of such polynucleotides, wherein fragment, derivative or the analog of the polypeptide of cDNA coding cloned in the polypeptide in the variant code pattern 1 or preservation. Such nucleotide diversity body comprises the deletion mutation body, replaces variant and interpolation or inserts variant.
As what above point out, polynucleotides can have the coded sequence of naturally occurring allelic variant of the coded sequence that is coded sequence shown in Figure 1 or preservation clone. As it be known to those skilled in the art that allelic variant is the another kind of optional form of polynucleotide sequence, it can have the replacement of one or more nucleotides, disappearance or stack, and it does not change the function of coded polypeptide basically.
The present invention also comprises such polynucleotides, wherein the coded sequence of mature polypeptide can merge at same single open reading frame and a polynucleotide sequence, this polynucleotide sequence helps polypeptide from host cell expression and secretion, for example, targeting sequencing, its function are from transcellular secretion sequence as the control polypeptide. Polypeptide with targeting sequencing is front albumen and can excises the polypeptide that targeting sequencing forms mature form by host cell. The all right encoding proteins of polynucleotides is former, and it is that maturation protein adds 5 ' additional amino acid residue. Having the former mature protein of sequence is proteinogen, is the inactive form of protein. In case sequence is former cut, then stay activated mature protein.
So for example, polynucleotides of the present invention can encoding mature protein, perhaps one has the former protein of sequence, perhaps has the protein of the former and presequence (targeting sequencing) of sequence.
Polynucleotide of the present invention also can have the encoding sequence that merges with flag sequence in single open reading frame, this flag sequence can make peptide purification of the present invention.Flag sequence can be one the six Histidine sign that the pQE-9 carrier provides, thereby be convenient to the mature polypeptide that purifying and mark merge in host bacterium, perhaps for example, when using mammalian hosts, for example during the COS-7 cell, flag sequence can be a hemagglutinin (HA) sign.The HA sign is corresponding to originating from the proteinic epitope of influenza hemagglutinin (Wilson, L., etal., cell 37:767 (1984)).
The invention further relates to such polynucleotide, 70% identity is also preferably arranged, so described polynucleotide and the sequence hybridization of above narrating as having 50% between the infructescence at least.The polynucleotide of the multi-nucleotide hybrid that The present invention be more particularly directed under stringent condition and above narrate.As used herein, term " stringent condition " is meant to have only to have 95% and preferably have at least 97% identity just to hybridize at least between sequence.In a preferred embodiment, kept identical biological function or the activity of mature polypeptide with cDNA or the preservation cDNA coding of Fig. 1 basically with the polypeptide of polynucleotide encoding of the multi-nucleotide hybrid of narration above.
This paper indication preservation thing is according to about the budapest treaty of the microbial preservation that is used for patented procedure of international recognition and preservation.These preservation things provide as just convenient those skilled in the art, rather than to according to the permission of 35U.S.C.1.2 to the requirement of preservation thing.Be included in the polynucleotide sequence in the preserved material, and the aminoacid sequence of encoded polypeptides thus, be incorporated into this paper and contrast as a reference and when any conflict appears in the sequence with this paper narration.Make, utilize or sell preserved material to obtain permission, but this paper does not authorize such permission.
The invention further relates to TNF-γ polypeptide, it has aminoacid sequence or the preservation cDNA amino acid sequence coded that Fig. 1 infers, the invention still further relates to fragment, analogue and the derivative of this polypeptide.
When referring to Fig. 1 or preservation cDNA encoded polypeptides, term " fragment ", " derivative " is meant with " analogue " and kept biological function identical with this polypeptide or active polypeptide substantially, so analogue comprises proteinogen, it can activate activated mature polypeptide of generation by downcutting the proteinogen part.
Polypeptide of the present invention can be a recombinant polypeptide, natural polypeptides or synthetic polypeptide, preferably recombinant polypeptide.
The fragment of the polypeptide of Fig. 1 or preservation cDNA encoded polypeptides, derivative or analogue can be that (i) wherein one or more amino-acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues) and such to be substituted amino-acid residue can the yes or no genetic code coded, or (ii) wherein one or more amino-acid residues comprise substituting group, or (iii) wherein mature polypeptide and another compound merge, as the compound that increases the polypeptide half life (for example, polyoxyethylene glycol), or (iv) wherein additional amino acid and mature polypeptide fusion, as leading or secretion sequence or be used for the sequence or the proteinogen sequence of mature polypeptide purifying.These fragments, derivative and analogue are considered to be in those skilled in the art according within this paper instruction category as can be known.
Preferably, polypeptide of the present invention and polynucleotide can provide with unpack format, and preferably are purified to homogeneity.
Term " isolating " is meant in the primal environment (if natural existence also is a natural surroundings) from it removes this material.For example, the naturally occurring polynucleotide or the polypeptide that are present in the Live Animals are not isolating, and still, the same polynucleotide or the polypeptide that separate in the some or all of coexistence materials from natural system are isolating.Such polynucleotide can be that the part of a carrier and/or such polynucleotide or polypeptide can be the parts of a composition.And if such carrier or composition be not the part of its natural surroundings, then it remains isolating.
The invention still further relates to the carrier that comprises polynucleotide of the present invention, carry out the host cell of genetic engineering processing and by recombinant technology production polypeptide of the present invention with carrier of the present invention.
Utilize carrier of the present invention that host cell is carried out genetically engineered (transduction or conversion or transfection), carrier for example can be, cloning vector or expression vector.Carrier can be a plasmid for example, virion, phage or the like form.Genetically engineered host cell can be for being suitable for activating promotor, screening transformant or amplification TNF-γ gene and cultivate in traditional nutritional medium of modifying.Culture condition, as temperature, PH is like that, be before to be used for the host cell expression screening, will be that the ordinary skill skilled person is conspicuous.
Can be used to produce polypeptide by recombinant technology polynucleotide of the present invention.So for example, for express polypeptide, polynucleotide can be included in various expression vectors any.Such carrier comprises karyomit(e), non-chromosome and synthetic DNA sequence, for example derivative of SV40; Bacterial plasmid; Phage DNA; Baculovirus; Yeast plasmid; The carrier that originates from the combination of plasmid and phage DNA; Viral DNA such as poxvirus, adenovirus, fowlpox virus and plan rabies virus.Yet, any other carrier as long as it in the host reproducible and the survival just can utilize.
Can insert carrier to suitable dna sequence dna by the whole bag of tricks.Generally speaking, dna sequence dna can be inserted a suitable restriction endonuclease sites by method known to those skilled in the art.Such method and other method are considered to be within the known scope of those skilled in the art.
Dna sequence dna in the expression vector operationally is connected with a suitable expression control sequenc (promotor) to instruct mRNA synthetic.As the representation example of these promotors, can mention: LTR or SV40 promotor, intestinal bacteria Lac or trp, phage P LThe promotor of promotor and other known may command genetic expression in prokaryotic organism or eukaryotic cells or their virus.Expression vector also contains the ribosome bind site and the transcription terminator of translation initiation.Carrier can also comprise the sequence that suitably is used to increase and expresses.
In addition, but expression vector preferably contains one or more selectable marker genes, with the phenotypic characteristic that provides to be used to screen transformed host cells, as be used for the Tetrahydrofolate dehydrogenase or the neomycin resistance of eukaryotic cells culture, perhaps as tsiklomitsin or amicillin resistance in intestinal bacteria.
The carrier that contains suitable dna sequence dna and suitable promotor or control sequence of narration can be used to transform suitable host so that this protein of host expresses as mentioned.
As suitable host's representation example, can mention: bacterial cell, as intestinal bacteria, streptomycete, Salmonella typhimurium; The fungal cell is as yeast; Insect cell such as Drosophila S2 and Sf9; Zooblast such as CHO, COS or Bowes melanoma; Adenovirus; Vegetable cell or the like.Selecting an appropriate host to be considered to be in those skilled in the art instructs in the known scope according to this paper.
In more detail, the present invention also comprises the recombinant precursor that contains one or more sequences of narrating above.These constructs comprise carrier, as plasmid or virus vector, wherein with forward or reverse insertion sequence of the present invention.This construct further contains and comprises for example adjusting sequence of a promotor that operationally links to each other with sequence.A large amount of suitable carriers and promotor are known for those skilled in the art, and are that commercial sources is obtainable.Following carrier is an illustrative.Bacterium: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, pSIX174, pbluescript SK, pbsks, pNH 8A, pNH16a, pNH18A, pNH46A (Stratagene); Ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), eucaryon: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia).Yet, also can use any other plasmid or carrier, as long as reproducible and can survive among its host.
The carrier that utilizes CAT (CAT) carrier or other to have selective marker can screen promoter region from any desired gene.Two suitable carriers are pKK232-8 and PCM7.The bacterium promotor of mentioning especially comprises lacI, lacZ, T3, T7, gpt, λ P R, P LAnd trp.Eukaryotic promoter comprises that CMV is promptly early stage, HS V thymidine kinase, early stage and late period SV40, from retroviral LTR and mouse metallothionein(MT)-I.Selecting appropriate carriers and promotor is in the level of those of ordinary skill.
In another embodiment, the present invention relates to contain the host cell of the construct of narrating above.Host cell can be a higher eucaryotic cells, as mammalian cell, and perhaps lower eukaryotes cell, as yeast cell, perhaps host cell can be a prokaryote, as bacterial cell.By calcium phosphate transfection, the transfection of DEAE-Dextran mediation, or electroporation method construct can be imported the host (Davis, L,, Dibner, M,, Battey, I., Basic Methods in Molecular Biology, (1986)).
These constructs in the host cell can be used to produce the gene product by the recombination sequence coding in a usual manner.Perhaps, polypeptide of the present invention can be produced by conventional peptide synthesizer is synthetic.
Under the control of suitable promotor, maturation protein can be at mammalian cell, and yeast is expressed in bacterium or other cells.Utilization originates from the RNA of DNA construct of the present invention, and cell free translation system also can be used to produce such protein.Sambrook, etal., the molecular cloning laboratory manual, second edition, Cold Spring Harbor, N.Y., (1989) have narrated suitable clone and the expression vector that uses with protokaryon and eucaryon host, and offer the described publication side of being incorporated herein by reference.
In carrier, insert enhancer sequence and can improve higher eucaryote transcribing the DNA of code book invention polypeptide.Enhanser is the cis acting factor of DNA, is generally 10 to 300 bp, acts on promotor to strengthen transcribing of it.Example is included in the SV40 enhanser of the bp100 to 270 that duplicates source point one side in late period, and the sub-enhanser of cytomegalovirus early promoter is at the polyoma enhanser and the adenovirus enhanser that duplicate source point side in late period one.
In general, but recombinant expression vector comprises the selective marker of duplicating source point and allowing host cell to transform, as, intestinal bacteria ampicillin resistance gene and cereuisiae fermentum TRP1 gene and a promotor that originates from cance high-expression gene instruct the downstream configurations gene transcription.Such promotor can originate from coding glycolytic ferment such as glycerol 3-phosphate kinases (PGK), α-factor, and acid phosphatase, or heat shock protein(HSP) etc.The allos structure sequence assembles with translation initiation and terminator sequence suitable, and preferably, assemble with the leader sequence that can guide the protein secreting of being translated to enter in kytoplasm space or the extracellular substratum, randomly, heterologous sequence can be encoded and be comprised and can give desired characteristic, for example the fused protein of the terminal identification polypeptide of the N-of the stability of the recombinant products of Biao Daing or purifying simplicity.
By reading that sign indicating number inserts the desired proteinic structural DNA sequence of coding mutually and suitable translation initiation and termination signal can make up the used useful expression vector of bacterium having operating of function on.Carrier comprises one or more Phenotypic Selection marks and one and guarantees carrier existence and if desired, is provided at the source point that duplicates of amplification in the host.Suitable conversion comprises intestinal bacteria with the prokaryotic organism host, subtilis, Salmonella typhimurium and Rhodopseudomonas, each kind in streptomyces and the Staphylococcus, certainly other also can select use.
As a representativeness but unrestriced example, the useful expression vector that is used for bacterium can comprise the selective marker and the bacterium that derive from the obtainable carrier of genic commercial sources that comprises known cloning vector PBR322 (ATCC 37017) and duplicate source point.Commercial carrier so for example comprises, p KK223-3 (PharmaciaFine Chemicals, Uppsala, Sweden) and GEMI (Promega Biotec, Madison, WI, USA).These pBR322 " skeleton " part combines with suitable promotor and structure sequence to be expressed.
Transform and after host strain grows into suitable cell density at the suitable host bacterial strain, induce selected promotor and with other for some time of cell cultures by suitable method (as temperature transition or chemical induction).
Usually by centrifugal collecting cell, by physics or chemical process smudge cells, and the crude extract that reservation obtains is to be further purified.
Can come fragmentation to be used for the microorganism cells of marking protein by the method for any routine, comprise freeze-thaw cycle, ultrasonication, Mechanical Crushing, or use the lysis agent, these methods are well known to those skilled in the art.
Various mammalian cell culture systems also can be used for express recombinant protein matter.The example of mammalian expression system comprises the fibroblastic COS-7 of monkey kidney system, as Gluzmam, and cell, 23:175 (1981) narrates and other can express the clone of compatible carrier, for example, C127,3T3, CH0, HeLa and bhk cell system.Mammalian expression vector comprises and duplicates source point, and suitable promotor and enhanser also have the ribosome bind site of any necessity, poly-adenosine site, splicing donor and acceptor site, transcription termination sequence and 5 ' side joint non-transcribed sequence.Derive from SV40 splicing and gather the dna sequence dna that adenosine turns into the site and can be used to provide needed non-transcribed genetic elements.
Can from the reconstitution cell culture, reclaim and purifying TNF-γ polypeptide by several different methods, described method comprises ammonium sulfate or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, the phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and phytohemagglutinin chromatography.If desired, when finishing the mature protein configuration, can use the protein refolding step.At last, high performance liquid chromatography (HPLC) can be used for last purification step.
Polypeptide of the present invention can be the product of natural purifying, and the product of perhaps chemosynthesis process perhaps produces from prokaryotic organism or eukaryote host (for example, bacterium, yeast, higher plant, the insect of cultivation and mammalian cell) by recombinant technology.According to host used in the recombinant production process, polypeptide of the present invention can be glycosylated or nonglycosylated.Polypeptide of the present invention can comprise an initial methionine amino-acid residue.
TNF-γ polypeptide of the present invention can be used to suppress growth of tumour cell or tumorigenesis.TNF-γ polypeptide may have effect to the destruction of the tumour by apoptosis, it is characterized in that film blebbin (Zeiosis), and tenuigenin concentrates and the activation (Fig. 7) of endogenous nucleic acid restriction endonuclease.Just as shown in table 1, TNF-γ has the intensive cytotoxic activity to the clone of being tested, and the clone of surveying has unusual cell proliferation and adjusting, for example, and fibrosarcoma and cancerous cell line.This point can find out also among the 6B and 8 that at Fig. 6 A the γ of TNF-shown in it has the ability to suppress L 929 and the growth of WEHI164 cell by cytotoxic activity.The WEHI164 cell is the mouse fibrosarcoma cell.The medication of preferred TNF-γ is to enter tumour by direct injection.
The cytoadherence activity of TNF-γ can be used for wound healing.As table 1 and shown in Figure 9, TNF-γ has the effect of intensive endothelial cell proliferation, and it is the sign that TNF-γ works in wound healing.The cytoadherence effect of TNF-γ also can be worked in wound healing.
TNF-γ also can be used for the treatment of the disease that needs growth-promoting activity, for example, and restenosis.As mentioned above, TNF-γ has shown the intensive proliferation function to endothelial cell growth.Therefore, TNF-γ also can be used to regulate hemopoiesis and endotheliocyte growth.
By its ability of stimulation T-cell activation, TNF-γ polypeptide is an immunoreactive important medium.Therefore, this polypeptide can be used for stimulating anti-various parasites, the immune response of bacterium and virus infection.TNF-γ can the lytic virus cells infected, so, can be used to catch the HIV cells infected.
TNF-γ polypeptide is replied by enhancing T-cell proliferation can be used for the treatment of autoimmune disease such as type i diabetes.
Table 1
The general introduction of TNF-gamma activity Active
Clone Source and type
Cytotoxicity Propagation Differentiation Adhesion
L?929 WE?HI?164 NRK-54E THP-1 HL-60 Raji Jurkat Primary Primary A-431 293 L cell mouse fibrosarcoma Rat renal class epithelium person monocytic cell leukaemia people progranulocyte leukaemia people Burkitt lymthoma HTL people venous endothelial cell people parteriole endothelial cell people epidermal carcinoma human embryo kidney + +++ + + - - ++ - +* ++ - - - - - - - - ++ ++ - ++ - - - ++ - - - - - - - - - - ++ ++ - - ？ ？ - -
* only when high dosage.+ number number refers to activity level relatively.-number finger does not detect activity in the test concentrations scope.
Polynucleotide of the present invention and polypeptide can be used as finder's treatment of diseases and Studies on Diagnosis reagent and material.
The invention provides the authentication method of TNF-γ acceptor.By the known method of many those skilled in the art can the identification code acceptor gene, for example, art (Coligan, etal, Current Protocols in Immun., 1 (2) Chapter 5, (1991)) that part is selected and FACS classifies.Preferably, utilized cloning by expression, wherein gathered adenosine RNA, and a cDNA library that produces from this RNA is divided into aggregate from TNF-γ being produced the cell preparation of replying.And be used for rotaring redyeing COS cell or other is to the responseless cell of TNF-γ.Being grown in the TNF-γ that transfectional cell on the sheet glass is exposed to mark, TNF-γ can pass through the whole bag of tricks mark, comprises iodate or introduces the kinase whose recognition site of site-specific protein.After fixing and incubation, slide is carried out radioautographic analysis.Identify positive set and utilize to repeat inferior set and rescreen the choosing method preparation and the inferior aggregate of transfection again, finally produced the mono-clonal that a coding is inferred acceptor.
As another approach selected that acceptor is identified, mark TNF-γ can or extract preparation affine connection of light with the cytolemma of expressed receptor molecule.Separate the material of crosslinked connection and be exposed to x-ray film by PAGE.The labeled complex that contains TNF-γ acceptor can be cut, resolves into peptide fragment, and carry out the analysis of protein micrometering preface.The aminoacid sequence that the analysis of micrometering preface obtains will be used to design a cover degenerate oligonucleotide probe and screen the cDNA library, so that the gene of the acceptor that identification code is inferred.
TNF-γ and two solvable TNF acceptors, sTNF-RI (p55) and sTNF-RII (p75) do not have obvious combination.Therefore, TNF-γ may have known TNF activity of proteins and the activity (referring to Figure 11) except that known TNF protein active.
The invention still further relates to the compound method of the effect of those imitation TNF-γ (stimulant) of Screening and Identification or obstruction TNF-γ.A kind of like this example of method has utilized the advantage of TNF-γ ability of energy significant stimulation human endothelial cell propagation when having mitogen Con A.Obtain endotheliocyte and (Calbiochem, LaJolla are incubated in the time of CA) to have and have replenished 10% heat inactivation placenta bovine serum (Hyclone Labs at the Con-A that has 2 μ g/ml, Logan, UT), 1%L-L-glutamic acid, 100u/ml penicillin, 100 μ g/ml Streptomycin sulphates, 0.1% gentamicin (Gibco Life Technologies, Grand Island, (Costar in the flat culture plate in 96 holes of RPMI1640 NY), Canbridge, MA).Adding Con-A and compound to final volume to be screened is 0.2ml.37 ℃ after 60 hours, with 1 μ Ci's 3H thymus pyrimidine (5Ci/mmol; 1Ci=37BGq; NEN) pulse 12-18 hour, and results (PhD on glass fiber filter; Cambridge Technology, Watertown, MA). (Beckman instrument, Irvine CA) determine that three are repeated the average of culture to utilize a liquid scintillation counter 3H thymus pyrimidine incorporation (cpm).Significantly 3The H thymus pyrimidine mixes the hormesis of indication to endothelial cell proliferation.
But the another kind system of selection is measured replying of known second messenger system under this compound and is compared existing or lack after the interaction of TNF-γ and acceptor.Described second messenger system includes but not limited to the cAMP guanylate cyclase, ionic channel or phosphoinositide hydrolysis.
In order to measure antagonist, carried out the test narrated above, but in this test, TNF-γ adds with compound to be screened and compound when having TNF-γ, suppress [ 3H] ability of mixing of thymus pyrimidine shows that this compound is the antagonist of TNF-γ.Another kind of selectable method, by under suitable being at war with property inhibition test condition, the antagonist that TNF-γ and potential are had membrane-bound TNF-γ acceptor or a recombinant receptor combines can measure TNF-γ antagonist.Can be with TNF-γ mark, as by radio-labeling, so that according to determining the effectiveness of potential antagonist with the number of the TNF-γ molecule of receptors bind.
Another kind of selectable method will be expressed the Mammals thing cell of TNF-γ acceptor or membrane prepare thing with mark TNF-γ incubation when having this compound.Measure the ability that compound strengthens or stop this effect then.
By combining with TNF-γ and stoping it and it receptors bind will be specific to the antibody of TNF-γ as antagonist.Effective especially about this point monoclonal antibody.But the antibody that is specific to TNF-γ acceptor can mediate unique cell response, thisly replys the interaction that trends towards stimulating TNF-γ and its acceptor.
Potential TNF-γ antagonist also comprises TNF-γ mutant, and it and TNF-γ receptors bind can't cause that effective prevention acceptor and its native ligand bonded second messenger reply.Specially designed oligonucleotide and small molecules also can and stop it to combine with TNF-γ with TNF-γ receptors bind.Micromolecular example includes but not limited to little peptide or class peptide molecule.
Another potential TNF-γ antagonist is the soluble form of TNF-γ acceptor, and it combines and prevent it and membrane-bound TNF-γ acceptor interaction with TNF-γ.In this mode, acceptor is not subjected to the stimulation of TNF-γ.
Another potential TNF-γ antagonist is the antisense construct that utilizes the antisense technology preparation.By formation or the antisense DAN or the RNA of triple helices, antisense technology can be used for controlling gene and express, two kinds of methods all are basic with being combined into of polynucleotide and DNA or RNA.For example, the encode 5 ' coding region of polynucleotide sequence of mature polypeptide of the present invention is used to design the approximately antisense rna oligonucleotide of from 10 to 40 base pairs of a length.Design that a DNA oligonucleotide makes it and complementary (triple helices one is referring to Lee etal., Nucl.Acids Res., 6:3073 (1979) in a district of the gene that participates in transcribing; Cooneyetal, Science, 241:456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby stop transcribing and producing of TNF-γ.Antisense rna oligonucleotide and mRNA are hybridized in vivo and have been stoped the mRNA molecule to translate into TNF-γ polypeptide (antisense-Okamo, J, Neurochem., 56:560 (1991); As the oligodeoxynucleotide of the antisense inhibitor of genetic expression, CRC Press Boc a Raton, FL (1988)).The oligonucleotide of narrating above also can be sent to cell so that sense-rna or DNA can express the production that suppresses TNF-γ in vivo.
TNF-γ antagonist also can be in order to the treatment emaciation, and it is the lipid removal of defects type by the system defect generation of the lipoprotein lipase that is suppressed by TNF-γ.TNF-γ antagonist can also be used for the treatment of cerebral malaria, and wherein TNF-γ has seemed morbific effect.Antagonist can also be used for the treatment of rheumatoid arthritis by the generation that suppresses the IL-1 in TNF-γ inductive inflammatory cytokine such as the synovial fluid cell.When treatment of arthritis, intra-articular injection TNF-γ preferably.
TNF-γ antagonist can also be used to stop transplant rejection by prevent TNF-γ stimulating immune system when having graft.
Because TNF-γ can induce bone resorption, TNF-γ antagonist can also be used for the treatment of osteoporosis.
Because TNF-γ mediation enhanced inflammatory response, thereby TFN-γ antagonist can also be used as anti-inflammatory agent.
Antagonist also can be used for treating endotoxin shock, is also referred to as the sepsis shock.This severe is to be caused by excessively the replying of infection to bacterium or other type.Thereby this is replied the rising that causes TNF-γ level and causes shock and tissue injury.
Antagonist can be used in and contain pharmaceutical acceptable carrier (for example hereinafter described in) the composition.
The fragment of total length TNF-γ gene can be separated full-length gene and separate other gene that sequence similarity highly or similar biologic activity are arranged to this gene as the hybridization probe in cDNA library.The probe of this type can be, for example between 20 and 2000 bases.But preferably probe has 30 to 50 base pairs.Probe also can be used for identifying one corresponding to the cDNA clone of total length transcript with contain and comprise and regulate and promoter region exon, the genomic clone of the complete TNF-γ gene of intron.An example as screening comprises the coding region that utilizes the synthetic oligonucleotide probe of known dna sequence dna to separate TNF-γ gene.Have oligonucleotide with the mark of the sequence of gene complementation of the present invention and be used to screening and go into cDNA, which member genomic dna or mRNA library decide in probe and the library hybridize.
TNF-γ polypeptide of the present invention and stimulant and antagonist can with suitable pharmaceutical carriers combined utilization.Such composition comprises compound and the pharmaceutical acceptable carrier or the vehicle for the treatment of significant quantity.This carrier includes but not limited to salt solution, buffer saline, glucose, water, glycerine, ethanol, and combination.The medicament of preparation should be suitable for administering mode.
The present invention also provides pharmaceutical pack or medicine box, and they contain one or more container that is filled with one or more compositions of pharmaceutical composition composition of the present invention.Being attached in such container is by control medicine or the manufacturing of biology product, uses or the announcement of the statutory regulation sold.Such notice reflects manufacturing, uses or sell this medicine to obtain government department's agreement for people's use.In addition, pharmaceutical composition of the present invention can be used in combination with other compound for the treatment of usefulness.
Pharmaceutical composition method easily is local as passing through, intravenously, and intraperitoneal, intramuscular, subcutaneous, the interior or intradermal routes administration of nose.This pharmaceutical composition can treat and/or prevent the significant quantity of specified disease and use.In general, they are with the amount administration of about at least 10 μ g/kg body weight, and in most of the cases, they are to be no more than the amount administration of 8mg/kg body weight every day.In most cases, consider the approach of administration, symptom or the like, dosage approximately be every days 10 μ g/kg to the 1mg/kg body weight.
By expressing stimulant and the antagonist that described polypeptide utilizes TNF-γ polypeptide and belongs to polypeptide in vivo, this often is called as " gene therapy " according to the present invention.
So, for example, can carry out genetic engineering to cell at the polynucleotide (DNA or RNA) of external use coded polypeptide and handle from patient, subsequently engineering cell is offered the patient of stand-by polypeptide treatment.These methods all are well known by persons skilled in the art, and conspicuous according to the instruction of this paper.For example, can utilize the counter-transcription-ing virus particle pair cell of the RNA that contains the polypeptide of the present invention of encoding to carry out the genetic engineering processing.
Similarly, for express polypeptide in vivo, can by program well known by persons skilled in the art in vivo pair cell carry out the genetically engineered operation.For example, for through engineering approaches cell and express polypeptide in vivo in vivo, can use the production cell of the counter-transcription-ing virus particle of producing the RNA that contains code book invention polypeptide to patient.According to instruction of the present invention, by these and other methods of such method afford polypeptide of the present invention, those skilled in the art are conspicuous.For example, the expression vector that is used for engineering cell for example, can be an adenovirus except that retrovirus, and it can be used for through engineering approaches cell in the body with suitable after transporting carrier and combining.
The invention still further relates to the diagnostic test method that has diseases associated or disease susceptibility that utilizes TNF-γ gene test and mutation T NF-γ.These diseases are relevant with the expression deficiency of TNF-γ, for example, and unusual cell proliferation such as tumour and cancer.
Can detect the individuality that has sudden change in the people TNF-γ gene at dna level by various technology.Can as blood, urinate from patient's cell in order to diagnose, saliva organizes the material of biopsy and ptomatopsia to obtain nucleic acid.Genomic dna can be directly used in detection or can utilize PCR (Saiki et al., Nature, 324:163-166 (1986)) to carry out enzymatic amplification before analyzing.RNA or cDNA also can be used for same purpose.As an example, the PCR primer that is complementary to the nucleic acid of coding TNF-γ can be used for identifying and analysis TNF-γ sudden change.For example, the variation of comparing the size of amplified production with normal genotype can detect disappearance and insertion.Can identify point mutation by hybridizing DNA and the radio-labeling TNF-γ RNA of amplification or with radiolabeled TNF-γ antisense dna sequence.Difference by RNase A digestion or melting temperature(Tm) can come mispairing duplex and correct matched sequence difference.
Can finish heredity test by the change that when being with or without denaturing agent, detects the electrophoretic mobility of dna fragmentation on the gel based on dna sequence dna difference.Can manifest the insertion of becoming estranged of foreword Lieque by the high resolution gel electrophoresis.On the methane amide gradient glue of sex change, can distinguish not homotactic dna fragmentation, wherein according to they distinctive unwinding or the part melting temperature(Tm), different dna fragmentation migrations rests on the different positions of gel (referring to, i.e. Myers et al., Science, 230:1242 (1985)).
Measure by the nuclease protection, resemble the sequence variation (being Cotton et al., PNAS, USA, 85:4397-4401 (1985)) that RNase and S1 protection or chemical cracking method also can be found specific position.
So, by as hybridization, RNase protection, chemical cracking, directly dna sequence analysis or use Restriction Enzyme (for example, the Southern engram analysis method of restrictive fragment length polymerphism (RFLP) and genomic dna can be finished the detection of specific dna sequence.
Except more convenient gel electrophoresis and dna sequence analysis, can also be by the sudden change of original position analyzing and testing.
Owing to compare with the normal control tissue sample, the proteinic overexpression of TNF-γ for example can detect disease or disease susceptibility, the existence of tumour and cerebral malaria is used for detecting the diagnostic test method that various tissue TNF-γ protein levels change so the invention still further relates to.Being used in the testing method that the sample that derives from the host detects TNF-γ protein level is that those skilled in the art are known and comprise radioimmunoassay, competition is in conjunction with test, the Western engram analysis, ELISA test and " sandwich " test, ELIAS tests (Coligan, et al., Current Protocols in Immunology, 1 (2), Chapter 6, (1991) comprise that at first one of preparation is specific to the antigenic antibody of TNF-γ, preferably monoclonal antibody.Preparation resists the report antibody of this monoclonal antibody in addition.But being attached to report antibody is detection reagent such as radioactivity, fluorescence or horseradish peroxidase in this example.Now, from the host, take out sample and, incubation on the polystyrene ware for example, the protein bound in ware and the sample at a solid support.Then, by covering any free protein binding site with non-specific protein such as BSA incubation.Then, with monoclonal antibody incubation in dish, monoclonal antibody is attached to the TNF-γ albumen that any and TNF-γ bonded monoclonal antibody are attached to the polystyrene dish in this process.All unconjugated monoclonal anti body and function damping fluid flush awaies.To place ware with horseradish peroxidase bonded report antibody, cause reporting that antibody combines with TNF-γ bonded monoclonal antibody with any, then the report antibody that do not adhere to of flush away.Peroxidase substrate is added in the ware subsequently, with typical curve relatively, can measure the proteic amount of TNF-γ in the patient samples that is present in specified rate according to the amount of the color that manifests in given period.
Can use competitive test, the antibody that wherein is specific to TNF-γ is attached to a solid support, the TNF-γ of mark passes through solid support with the sample that derives from the host, and for example the amount by the detected mark of liquid scintillation chromatogram is relevant with the amount of TNF-γ in the sample.
" sandwich " test is similar to the ELISA test." sandwich " test in, TNF-γ by a solid support and with the antibodies that is attached on the solid support.Second antibody combines with TNF-γ then.Then, the 3rd an antibody mark and that be specific to second antibody by solid support and with second antibodies, quantitative assay then.
Sequence of the present invention identifies it also is valuable for karyomit(e).This sequence specifically target hit and with the specific position hybridization of the human chromosome of uniqueness.In addition, the present specific site that needs to identify on the karyomit(e).Have only a few chromosome marking reagent to can be used for the marker chromosomes position at present according to actual sequence data (repetition polymorphism).The present invention is the important the first step that the gene with these sequences and disease-related links to the physical mapping of chromosomal DNA.
In brief, can be positioned sequence on the karyomit(e) by prepare PCR primer (preferably 15-25bp) from cDNA.The Computer Analysis in 3 of this sequence ' untranslated district is used to the rapid screening primer, thereby primer is not crossed over the length of an above exon in the genomic dna and made amplification procedure complicated.These primers are used for the somatic hybridization body that the PCR screening contains single human chromosome then.Have only those crossbreds that contain corresponding to the people's gene of primer will produce amplified fragments.
The PCR mapping of somatic hybridization body is that specific DNA is navigated to specific chromosomal fast method.Utilize same Oligonucleolide primers in the present invention, can finish inferior location by a group chromosome slice groups or big genomic clone storehouse with similar method.Can be used for equally its other method of chromosome mapping is comprised in situ hybridization, with the streaming of mark classify chromosomal prescreen and the special cDNA of the preselected structure karyomit(e) library by hybridization.
The cDNA clone can be used to provide an accurate chromosomal localization that goes on foot with the fluorescence in situ hybridization (FISH) of Metaphase Chromosome smear.This technology can be used with the cDNA that is as short as 500 or 600 bases; But, have and unique chromosomal position bonded high likelihood more greater than the clone of 2000bp, and have to make and measure simple required enough strength of signal.FISH need utilize the clone of expressed sequence sign (EST) that derive, and the longer the better.For example, 2,000bp is good, 4,000 is better, surpass 4,000 for the result who obtains and reasonably time percentage ratio may be unnecessary.Look back this technology and can see Verma et al., human chromosome basic fundamental handbook, Pcrgamon Press, New York (1988).
In case sequence is positioned accurate chromosome position, the physical location of sequence on karyomit(e) just can be relevant with the genetic map data.Such data can be at for example Mckusick of V.Jomh Hopkins university, finds the Mendelian Inheritance in Man (online from the Welch of Johns Hopkins University medical library).Be positioned the gene of same chromosomal region and the relation of disease by linkage analysis (the common heredity of physical abutment gene) then.
Then, the difference of cDNA or genome sequence between the individuality that must determine to infect and do not infect.If at some or all infected individuals but do not observe sudden change in any normal individual, this sudden change is likely the reason that causes disease so.
According to the resolving power and the genetic map analytical technology of present physical map, accurately be positioned cDNA with the chromosomal region of disease-related and can be a kind of in 50-500 the potential Disease-causing gene.(this supposes that 1 megabasse mapping resolving power and every 20kb are a gene).
Polypeptide, their fragment or other derivative, or its analogue, or express their cell can be as immunogen to produce antibody.These antibody can for example be polyclone or monoclonal antibody.The present invention also comprises chimeric, strand and humanized antibody, and Fab fragment, or the product of Fab expression library.Various techniques known in the art can be used for these antibody and segmental production.
By the polypeptide direct injection being entered animal or using this polypeptide for animal (preferably non-human), can obtain at antibody corresponding to polypeptide of sequence of the present invention.The antibody that obtains like this combines with polypeptide is own then.By this way, even only coded polypeptide fragments sequence also can be used to produce antibody in conjunction with whole natural polypeptides.So then antibody can be used for from expressing the tissue isolated polypeptide of this polypeptide.
In order to prepare monoclonal antibody, can utilize any technology that can cultivate production antibody by continuous cell line.Example comprises hybridoma technology (Kohler and Milstein, 1975, Nature, 256:495-497), three knurl technology, people B-quadroma technology (Kozbor et al., 1983, Immunology Today 4:72), the EBV-hybridoma technology (Cole of generation human monoclonal antibodies, et al., 1985, monoclonal antibody and cancer therapy, Alan R.Liss, Ins., PP, 88-96).
Can adopt the technology (U.S. patent 4,946,778) of manufacture order chain antibody to come the single-chain antibody of production immunogenic polypeptide product of the present invention.Equally, transgenic mouse can be used to express the antibody of the change of immunogen polypeptide product of the present invention.
To further narrate the present invention with reference to the following examples; What however, it should be understood that is that the present invention is not limited to these embodiment.All parts or amount unless stated otherwise, all are by weight calculation.
In order to help understanding the following examples, with some method and/or term that often occurs of narration.
" plasmid " is by the small letter p of front and/or capitalization of following and/or numeral name., it is obtainable that the initial plasmid of this paper is that commerce can obtain with the unrestricted public, perhaps can be according to the method for public publication from obtainable plasmid construction.In addition, described plasmid to be equal to plasmid be known in the art and will be that common those of skill in the art are conspicuous.
" digestion " of DNA refers to that restriction enzyme is only worked with restriction enzyme catalyze cleavage DNA in some sequence of DNA.Various restriction enzyme used herein is commercial obtainable and their reaction conditions, and cofactor and other requirement are that the general technology skilled person is known.For analysis purposes, usually 1 μ g plasmid or the dna fragmentation enzyme with 2 units is used in about 20 μ l buffer solns.For the DNA isolation fragment makes up plasmid, common 5 to 50 μ g DNA digest with the enzyme of 20 to 250 units in comparatively large vol.Suitable damping fluid and amount of substrate for specific restriction enzyme are by manufacturers's special stipulation.The general use 1 hour of incubation time, 37 ℃, but can change according to supplier's explanation.After the digestion, directly coagulate on the amine electrophoresis reactant to separate needed fragment at polyacrylamide.
Utilize 8 percent polyacrylamide gel to carry out the size separation of crack fragment, be described in Goeddel, D, et al., Nucleic Acids Res., 8:4-057 (1980).
" oligonucleotide " refer to can chemosynthesis the poly-deoxy polynucleotide of strand or two complementary deoxy polynucleotide chains.This synthetic oligonucleotide does not have 5 ' phosphoric acid, just can not be connected with another oligonucleotide with phosphoric acid so do not add ATP when having kinases.The synthetic oligonucleotide will be connected to not to be had on the dephosphorylized fragment.
" connection " be meant between two double stranded nucleic acid fragments the process that forms phosphodiester bond (Maniatis, T., et al., Id., P.146).Unless provide in addition, utilize known damping fluid and condition, the dna fragmentation to be connected of about equimolar amount of per 0.5 μ g is with the T of 10 units 4Dna ligase can be finished connection.
Unless otherwise stated, conversion will be as Graham, F. and Van der Eb, and A., Virology, the method for 52:456-457 (1973) is carried out.
Bacterial expression and the purifying of embodiment 1 TNF-γ
Utilization corresponding to TNF-γ proteinic 5 '-sequence and TNF-γ gene 3 ' the PCR Oligonucleolide primers dna sequence dna of amplification coding TNF-γ at first of carrier sequence, ATCC#75927.To be added to 5 corresponding to the Nucleotide of TNF-γ separately ' and 3 ' sequence on.5 ' Oligonucleolide primers sequence is 5 '-GCGCGGATCCACCATGAGACGCTTTTTAAGCAAAGTC-3 ', it contains a Bam HI restriction enzyme sites, then and then starts from 24 Nucleotide of TNF-γ encoding sequence beginning of the end amino acid of the processed albumen codon supposed.3 ' sequence is 5 '-CGCGTCTAGACTATAGTAAGAAGGCTCCAAAGAAGG-3 ', it contains the complementary sequence in xbaI site, and after connect the Nucleotide of 22 TNF-γ and be positioned at the complementary sequence of 3 ' pQE-99 carrier sequence of TNF-γ DNA inset.Restriction enzyme sites is corresponding to the restriction enzyme sites among the bacterial expression vector pQE-9 (Qiagen).Then with Bam HI and XbaI digestion pQE-9.With extension increasing sequence connect enter pQE-9 and insert sequence with encoding histidine sign and the framework of RBS in.Use Sambrook then, J, et al., the molecular cloning laboratory manual, the method for Cold Spring Laboratory Press (1989) narration will connect mixture and transform the E.coli bacterial strain of buying with trade mark M15/rep4 from Qiagen.M15/rep4 contains a plurality of copies of plasmid pREP4, and plasmid pREP4 expresses the lacI repressor and has kalamycin resistance (Kan r).Identify transformant and screening penbritin/kalamycin resistance bacterium colony by its energy for growth on the LB flat board.Isolated plasmid dna is also verified by restriction analysis.Contain being cloned in of required construct and replenished grow overnight (O/N) in the LB liquid nutrient medium of Amp (100 μ g/ml) and Kan (25 μ g/ml).The O/N culture is used to inoculate a large-scale culture, and ratio is 1: 100 to 1: 250.Cell grows into optical density(OD) 600 (0.D. 600) be between 0.4 and 0.6.Add IPTG (" sec.-propyl-β-D-thio-galactose pyran-glucoside ") then to final concentration 1mM.IPTG induces and makes lacI repressor inactivation, removes P/O, causes improving genetic expression.Centrifugal cell harvesting is passed through in long 3 to 4 hours of cell regeneration then.Cell precipitation is dissolved in the 6 mole hydrochloride guanidine chaotropic agents.After the clarification, under the condition of the protein mortise that allows to contain the 6-His sign by chromatography on a nickel chelate column dissolved TNF-γ purifying from this solution is come out (Hochuli, E.et al., chromatography magazine 411:177-184 (1984)).By being further purified TNF-γ by the nickel chelate column for the second time.With 6 mole hydrochloride guanidine pH5.0 wash-out TNF-γ (90% purity) and for renaturation from post, in the PBS damping fluid, dialyse.By SDS-PAGE electrophoresis expression product, the visible Fig. 4 of result, wherein M is a molecular weight marker; The 1st swimming lane is the inductive cell lysates, and the 2nd swimming lane is an inductive cell lysates not; The 3rd swimming lane is the twice TNF-γ protein behind the nickel chelate column purifying; The 4th swimming lane is 1 the TNF-γ protein behind the column purification.
Embodiment 2 utilizes the baculovirus expression system clone and expresses TNF-γ
Utilization is corresponding to 5 of described gene ' and the PCR Oligonucleolide primers of 3 ' sequence, the coding total length that increased TNF-γ protein DNA sequence, ATCC#75927.
5 ' primer have sequence 5 '-GCGCGGATCCACC ATGAGACGCTTTTTAAGCAAAGTC-3 ' also contains a BamHI restriction enzyme sites (black matrix), and the back is the Nucleotide of 24 TNF-γ genes (line under the translation initiation codon " ATG ") then.
3 ' primer have sequence 5 '-CGCGTCTAGACTATAGTAAGAAGGCTCCAAAGAAGG-3 ' and contain the cleavage site of limiting acid endo enzyme XbaI and 22 be complementary to TNF-γ gene 3 '-Nucleotide of non-translated sequence.Utilization is purchased test kit, and (" Geneclean " BIO 101 Inc., La Jolla Ca.) separate the sequence that increases from 1% sepharose.Digest this fragment with restriction endonuclease BamHI and XbaI then, then another purifying on 1% sepharose.This fragment is named as F2.
Utilize baculovirus expression system, carrier pA2 (the pVL941 carrier of modification, discuss below) (relevant summary is referring to Summers to be used for TNF-γ protein expression, M.D. with Smith G.E, 1987, the method handbook of baculovirus vector and insect cell culture procedure, Texas agricultural experiment centre bulletin No.1555).This expression vector contains the strong polyhedron promotor of autographa california nuclear polyhedrosis virus (Ac MNPV), and the recognition site of restriction enzyme BamHI and XbaI is followed in the back.The poly-adenosine site of simian virus (SV) 40 is used to effectively poly-adenosine and turns usefulness into.For recombinant celo virus easily, to insert with the direction in the polyhedron promotor equally from colibacillary beta-galactosidase gene, the poly-adenosine signal of polyhedrosis gene is followed in polyhedron promotor back.In order to carry out the homologous recombination of cell-mediated cotransfection wild-type virus DNA, virus sequence is followed in polyhedron sequence both sides.Many other baculovirus vectors can be used to replace pA2, as pRG1, and pAc373, pVL941 and pAcIM1 (Luckow, V.A and Summers, M.D., virusology, 170:31-39).
With restriction enzyme BamHI and XbaI digested plasmid, use calf intestinal phosphatase enzyme and program dephosphorylation well known by persons skilled in the art then.(" Geneclean " BIO 101Inc., La Jolla Ca.) separate this DNA from 10% sepharose to utilize the test kit that is purchased then.This carrier DNA is named as V2.
With T4DNA ligase enzyme junction fragment F2 and dephosphorylated plasmid V2.Transformed into escherichia coli XL1 large cortical cells then.Determined the sequence of cloned sequence by dna sequence analysis.
Utilize fat transfection method (Felgner et al.Proc Natl.Acad.Sci.USA, 84:7413-7417 (1987)) that 1.0 μ g commerce can be obtained linearized baculovirus (" BaculoGold TMBaculovirus DNA ", Pharmingen, San Diego is CA.) with 5 μ g plasmid pBacTNF-γ cotransfections.
Contain the serum-free Grace ' S substratum of 50 μ l (Life Technologies Inc., Gaithersburg, in the aseptic hole of droplet plate MD) with the BaculoGold of 1 μ g TMThe plasmid pBacTNF-γ of viral DNA and 5 μ g mixes.Afterwards 10 μ l Lipofectin and 90 μ l Grace ' s substratum are added, mix and incubation 15 minutes at room temperature.Then, transfection mixture is added dropwise on the Sf9 insect cell (ATCC CRL 1711) of planting in having the 35mm tissue culturing plate of 1ml Grace ' serum free medium.With jog before and after the flat board, so that initiate solution is mixed.Dull and stereotyped then 27 ℃ of incubations 5 hours.After 5 hours, from flat board, remove transfection solution, add the 1ml Grace ' s insect substratum that has replenished 10% foetal calf serum.Flat board is put back to an incubator and is continued to cultivate 4 days at 27 ℃.
After 4 days, collect supernatant liquor, and as described in Summers and Smith (above-mentioned), carry out plaque measurement.As a modification protocols, used and had " Blue Gal " that (it can make dyes blue plaque and be easy to separate for LifeTechnologies Inc., sepharose Gaithersburg).(detailed description of " plaque test " also can be referring to Life Technolgies Inc., and the insect cell of Gaithesburg distribution is cultivated and baculovirus is learned user's guidance, 9-10 page or leaf).
After the serial dilution four days, virus is added cell, choose with the tip of an Eppendorf suction pipe and dye blue plaque.The agar that will contain recombinant virus then suspends in an Eppendorf pipe that contains 200ml Grace ' s substratum again.The centrifugal in short-term agar of removing, the supernatant liquor that contains recombinant baculovirus is used for infecting the Sf9 cell that is seeded in the 35mm ware.After four days, collect the clear liquid in these culture dish, be stored in 4 ℃ then.
The Sf9 cell grows in the Grace ' s substratum that has replenished 10% heat inactivation FBS.Is 2 cells infecteds with recombinant baculovirus V-TNF-γ with infection multiplicity (MI).Remove substratum after six hours and add again the SF900II substratum do not have methionine(Met) and halfcystine (LifeTechnologies Inc., Gaithersburg).Add 5 μ Ci's after 42 hours 35S-methionine(Met) and 5 μ Ci's 35S halfcystine (Amersham).The further incubation of cell 16 hours, centrifugal then collection is by SDS-PAGE and radioautograph witness marking protein.Fig. 5 has shown gel, and wherein swimming lane 1 and 3 is TNF-γ substratum and contrast, and swimming lane 2 and 4 is TNF-gamma cells lysate and contrast.
Embodiment 3 expression of reorganization TNF-γ in the COS cell
Expression plasmid, TNF-γ HA derives from carrier pcDNAI/Amp (Invitrogen), it contains 1) SV40 duplicates source point, 2) ampicillin resistance gene, 3) intestinal bacteria are duplicated source point, 4) the CMV promotor, the back is a polylinker district then, a SV40 intron and poly-adenosine site.The dna fragmentation of the HA sign of coding total length TNF-γ precursor and merge 3 ' terminal is entered the polylinker district of this carrier by the clone, so Recombinant Protein Expression is carried out under the CMV promotor instructs.HA sign is corresponding to by an epi-position of the influenza hemagglutinin protein derived of previous narration (A.Cherenson, M.Connolly, and R.Lerner, 1984, Cell 37,767 for I.Wilson H.Niman, R.Heighten).Incorporate the HA sign in our target protein, making can be easily with the antibody test recombinant protein of discerning the HA epi-position.
The plasmid construction scheme is described below:
On the original EST that clones with two primers, make up the dna sequence dna of coding TNF-γ by PCR, ATCC#75927, two primers are: 5 ' primer (with among the baculovirus embodiment identical) contains a BamHI site, and the back and then starts from 24 Nucleotide of the TNF-γ encoding sequence of initiation codon; 3 ' sequence 5 '-CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGATAGTAAGAAGGCTCCAAAG-3 ' contains and XbaI site complementary sequence, rotaring inter-transtating terminating cipher, HA sign and last 18 Nucleotide (not comprising terminator codon) of TNF-γ encoding sequence.So the PCR product contains a BamHI site, TNF-γ encoding sequence, the HA sign in framework is then merged in the back, in abutting connection with translation stop codon and XbaI site of HA sign.With the dna fragmentation and the carrier pcDNAI/Amp of BamHI and XbaI restriction enzyme digestion pcr amplification, and connect.To connect mixture is entered coli strain SURE by conversion and (obtains from StratageneCloning Systems, 11099North Torrey Pines Road, La Jolla CA 92037), will transform culture and be layered on the ampicillin medium flat board and cultivate, filter out the resistance bacterium colony.Isolated plasmid dna and detect the existence of suitable fragments by restriction analysis from transformant.For express recombinant TNF-γ, with expression vector and DEAE-DEXTRAN method rotaring redyeing COS cell (J.Sambrook, E.Fritsch, T.Maniatis, molecular cloning laboratory manual, Cold Spring Laboratory Press, (1989)).Detect TNF-γ HA protein expression (E.Harlaw, D.Lane, antibody laboratory manual, Cold Spring Harbour Laboratory Press, (1989)) by radio-labeled and immunoprecipitation method.Transfection is used two days later 35S-halfcystine labeled cell 8 hours.Collect substratum then, with stain remover (RIPA damping fluid (150mm NaCl, 1%NP-40,0.1%SDS, 1%NP-40,0.5%DOC, 50mM Tris, PH7.5) dissolved cell.(Wilson，I.et?al.，Id.37：767(1984))。Cell lysates and substratum precipitate with the HA monoclonal antibody specific.On the 15%SDS-PAGE gel, analyze sedimentary protein.
The phraseology of embodiment 4 TNF-γ in people's tissue
Carry out rna blot analysis and check the expression level of TNF-γ in people's tissue.Use RNAzol TMB system (TX 77033 for Biotecx Laboratories Inc.6023 South Loop East, Houston) separates total cell RNA sample.On 1% agarose-formaldehyde gel, separate the total RNA of isolating about 2 μ g (the RNA trace by Fig. 3 A draws) from each specific people's tissue and xerox (Sambrook on a nylon leaching film, Fritsch, and Maniatis, molecular cloning, Cold SpringHarbar Press, (1989)).According to Stratagen Prime-It test kit, carry out labeled reactant to produce with 50ngTNF-γ cDNA 32The TNF-γ cDNA of P-mark.DNA with Select-G-50 post (5603Arapahoe Road, Boulder, CO 80303 for 5Prime-3Prime, Inc) purifying mark.At 0.5M NaPO 4With 1,000, radiolabeled total length TNF-γ gene and the filter membrane of 000cpm/ml spend the night 65 ℃ of hybridization among pH7.4 and the 7%SDS.Use 0.5XSSC, 0.1%SDS at room temperature and at 60 ℃ respectively washes after twice, filter membrane is placed under-70 ℃ of intensifying screens and spends the night.(Fig. 3 A) has the mRNA of abundant TNF-γ in kidney.
Carry out same reaction and obtain the result and be presented at Fig. 3 B, unique variation is the 10 μ g poly A RNA that are used to from specified.The mRNA of TNF-γ mainly expresses (Fig. 3 B) in the HUVEC cell.
Embodiment 5 reorganization TNF-γ suppress WEHI164 and the growth of L929 cell, induce the ability of HL-60 cytoadherence and promotion endothelial cell growth
From the culture that is paved with, prepare the target cell of adhesion by trysinization in PBS, results adhesion target cell and wash once from static culture with substratum.Target cell is with 3 * 10 5Cell/ml is suspended in the substratum that contains 10%FCS.Distribute the 0.1ml aliquots containig in 96 hole flat-bottom microtiter plates of the cell that contains the 0.1ml serial dilution (WEHI164 and L929) sample.Incubation continues 70 hours.Concentration with 0.5 μ g/ml adds TNF-α, TNF-β, TNF-γ.Utilize MTS test detection by quantitative cytotoxicity and proliferation activity, the MTS test is by adding 20 μ l MTS and Phenelzine Methylsulfate (PMS) solution carries out in cell.After 3 hours incubation, measure the OD value at 492nm place with the ELISA plate reader.OD 492Proportional with survival cells in the hole.Cytotoxic per-cent is pressed following calculating: % cytotoxicity=(100-OC Experiment/ OD Contrast* 100.Take photo after 72 hours, as Fig. 6 A and shown in Figure 8, TNF-γ has induced morphological change, and described variation shows that with black circle cell this is killed cell.
In the photo in Fig. 6 B, test is to carry out as mentioned above, still, adds the TNF amount that increases.The result shows that TNF-γ is a WEHI164 cell inhibiting thing.
In order to measure the adhesion ability of TNF-γ, used the HL-60 cell, measure contacting of cytoadherence and cell-cell by examining under a microscope, and subjectively by two independently researchist's evaluations.Figure 10 has illustrated the ability of TNF-γ inducing cell adhesion.
In the test of the ability of measuring TNF-γ promotion endothelial cell growth, proliferation index (PI) following calculating: PI=OD Experiment/ OD ContrastFig. 8 illustrates that TNF-γ is the promotor of endothelial cell growth.
The mensuration of the apoptosis ability of embodiment 6 TNF-γ
In first incubation step, histonic antibody absorption is fixed on the wall of microtitration flat board.Subsequently, by handle the non-specific combination site on the saturated wall with incubation buffering liquid (that is lock solution).In second incubation step, use TNF-α, the nucleosome that contains in the WEHI104 cell sample that TNF-β or TNF-γ handle is incorporated on the fixed histonic antibody by their histone composition.In the 3rd incubation step, the DNA partial reaction of anti-DNA peroxidase (POD) and nucleosome.Remove all unconjugated peroxidase conjugated things by washing step after, with ABTS (2,2 '-azine-two [3-ethylphenyl thiazole sulfonic acid] is as substrate, with the amount of the peroxidase of the reservation in the photometric determination immunocomplex.Histone HI in histonic antibody and the sample, H2A, H2B, H3 and H4 reaction.Anti-DNA PQD antibody combines with strand and double-stranded DNA.So ELISA can measure mononucleosome and oligoneucleosomes and can be applied to measure apoptosis.By level (referring to Boehringer manheim Catalogue, 0990C 93 2 1541170) (referring to Fig. 7) indication by the A405nm/A490 absorption value and flow measurement necrocytosis tenuigenin histone bonded dna fragmentation.
Embodiment 7 utilizes the receptors bind of TNF-γ to measure
Utilize 6-His sign to carry out Ni-NTA affinitive layer purification TNF-α and TNF-γ, and add 96 hole flat board (XenopokeCorp) incubations 2 hours of 1 μ g/ hole to a Ni chelating bag quilt.After giving a baby a bath on the third day after its birth time, in every hole, added 100ng people solvable TNF acceptor sTNF RI or sTNF RII and incubation 2 hours.Wash the polyclonal antibody (200 μ l) that adds the alkali phosphatase enzyme mark of anti-sTNFRI or sTNF RII behind the plate three times.In each hole, add 200 μ l substrate solutions, dull and stereotyped 2 hours of incubation.Utilize the ELISA reader to measure OD value (test wavelength 450nm, tuning wavelength 590nm).The result of Figure 11 shows that TNF-γ does not have obvious the combination with the sTNF acceptor.
Can carry out a large amount of modifications and variation to the present invention according to above-mentioned instruction, so, its also within the scope of the appended claims, the present invention can be different from above-described mode and implement.
Sequence table
(1) general information:
(i) applicant: Yu, ETAL.
(ii) denomination of invention: human tumor necrosis factor-γ
(iii) sequence number: 2
(iv) address:
(A) contact person: CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI STEWART ﹠amp; OLSTEIN
(B) street: 6BECKER FARM ROAD
(C) city: ROSELAND
(D) state: New Jersey
(E) country: the U.S.
(F) postcode: 07068
(v) computer-reader form:
(A) medium type: 3.5 inches dishes
(B) computer: IBM PS/2
(C) operating system: MS-DOS
(D) software: WORD PERFECT 5.1
(vi) present application materials:
(A) application number:
(B) applying date:
(C) classification number:
(vii) application materials formerly:
(A) application number:
(B) applying date:
(viii) proxy/lawyer's data:
(A) name: FERRARO, GREGORY D
(B) registration number: 36,134
(C) reference/file number: 325800-256
(ix) telecommunications situation:
(A) phone: 201-994-1700
(B) fax: 201-994-1744
(2) information of SEQ ID NO:1:
(i) sequence signature:
(A) length: 2442 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: SEQ ID NO:1
CCCAATCAAG?AGAAATTCCA?TACTATCACC?AGTTGGCCGA?CTTTCCAAGT?CTAGTGCAGA 60
AATCCAAGGC?ACCTCACACC?TAGAGTTCCT?ATACCTCTGA?GACTCCAGAG?GAAAGAACAA 120
GACAGTGCAG?AAGGATATGT?TAGAACCCAC?TGAAAACCTA?GAAGGTTGAA?AAGGAAGCAT 180
ACCCTCCTGA?CCTATAAGAA?AATTTTCAGT?CTGCAGGGGG?ATATCCTTGT?GGCCCAAGAC 240
ATTGGTGTTA?TCATTTGACT?AAGAGGAAAT?TATTTGTGGT?GAGCTCTGAG?TGAGGATTAG 300
GACCAGGGAG?ATGCCAAGTT?TCTATCACTT?ACCTCATGCC?TGTAAGACAA?GTGTTTTGTT 360
CCAATTGATG?AATGGGGAGA?AAACAGTTCA?GCCAATCACT?TATGGGCACA?GAATGGAATT 420
TGAAGGGTCT?GGTGCCTGCC?CTTGTCATAC?GTAAACAAGA?GAGGCATCGA?TGAGTTTTAT 480
CTGAGTCATT?TGGGAAAGGA?TAATTCTTGC?ACCAAGCCAT?TTTCCTAAAC?ACAGAAGAAT 540
AGGGGGATTC?CTTAACCTTC?ATTGTTCTCC?AGGATCATAG?GTCTCAGGAT?AAATTAAAAA 600
TTTTCAGGTC?AGACCACTCA?GTCTCAGAAA?GGCAAAGTAA?TTTGCCCCAG?GTCACTAGTC 660
CAAGATGTTA?TTCTCTTTGA?ACAAATGTGT?ATGTCCAGTC?ACATATTCTT?CATTCATTCC 720
TCCCCAAAGC?AGTTTTTAGC?TGTTAGGTAT?ATTCGATCAC?TTTAGTCTAT?TTTGAAAATG 780
ATATGAGACG?CTTTTTAAGC?AAAGTCTACA?GTTTCCCAAT?GAGAAAATTA?ATCCTCTTTC 840
TTGTCTTTCC?AGTTGTGAGA?CAAACTCCCA?CACAGCACTT?TAAAAATCAG?TTCCCAGCTC 900
TGCACTGGGA?ACTAGAACTA?GGCCTGGCCT?TCACCAAGAA?CCGAATGAAC?TATACCAACA 960
AATTCCTGCT?GATCCCAGAG?TCGGGAGACT?ACTTCATTTA?CTCCCAGGTC?ACATTCCGTG 1020
GGATGACCTC?TGAGTGCAGT?GAAATCAGAC?AAGCAGGCCG?ACCAAACAAG?CCAGACTCCA 1080
TCACTGTGGT?CATCACCAAG?GTAACAGACA?GCTACCCTGA?GCCAACCCAG?CTCCTCATGG 1140
GGACCAAGTC?TGTATGCGAA?GTAGGTAGCA?ACTGGTTCCA?GCCCATCTAC?CTCGGAGCCA 1200
TGTTCTCCTT?GCAAGAAGGG?GACAAGCTAA?TGGTGAACGT?CAGTGACATC?TCTTTGGTGG 1260
ATTACACAAA?AGAAGATAAA?ACCTTCTTTG?GAGCCTTCTT?ACTATAGGAG?GAGAGCAAAT 1320
ATCATTATAT?GAAAGTCCTC?TGCCACCGAG?TTCCTAATTT?TCTTTGTTCA?AATGTAATTA 1380
TAACCAGGGG?TTTTCTTGGG?GCCGGGAGTA?GGGGGCATTC?CACAGGGACA?ACGGTTTAGC 1440
TATGAAATTT?GGGGCCAAAA?TTTCACACTT?CATGTGCCTT?ACTGATGAGA?GTACTAACTG 1500
GAAAAAGGCT?GAAGAGAGCA?AATATATTAT?TAAGATGGGT?TGGAGGATTG?GCGAGTTTCT 1560
AAATATTAAG?ACACTGATCA?CTAAATGAAT?GGATGATCTA?CTCGGGTCAG?GATTGAAAGA 1620
GAAATATTTC?AACACCTCCC?TGCTATACAA?TGGTCACCAG?TGGTCCAGTT?ATTGTTCAAT 1680
TTGATCATAA?ATTTGCTTCA?ATTCAGGAGC?TTTGAAGGAA?GTCCAAGGAA?AGCTCTAGAA 1740
AACAGTATAA?ACTTTCAGAG?GCAAAATCCT?TCACCAATTT?TTCCACATAC?TTTCATGCCT 1800
TGCCTAAAAA?AAATGAAAAG?AGAGTTGGTA?TGTCTCATGA?ATGTTCACAC?AGAAGGAGTT 1860
GGTTTTCATG?TCATCTACAG?CATATGAGAA?AAGCTACCTT?TCTTTTGATT?ATGTACACAG 1920
ATATCTAAAT?AAGGAAGTTT?GAGTTTCACA?TGTATATCCC?AAATACAACA?GTTGCTTGTA 1980
TTCAGTAGAG?TTTTCTTGCC?CACCTATTTT?GTGCTGGGTT?CTACCTTAAC?CCAGAAGACA 2040
CTATGAAAAA?CAAGACAGAC?TCCACTCAAA?ATTTATATGA?ACACCACTAG?ATACTTCCTG 2100
ATCAAACATC?AGTCAACATA?CTCTAAAGAA?TAACTCCAAG?TCTTGGCCAG?GCGCAGTGGC 2160
TCACACCTGT?AATCCCAACA?CTTTGGGAGG?CCAAGGTGGG?TGGATCATCT?AAGGCCGGGA 2220
GTTCAAGACC?AGCCTGACCA?ACGTGGAGAA?ACCCCATCTC?TACTNAAAAT?ACNAAATTAG 2280
CCGGGCGTGG?TAGCGCATGG?CTGTAANCCT?GGCTACTCAG?GAGGCCGAGG?CAGAANAATT 2340
NCTTGAACTG?GGGAGGCAGA?GGTTGCGGTG?AGCCCAGANC?GCGCCATTGC?ACTCCAGCCT 2400
GGGTAACAAG?AGCAAAACTC?TGTCCAAAAA?AAAAAAAAAA?AA 2442
(2) information of SEQ ID NO:2:
(i) sequence signature
(A) length: 174 amino acid
(B) type: amino acid
(C) chain:
(D) topological structure: linearity
(ii) molecule type: protein
(xi) sequence description: SEQ ID NO:2
Met?Arg?Arg?Phe?Leu?Ser?Lys?Val?Tyr?Ser?Phe?Pro?Met?Arg?Lys
-25 -20 -15
Leu?Ile?Leu?Phe?Leu?Val?Phe?Pro?Val?Val?Arg?Gln?Thr?Pro?Thr
-10 -5 1 5
Gln?His?Phe?Lys?Asn?Gln?Phe?Pro?Ala?Leu?His?Trp?Gly?His?Glu
10 15 20
Leu?Gly?Leu?Ala?Phe?Thr?Lys?Asn?Arg?Met?Asn?Tyr?Thr?Asn?Lys
25 30 35
Phe?Leu?Leu?Ile?Pro?Glu?Ser?Gly?Asp?Tyr?Phe?Ile?Tyr?Ser?Gln
40 45 50
Val?Thr?Phe?Arg?Gly?Met?Thr?Ser?Glu?Cys?Ser?Glu?Ile?Arg?Gln
55 60 65
Ala?Gly?Arg?Pro?Asn?Lys?Pro?Asp?Ser?Ile?Thr?Val?Val?Ile?Thr
70 75 80
Lys?Val?Thr?Asp?SEr?Tyr?Pro?Glu?PRo?Thr?Gln?Leu?Leu?Met?Gly
85 90 95
Thr?Lys?Ser?Val?Cys?Gln?Val?Gly?Ser?Asn?Trp?Phe?Gln?Pro?Ile
100 105 110
Tyr?Leu?Gly?Ala?Met?Phr?Ser?Lau?Gln?Glu?Gly?Asp?Lys?Leu?Met
115 120 125
Val?Asn?Val?Ser?Asp?Ile?Ser?Leu?Val?Asp?Tyr?Thr?Lys?Glu?Asp
130 135 140
Lys?Thr?Phe?Phe?Gly?Ala?Phe?Leu?Leu
145

Claims (23)

1. isolating polynucleotide are selected from following one group:
(a) polypeptide formed by the aminoacid sequence of the deduction of Fig. 1 of coding or the segmental polynucleotide with TNF-gamma activity of said polypeptide,
(b) polypeptide formed of the cDNA amino acid sequence coded that comprises by ATCC preserving number 75927 of coding or the segmental polynucleotide with TNF-gamma activity of said polypeptide.
2. polynucleotide according to claim 1, wherein polynucleotide are DNA.
3. polynucleotide according to claim 1, wherein polynucleotide are RNA.
4. polynucleotide according to claim 1, wherein polynucleotide are genomic dna.
5. polynucleotide according to claim 2, the polypeptide that wherein said polynucleotide encoding is made up of the aminoacid sequence of the deduction of Fig. 1.
6. polynucleotide according to claim 2, wherein said polynucleotide encoding is by the cDNA encoded polypeptides of ATCC preserving number 75927.
7. polynucleotide according to claim 1, it is made up of the encoding sequence of polypeptide shown in Figure 1.
8. polynucleotide according to claim 2, it is made up of the encoding sequence with the polypeptide of ATCC preserving number 75927 preservations.
9. carrier that contains the described DNA of claim 2.
10. one kind with the genetically engineered host cell of the described carrier of claim 9.
11. a method that produces polypeptide comprises: the polypeptide of the said dna encoding of host cell expression of Accessory Right requirement 10.
12. the method for the cell that a generation can express polypeptide comprises the genetically engineered cell of carrier with claim 9.
13. the DNA with claim 2 has at least 90% homogeny and coding to have the separated DNA of the polypeptide of TNF-gamma activity.
A 14. peptide species, be selected from polypeptide that following one group (i) be made up of the amino acid whose sequence of the deduction of Fig. 1 and said polypeptide a kind of TNF-of having gamma activity fragment and (ii) by the fragment of a kind of TNF-of having gamma activity of the cDNA encoded polypeptides of ATCC preserving number 75927 and said polypeptide.
15. polypeptide according to claim 14, wherein said polypeptide is made up of the aminoacid sequence of the deduction of Fig. 1.
16. the antibody of the described polypeptide of anti-claim 14.
17. the antagonist of right 14 described polypeptide, wherein said antagonist is an antibody.
18. the polypeptide of claim 14 is used for the treatment of application in the patient's who needs TNF-γ the medicine in preparation.
19. the antagonist of claim 17 is used for the treatment of application in patient's the medicine that needs suppress TNF-γ in preparation.
20. the DNA of the polypeptide of coding claim 14 is used for the treatment of application in the patient's who needs TNF-γ the medicine in preparation.
21. a method of differentiating the stimulant and the antagonist of the described polypeptide of claim 14 comprises:
When existing or lack TNF-γ selectively with endotheliocyte, Concavilin-A, compound to be screened, [ 3H] the thymus pyrimidine combination;
Measure that endotheliocyte mixes [ 3H] amount of thymus pyrimidine; With
Determine this compound whether strengthen or stop [ 3H] the mixing of thymus pyrimidine.
22. a pharmaceutical composition that suppresses growth of tumour cell in patient comprises: the TNF-γ protein of treatment significant quantity and pharmaceutical acceptable carrier randomly.
23. the polynucleotide of claim 1 are used for the application of the reagent of diagnosing tumour or tumor susceptibility in preparation.
CNB941952118A 1994-11-07 1994-11-07 Tumor necrosis factor-Gamma Expired - Fee Related CN1304410C (en)

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CN1304410C true CN1304410C (en) 2007-03-14

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