CN1173033C - Modulator of TNF/NGF superfamily receptors and soluble oligomeric TNF/NGF superfamily receptors - Google Patents

Abstract

The present invention generally concerns novel proteins which bind to the intracellular domains of the p55 and p75 TNF-Rs and the Fas-R, which are capable of modulating the function of the p55 and p75 TNF-Rs and the Fas-R, and the DNA sequences which encode them. The present invention also concerns new soluble oligomeric TNF-Rs, oligomeric Fas-Rs and oligomeric receptors having a mixture of TNF-Rs and Fas-Rs. In addition, the present invention concerns methods of preparation and uses of all of the aforementioned.

Description

The conditioning agent of TNF/NGF superfamily receptors and soluble oligomeric TNF/NGF superfamily receptors

Invention field

All things considered of the present invention relates to some acceptors in the TNF/NGF receptor superfamily, and the adjusting of biological function.The TNF/NGF receptor superfamily comprises some acceptors, for example P55 and P75 Tumor Necrosis Factor Receptors (TNF-Rs) and FAS aglucon acceptor (also be called FAS/APOI or FAS-R, below be called FAS-R) etc. like this.More particularly, the present invention relates to some novel proteins, they can be incorporated into the intracellular region (IC) (these intracellular regions are called P55IC respectively, P75IC and Fas-IC) of P55 and P75TNF-Rs and Fas-R, and these novel proteins can be regulated the function of P55 and P75TNF-Rs and Fas-R.Can be the part of P55IC itself or this molecule wherein, for example be called " dead functional domain " (death domain is abbreviated as DD) of P55IC in conjunction with a kind of protein of the P55IC of whole P55-TNF-R.Thereby, the invention still further relates to a kind of novel TNF correlation effect, this effect can be induced generation in the mode that does not rely on aglucon (TNF) by P55TNF-R or its segmental intracellular region (P55IC) in cell.The invention still further relates to the preparation and the purposes of these novel proteins, comprise P55 and P75TNF-R conjugated protein and FAS-R conjugated protein, be called P55IC conjugated protein, P75IC conjugated protein and FAS-IC conjugated protein herein.

On the other hand, the invention still further relates to novel soluble oligomerization (oligomeric) TNF-Rs, oligomerization FAS-Rs and contain TNF-Rs and the oligomerization acceptor of FAS-Rs mixture, their purposes and production method.

Background of invention and prior art

Tumour necrosis factor (TNF-α) and lymphotoxin (TNF-β) (below mention TNF be meant TNF-α and TNF-β) mainly are by the formed multi-functional inflammatory cells kinetin of mononuclear macrophage former (pro-inflammatory cytokine), its pair cell has multiple effect (Wallach, D. (1986)): Interferon 7 (Ion Gresser volume), PP83-122, Academic press, London; And Beutler and Cerami (1987)).TNF-α and TNF-β bring into play their effect by being incorporated into the specific cells surface receptor.Perhaps, their some effect is useful to organism: for example they can eliminate the cell of tumour cell and virus infection, and increase granulocytic anti-microbial activity.Like this, TNF just can have effect to the organism defence system of opposing tumour and infectious agent, and the recovery to damage simultaneously also has effect.Therefore, TNF can be used as antineoplastic agent, in this uses TNF be incorporated on the tumor cell surface acceptor and with this and the lip-deep acceptor of triggering tumor cell and cause the death of tumour cell with this.TNF can also be used as anti-infection agent.

Yet TNF-α and TNF-β also have deleterious effects.Verified, too much synthetic TNF-α can play main pathogenic effects to several diseases.TNF-α is to the effect of vascular system, and now known is the major cause (Tracey etc., 1996) that causes sepsis shock disease.In some disease, TNF can and cause that apositia causes over-drastic to lose weight (emaciation) by inhibition lipocyte activity, thereby TNF-α is called cachectin.TNF also is described to tissue necrosis regulatory factor in the rheumatism (Beutler and Cerami, 1987), and the main regulatory factors (Piquet etc., 1987) of the untoward reaction that is occurred in transplant antagonism host response.In addition, known TNF is relevant with many other diseases with inflammation.

Whether or not acceptor different, independent statement for two kinds, P55TNF-Rs and P75TNF-Rs can be single-mindedly in conjunction with TNF-α and TNF-β, and cause and/or mediate the biological effect of above-mentioned TNF.These two kinds of acceptors have intracellular regions different on the structure, so the signal mode of action is also different, (referring to Hohmann etc., 1989; Engelmann etc., 1990; Brockhaus etc., 1990; Leotscher etc., 1990; Schall etc., 1990; Nophar etc., 1990; Smith etc., 1990; And Heller etc., 1990).But its cytologic mechanism, for example relate to the various related proteins of intracellular signal effect of P55 and P75TNF-Rs and other possible factor, wait to illustrate that (what propose below is to describe first, this novel protein that can be incorporated into P75IC and P55IC is this intracellular signal effect, normally betiding aglucon is that TNF (α or β) is incorporated into after the acceptor, reaction is amplified in the cascade that causes, and finally causes the reaction of cell to TNF.

About the cytocidal effect (cytocidal effect) of above-mentioned TNF, in the most cells of being studied so far, this effect should be triggered by P55TNF-R.The antibody of the extracellular region of anti-P55TNF-R (ligand-binding domain), itself can trigger cytocidal effect (referring to EP412486), thereby cytocidal effect is that validity with antibody and receptors bind interrelates, and is considered to produce the first step of intracellular signal mechanism.In addition, mutation research (Brakebusch etc., 1992; Tartaglia etc., 1993) show, therefore the biological function of P55TNF-R is decided by the integrity of its intracellular region, and having the people to propose starting owing to intracellular signal, to cause the cytocidal effect of TNF be the result of two or more intracellular region generation associations of P55TNF-R.And, the appearance of TNF (α or β) is as a kind of homotrimer (homotrimer), and, be incorporated into acceptor molecule and congregation (aggregation) crosslinked with it that is that cause acceptor induces intracellular signal to take place by P55TNF-R as proposed.Ubi infra P55IC and P55DD be how self-association and with in the mode inducing cell that does not rely on aglucon with the TNF related effect.

Another member of TNF/NGF receptor superfamily is FAS acceptor (FAS-R), and it also is called Fas antigen, promptly a kind of in various tissues all effable cell surface protein, and with many cell surface receptors, comprise TNF-R and NGF-R, the tool homology.FAS-R is with inclined to one side journey cell death (apoptosis) form mediated cell death (Itoh etc., 1991), and show as the negativity selector (negative selector) of id reaction T cell that is FAS-R mediation can be discerned the T cell of autoantigen in the T cell mature process programmatic death.Find that also the sudden change of FAS-R gene (IPr) can cause mouse lymphocyte hyperplasia illness, this illness is similar to people's autoimmune disease systemic lupus erythematous (SLE) (Watanabe-Fukunage etc., 1992).Seemingly a kind of cell surface binding molecule of the aglucon of FAS-R, it is by killer T cell (or cytotoxic T lymphocyte-CTLs) carry, thereby when such CTLs contact was carrying the cell of FAS-R, they can bring out the cell generation programmatic death that carries FAS-R.Further, prepared a kind of monoclonal antibody, it has specificity to FAS-R, and this monoclonal antibody can be brought out the cell generation programmatic death that carries FAS-R, the apoptosis (Itoh etc., 1991) that comprises the mouse cell that the cDNA by coding people FAS-R transforms.

Find that also except that the T lymphocyte, various other normal cells all can be expressed FAS-R on the surface of oneself, and can be killed by the triggering of this receptor.This immoderate the inducing of killing process may have been played effect to tissue injury, for example liver cell destruction of acute hepatitis in some disease.Thereby the cytotoxic activity that searce way suppresses FAS-R has very big medical potentiality.

On the contrary, owing to also find that some malignant cell and HIV cells infected have carried FAS-R on their surface, so the antibody of anti-FAS-R or FAS-R aglucon can be used to trigger the cytotoxic effect of FAS-R mediation in these cells, and provide a kind of method to resist this malignant cell or HIV cells infected (referring to Itoh etc., 1991) with this.Seek other method to strengthen the cellular cytoxicity activity of FAS-R, also have medical potentiality.

People feel for a long time need provide a kind of approach to regulate cell replying TNF (α or β) and FAS-R aglucon, for example under above-mentioned pathologic condition, TNF or FAS-R aglucon are overexpressions, thereby the cytosis extremely that needs inhibition TNF or FAS-R aglucon to bring out; And in other cases, for example in the wound healing process, need the enhance TNF effect, perhaps in tumour cell or HIV cells infected, need to strengthen the mediation of FAS-R.

The inventor has proposed the deleterious effect that certain methods (for example can referring to European application number: EP186833, EP308378, EP398327 and EP412486) is regulated TNF.For example suppress TNF and be incorporated into its acceptor, perhaps by using soluble TNF acceptor (being the extracellular soluble outskirt of acceptor basically) competitive inhibition TNF to combine with cell surface bonded TNF-Rs's by antibody with anti-TNF.Further, the acceptor that must be incorporated into it according to TNF just can bring out cytological effect, and the inventor has proposed some approach and regulated the TNF effect, promptly by regulating the activity of TNF-Rs.In brief, EP0568925 is signal transduction (signal transduction) about a kind of TNF-Rs of adjusting and/or the method sheared, can regulate the normal function of TNF-Rs with acceptor itself or with effector (effector) (this albumen mass-energy and acceptor interaction) interaction by this method peptide or other molecule.The structure and the evaluation of various P55TNF-Rs mutant (mutant) have been described in EP0568925, these sudden changes occur in P55TNF-R extracellular region, stride film (transmembranal) district and intracellular region.Use this method, the verified function to acceptor of some sections (regions) in the above-mentioned P55TNF-R district is essential, that is to the combination and the signal transduction subsequently of aglucon (TNF) and finally to cause the intracellular signal of TNF effect to be replied all be necessary.Further, also described many separation and identified protein, peptide class or other genic approach that can be incorporated into each section in the above-mentioned TNF-R functional domain, these protein, peptide class and other factor are all relevant with the activity of regulating TNF-R.In EP0568925, also proposed other many methods, comprised how separating and these protein of clones coding and peptide genoid; How to make up and be used to produce these proteic expression vectors; How to prepare these proteic antibody or can with the peptide class fragment of TNF-R effect, also have method prepare can with TNF-R different zones bonded peptide class fragment.Yet, in EP0568925, both do not address the actual protein and the peptide class (for example P55TNF-R) of the intracellular region that can be incorporated into TNF-Rs, do not addressed protein or peptide class how to utilize the two hybrid methods (yeast two-hybrid) of yeast to separate and identify the intracellular region that these can be incorporated into TNF-Rs again.Equally, up to now, to not appearing in the newspapers in conjunction with the protein or the peptide class of FAS-R intracellular region.

Therefore, when the time spent of doing that needs suppress TNF or FAS-R aglucon, with regard to TNF-Rs on the need minimizing cell surface or quantity or the activity of FAS-R; And, just need to increase quantity or the activity of TNF-Rs or FAS-R when the time spent of doing that needs enhance TNF or FAS-R aglucon.For reaching this purpose, the inventor carries out sequencing analysis to the promotor (promoters) of P55TNF-R and P75TNF-R recently, found multiple main key sequential element (sequence motifs), they are to various transcription regulaton factor tool specificitys, find that also being expressed on the promotor level of these TNF-Rs is modulated, that is the quantity and the enhancing promoter transcription of transcribing to reduce acceptor of inhibition promotor increase acceptor quantity (referring to the corresponding part of IL104355 with IL109633 and their corresponding relevant EP that does not deliver and PCT).About the corresponding research of regulation and control on FAS-R gene promoter level, wait report.

Further, also should mention, on the one hand known cancer necrosin (TNF) acceptor, with FAS-R acceptor relevant on the structure, when being subjected to the stimulation of the aglucon that white corpuscle produces, can triggering intracellular lytic activity and cause himself death, yet also know seldom this trigger mechanism.Mutation research points out that in FAS-R and P55TNF acceptor (P55-R), the expression of cytotoxicity signal relates to different sections (Brakebusch etc., 1992 of its intracellular region; Tartaglia etc., 1993; Itoh and Nagata, 1993).These sections, promptly so-called " dead functional domain " (death domain) has the homology of sequence." the dead functional domain " of FAS-R and P55-R is tending towards self and associates.Their self association (self-association) has promoted the congregation (aggregation) of acceptor significantly, and that congregation is the priming signal effect is needed (as described below, and referring to Song etc., 1994; Wallach etc., 1994; Boldin etc., 1995), the acceptor great expression will trigger with the irrelevant signal effect of aglucon and produce (as described below and Boldin etc., 1995).

Therefore, before the present invention, the someone did not provide such protein, can regulate the protein of the effect of the aglucon that belongs to the TNF/NGF superfamily, for example the effect of TNF or FAS-R aglucon pair cell is undertaken by the different intracellular signal mechanism that is situated between, arranged by the intracellular region (ICs) of some acceptors that belong to the TNF/NGF receptor superfamily, the for example intracellular region of TNF-Rs, that is P55 and p75TNF-R intracellular region (being respectively P55IC and P75IC) and FAS-IC.

Thereby, one of purpose of the present invention provides some protein, they can be incorporated into the intracellular region of TNF-Rs and FAS-R, it is relevant with the intracellular signal generating process that these protein are considered to now, and signal is because the acceptor that TNF is incorporated into it causes, or the acceptor that the FAS aglucon is incorporated into it causes.

Another object of the present invention provides some antagonists (antagonists) (for example antibody), resist these intracellular region conjugated proteins (IC-conjugated protein), these antagonists can be used to suppress the signal generating process, promptly when this IC-conjugated protein is the positive-effect factor (effectors) (that is inducement signal generation); Or be used for the enhancing signal generating process, promptly when these protein are the negative effect factor (that is suppress signal take place).

It is to utilize these IC-conjugated proteins to separate and protein or the effector of identifying that other are relevant that the present invention also has another purpose.These albumen and effector are participated in the downstream events of signal conduction and upstream incident and IC-conjugated protein (as other TNF-Rs or associated receptor) also must participate in these incidents.

Another one purpose of the present invention is to utilize above-mentioned IC-conjugated protein as corresponding polyclone of antigen prepd and/or monoclonal antibody.It is conjugated protein that these antibody also can be used for the IC-of the new different sources of purifying successively, for example conjugated protein from the IC-of cell extract or cell transformed system.

And these antibody can be used for diagnostic purpose, for example are used to identify the malfunction diseases associated with cytological effect, and said cytological effect is receptor-mediated by belonging to some of TNF/NGF receptor superfamily.

Further purpose of the present invention provides the pharmaceutical composition that some contain above-mentioned IC-conjugated protein, contain the pharmaceutical composition of IC-conjugated protein antagonist with some, be used for the treatment of or prevent the illness of that TNF brings out or FAS aglucon-bring out, the character of for example conjugated protein or its antagonist according to contained IC-in the medicine, these medicines can be used to enhance TNF effect or FAS aglucon effect, perhaps suppress TNF effect or FAS aglucon effect.

And, according to another object of the present invention, the invention discloses the other method, eliminate or the TNF or the FAS-R aglucon of antagonism endogenous or external application by the oligomer that uses soluble oligomeric TNF-Rs, oligomerization FAS-Rs or TNF-Rs and FAS-Rs mixture.Should be mentioned that in this respect that once the someone attempted to separate and recombinant production TNF conjugated protein, this protein is called TBP-1, it demonstrate can antagonism TNF effect.The mensuration of this antagonism is to reduce situation by the cytoactive of measuring TNF, and the situation (EP308378) of disturbing TNF to be incorporated into its acceptor realizes by measuring.TBP-1 has been proved to be in concentration and has been number during ng/ml and use these cytokinins simultaneously and can protect cell not to be subjected to the toxic effect of TNF and can disturb TNF-α and TNF-β is incorporated into cell.Further about the TBP-1 mechanism of action studies show that TBP-1 not with neuron target cell interaction, but by single-minded in conjunction with TNF, thereby the function of sealing TNF with TNF receptor competition TNF, thereby, with different purification techniques, find to have two kinds of active ingredients: a kind of is TBP-1, and second kind is that TNF-is conjugated protein, and we are called TBP-II (doing to describe) with the latter in EP398327.These two kinds of protein can shield, and in order to the external cytosis of killing of antagonism TNF, they are lower than validity in conjunction with TNF-α in conjunction with TNF-β validity.Though analyze this two kinds of protein (TBP-I and TBP-II) by sds gel electrophoresis, as if has very close molecular weight, but they can also clearly be carried out difference to lack immune cross-reactivity (cross reactivity) each other according to it, in addition, its-terminal amino acid row and amino acid are formed all variant.

Yet, above-mentioned early stage solvable TNF is conjugated protein to be monomer, and can only be in conjunction with a monomer in the homotrimer, i.e. a natural aglucon, like this because TNF-still has two reactive monomers not by the conjugated protein combination of TNF, TNF is still had an activity (that is not exclusively neutralization).And, still there is not report up to now about solubility FAS-Rs (solubility FAS-R ligand binding protein matter), said solubility FAS-Rs can be incorporated into the FAS-R aglucon, and FAS-R is known to be a kind of homotrimer, also is a kind of cell surface associated molecule.

" dead functional domain " (Tartaglia etc., 1993) of a kind of so-called P55-IC are found, but and undeclared.As of the present invention, P55-IC and its " dead functional domain " can produce self-association and use, and this self-association is the signal generation and causes bringing out Cytotoxic major cause.And this part report is not spoken of possible synthesizing soluble oligomerization TFN-Rs, or soluble oligomeric FAS-Rs, or their mixing oligomer, do not revealed other TNF correlation effect, i.e. P55-IC or its inductive effect of mentioning by the present invention, for example IL-8-genetic expression inductive effect yet.Equally, another part report, be published in after day of the present invention, reported the congregation (that is self-association) of P55-IC, but do not refer to, as described in the present invention, utilize the P55-IC self-association to prepare soluble oligomeric TNF-Rs or FAS-Rs, also do not refer to as described in the present invention by P55-IC or its part not rely on other TNF-correlation effect that the aglucon mode is brought out.

Summary of the invention

As of the present invention, some novel proteins have been we have found that, they maybe can be incorporated into the intracellular region (P55IC-conjugated protein) of P55TNF-R, the intracellular region (P75IC-conjugated protein) of P75TNF-R, maybe can be incorporated into the intracellular region (FAS-IC-conjugated protein) of FAS-R.These P55IC-, P75IC-and FAS-IC-are conjugated protein, can be used as mediated factor (mediators) or conditioning agent (modulators) that TNF or FAS-R pair cell produce coordination effect, the mode of action is mediation or regulates the intracellular signal generating process, this process normally is incorporated into P55 and/or P75TNF-R at TNF, or the FAS-R aglucon is incorporated into after the cell surface and takes place.And be astoundingly, P55IC and FAS-IC can produce self-association, and some fragments of P55IC and FAS-IC can be incorporated into P55IC equally, particularly are incorporated into the interior what is called " dead functional domain " of these acceptors ICS (DD), that is P55DD and FAS-DD.Therefore P55IC and PAS-IC and fragment thereof are also represented some protein like this, and promptly they can be incorporated into P55IC and FAS-IC, thereby can produce the conditioning agent of aglucon effect as TNF or FAS-R pair cell.

Further thrown a flood of light on one of novel protein that the present invention addresses, name is 55.11 protein herein, be incorporated into the essence (seeing embodiment 1) of P55-TNF-R intracellular region.

And on the other hand, the present invention is based on such discovery: the intracellular region of P55TNF acceptor (P55-IC), be a kind of zone, and wherein include so-called P55-IC " dead functional domain "; The intracellular region of FAS/AP01 acceptor (Fas-IC) is a zone, wherein includes so-called Fas-IC " dead functional domain ", and these intracellular regions can produce self-association.Thereby might make a kind of soluble oligomeric TNF acceptor according to conventional recombinant DNA technology, it is a kind of fusion rotein, the one end contains at least two extracellular regions of TNF acceptor, the other end contains at least two above-mentioned self-association intracellular regions, or a certain fragment, self-association can take place and provide a kind of oligomer, this oligomer to have at least two this fusion products that link together in these zones.Such soluble oligomeric TNF-R is can be in conjunction with two monomers in the spontaneous TNF homotrimer, thereby effectively and the activity of TNF.The active neutralization of TNF all is essential under all above-mentioned conditions, and promptly wherein TNF excessively produces in vivo, or externally uses with high dosage, and when producing undesirable side effect.Further, utilize soluble oligomeric acceptor of the present invention to come effectively also to can be used in conjunction with external source TNF, slowly discharge subsequently in the desired amount then, for example its application aspect the treatment tumour in conjunction with TNF.Equally, according to conventional recombinant DNA technology, also might make up a kind of oligomerization FAS-R fusion product, the one end contains the extracellular region of at least two FAS-R, and containing at least two above-mentioned self-association intracellular regions or its fragment at the other end, these regional self-association meetings produce a kind of oligomer that has at least two fusion products to connect together.This FAS-R oligomer can be in conjunction with two monomers in the spontaneous FAS-R aglucon homotrimer, thereby effectively and the activity of FAS-R aglucon.The active neutralization of FAS-R aglucon all is necessary under all above-mentioned conditions, because excessive in these cases FAS-R aglucon is all relevant with unfavorable side effect.Equally, in view of nearest report is pointed out may get in touch between TNF and the FAS-R aglucon inductive pair cell effect, thereby they also have contact at the cell surface of bind receptor.So also just may utilize conventional recombinant DNA technology to make up a kind of compound oligomer acceptor, have simultaneously the two specificity of TNF and FAS-R aglucon.This compound oligomer is a kind of mixture of above-mentioned fusion product, one end contains the extracellular region of at least one TNF-R and the extracellular region of at least one FAS-R, the other end contain at least two above-mentioned TNF-R and FAS-R from-associating intracellular region or its fragment, these zones can from-associate and produce a kind of mixing oligomer that has at least two kinds of fusion products to link together.Such mixing oligomer can be in conjunction with at least one monomer of TNF, while is in conjunction with a monomer of FAS-R aglucon, this compound oligomer also can effectively reduce under the condition below or in and the TNF and the FAS-R acceptor of cell surface, promptly aforesaid excessive two kinds of hormones situation relevant with unfavorable cytological effect.As top pointed, the FAS-R aglucon is normally mutually associating with cell surface, and the TNF of cell surface-association form has been described in nearest several parts of reports.Therefore these mix that the TNF-R/FAS-R oligomer is used for and cell surface on TNF and the activity of FAS-R aglucon be effective especially.

For adapting to these situations, the invention provides a kind of proteic dna sequence dna of coding, this albumen can be incorporated into the one or more intracellular region of one or more acceptors, and these acceptors belong to tumour necrosis factor/nerve growth factor (TNF/NGF) receptor superfamily.

Specifically, the invention provides a kind of dna sequence dna, is a big class DNA family that is selected from following composition:

(a) a kind of cDNA sequence derived from the protein-bonded coding section of natural TNF-R intracellular region;

(b) can under not really strict condition, hybridize certain segment DNA in (a), and the TNF-R intracellular region-protein-bonded dna sequence dna of coding biologically active; And

(c) because of the genetic code degeneracy, degeneracy is the dna sequence dna in the (a) and (b), the bioactive TNF-R intracellular region of they coding tools-conjugated protein.

The present invention also provides a kind of dna sequence dna, is a big class DNA family that is selected from following composition:

(a) a kind of cDNA sequence is derived from natural FAS-R intracellular region-protein-bonded coding section;

(b) some dna sequence dnas can be hybridized in the cDNA of (a) under mild conditions, and the bioactive FAS-R intracellular region of a kind of tool of encoding is conjugated protein; And

(c) some dna sequence dnas and since the degeneracy as a result of the degeneracy value of genetic code be (a) and (b) in defined dna sequence dna, their encode tool biological activity FAS-R intracellular regions-conjugated protein.

In the present invention, also comprise some coding P55TNF-R, P75TNF-R and the protein-bonded dna sequence dnas of FAS-R intracellular region, for example called after protein 55.1,55.3,55.11,75.3,75.16, F2, the dna sequence dna of F9 and DD11.

The present invention also provides a kind of and has come encoded protein matter according to above-mentioned any one sequence of the present invention, or analogue, or derivatives thereof, said these protein, analogue and derivative thereof are the one or more intracellular regions that can be incorporated into one or more TNF-Rs or FAS-R.The embodiment of this respect of the present invention comprises protein 55.1,55.3,55.11,75.3,75.16, F2, F9 and DD11, and analogue and derivative.

The present invention also provides and has been encoded to above-mentioned more proteinic carriers of the present invention (vectors), they contain above-mentioned dna sequence dna of the present invention, and can be expressed in the suitable eukaryotic host cell or in the prokaryotic host cell, a kind of conversion that contains this carrier also is provided eucaryon or prokaryotic host cell; And the method for producing protein of the present invention, analogue or derivative, promptly under the expression condition that is fit to, cultivate the host cell that these have transformed, said albumen translated post-treatment, from the cell culture medium that transformed or cell extract, extract cell expression product.

On the other hand, the present invention also provides some antibody that are directed to protein of the present invention and analogue and derivative or its reactive derivative or its fragment.

The present invention has provided above-mentioned dna sequence dna or proteinic various uses on the other hand, and its use comprises:

(i) a kind of method is used to regulate the effect to the cell that is loaded with TNF-R or FAS-R of TNF or FAS-R aglucon.Comprise with one or more protein, analogue or derivative are handled cell, these albumen, analogue or derivative are selected from P55IC, P55DD, FAS-IC or FAS-DD albumen and their analogue or derivative, the activity that all above-mentioned protein can both be incorporated into intracellular region and regulate described TNF-R or FAS-R, wherein said cell is handled and is comprised one or more protein, analogue or derivative are introduced cell, this kind protein of perhaps will encoding with introducing form in the born of the same parents that are fit to, the dna sequence dna of analogue or derivative is introduced in the said cell with the expression vector form that is fit to;

(ii) a kind of method is used to regulate the effect to the cell that is loaded with TNF-R or FAS-R of TNF or FAS-R aglucon, according to the present invention, comprises with antibody and reactive derivative thereof or fragment and handles said cell;

(iii) a kind of method is used to regulate the effect to the cell that is loaded with TNF-R or FAS-R of TNF or FAS-R aglucon, comprise with oligonucleotide sequence and handle said cell, according to the encode antisense sequences (antisensesequence) of this sequence of partial sequence at least in the oligonucleotide sequence of the present invention, or be the antisense sequences of coding P55IC, P55DD, FAS-IC or FAS-DD sequence, said oligonucleotide sequence can seal the expression of at least a TNF-R or FAS-R intracellular region conjugated protein;

(iv) a kind of method is used to regulate the effect to the cell that is loaded with TNF-R or FAS-R of TNF or FAS-R aglucon, comprising:

(a) make up a kind of recombinant animal virus vector, be loaded with the dna sequence dna of a coding virus capsid protein, this albumen can be incorporated into a certain specific cells surface receptor; And be loaded with another sequence, be to be selected from an oligonucleotide sequence, this oligonucleotide sequence wherein at least partial sequence be the antisense sequences of albumen coded sequence among the present invention, and the Antisensedigonucleotsequence sequence of sequence that is selected from coding P55IC, P55DD, FAS-IC or a FAS-DD, said oligonucleotide sequence is when sealing at least a TNF-R or the protein-bonded expression of FAS-R intracellular region by virus-mediated after entering cell; And

(b) infect said cell with said carrier in (a)

(v) a kind of method is used to regulate the effect to the cell that is loaded with TNF-R or FAS-R of TNF or FAS-R aglucon, comprise the said cell of vehicle treated with suitable coding one ribozyme, this ribozyme has a sequence, and it has specificity to the mRNA sequence of albumen, analogue or the derivative described in the code book invention; It also has specificity to the mRNA sequence of coding P55IC, P55DD, FAS-IC or FAS-DD.Said ribozyme sequence can interact with said mRNA sequence and can cut said mRNA sequence, the result suppressed described in the present invention and protein, the expression of analogue or derivative, or suppressed the expression of P55IC, P55DD, FAS-IC or FAS-DD;

(vi) a kind of method is used to handle tumour cell or HIV cells infected, or other ill cells, comprising:

(a) make up the recombinant animal virus vector, it is loaded with the dna sequence dna of coding virus capsid protein, and the cell surface receptor that this albumen can infect in conjunction with tumor cell surface acceptor or HIV-perhaps can be in conjunction with the cell surface receptor of the ill cell of other; The encode fragment of albumen, analogue or the derivative that also is loaded with the present invention and is mentioned and be loaded with coding P55IC, P55DD, FAS-IC, FAS-DD, or the sequence of its analogue or derivative.The said protein of the present invention, its analogue or derivative reach P55IC, P55DD, FAS-IC, FAS-DD, and analogue or derivative in the time of among being expressed in said tumour cell or HIV-cells infected or other ill cell, all can be killed these cells; And

(b) infect said tumour cell or HIV-cells infected or other infected cells with said carrier in (a).

(vii) a kind of method that is used to separate and identify albumen, the factor or acceptor, it is conjugated protein to be incorporated into intracellular region according to these protein of the present invention, the factor and acceptor.Separation and authentication method comprise the application affinity chromatography, said albumen can be attached on the matrix in the affinity chromatography in this method, these albumen that adhere to again with cell extract in some protein, the factor or acceptor be bonded with each other, conjugated protein then again by wash-out, separation and analysis;

(viii) a kind of method is used for separating and identification of protein.According to the present invention, it is conjugated protein that these protein can be incorporated into intracellular region.This method comprise using yeast two-hybrid (yeast two-hyhrid) method, the protein-bonded sequence of said intracellular region of wherein encoding is carried by a hybrid carrier, and another carries from the sequence of cDNA or genomic library and by the second hybrid carrier (secondhyhrid vector), and these carriers are used to the transformed yeast host cell then.Separate these positive transformants and extract the said second hybrid carrier subsequently and obtain a sequence, it is conjugated protein that its encoded protein matter can be incorporated into said intracellular region; And

(ix) a kind of method is used to separate and identify a kind of protein of the intracellular region that can be incorporated into TNF-Rs or FAS-R, comprises and uses gentle Sourthern hybridization (nonstringentsouthern hybridization), carries out pcr amplification subsequently.According to the present invention, make probe (probe) in cDNA or group group library with wherein a sequence or its part, angle out the sequence that has portion homologous at least.With said homologous sequence, pcr amplification just can obtain a large amount of and described sequence has the albumen of homology to clone.

The present invention also provides a kind of medicine to be used to regulate the effect of TNF or FAS aglucon pair cell, comprise following any material: (i) a kind of protein of the present invention as active ingredient, perhaps protein P55IC, P55DD, FAS-IC or FAS-DD, or its bioactive fragment, analogue, derivative or their mixture, (ii) a kind of recombinant animal virus vector of the virus capsid protein of encoding, the cell receptor that this coat protein can carry in conjunction with TNF-R or FAS-R or the special receptor of tumour cell; A kind of protein, analogue or the derivative that invention is addressed with code book or coding P55IC, P55DD, FAS-IC or the proteic one section sequence of FAS-DD; (iii) a kind of recombinant animal virus vector, coding as above-mentioned (ii) in said virus capsid protein, and the Antisensedigonucleotsequence sequence of P55IC, P55DD, FAS-IC or FAS-DD encoding sequence; And (iv) a kind of encoding ribozyme carrier, this ribozyme can be invented the protein of addressing with code book, and the mRNA sequence of the mRNA sequence of analogue or derivative etc. and coding P55IC, P55DD, FAS-IC or FAS-DD interacts.

One particular instance of the above-mentioned each side of the present invention is the use of P55-IC or himself the use of dna sequence dna of encoding.Present embodiment is based on P55IC can be with the TNF-correlation effect in the method inducing cell that does not rely on aglucon (TNF).Thereby provide a kind of method, be used for bringing out the TNF-correlation effect of cell or tissue, comprise with one or more protein or its analogue or derivative and handle said cell, said protein is to be selected from such class protein, it in fact all belongs to self-association intracellular region (P55-IC) or its part of P55TNF-R, and can induce said intracellular TNF effect in the mode that does not rely on aglucon, wherein said cell is handled and is comprised said more than one protein, analogue or derivative are introduced in the said cell with the form that is suitable for introducing in the born of the same parents, or with one section more than one protein of coding, analogue or derivative dna sequence dna are introduced in the said cell with suitable carriers, wherein carrier can be finished the process that aim sequence embeds target cell, thereupon, aim sequence can be expressed in target cell.

The embodiment of aforesaid method of the present invention comprises:

(i) a kind of method, it is with a kind of recombinant animal virus vector transfectional cell that wherein said cell is handled, and comprises the steps:

(a) make up a kind of recombinant animal virus vector that may be encoded as virus capsid protein (aglucon) sequence that is loaded with, this albumen can be incorporated into the specific cells surface receptor on the cell surface to be processed.This carrier also is loaded with second sequence, its encode P55-IC or its fragment, analogue and derivative.These protein are the energy self-association behind cell expressing, and can induce a series of TNF-correlation effects; And

(b) infect said cell with the carrier in (a).

(ii) a kind of method, the wherein said TNF effect of inducing in cell is meant inducing of IL-8 genetic expression, said carrier is loaded with a sequence, its encode all said P55-IC or its part and analogue and derivative in fact.When at cell expressing, their energy self-associations are also sent signal, bring out IL-8 genetic expression.

(iii) a kind of method is used to handle tumour cell or virus infected cell, or be used to increase granulocytic anti-microbial effect, wherein said virus vector is loaded with the sequence of the viral aglucon of a coding, and this aglucon can be in conjunction with tumour cell, virus infected cell and granulocytic specific cells surface receptor.It also is loaded with coding P55-IC or its part, analogue and derivative sequence, when they in tumour cell, virus infection cell or granulocyte in just bring out the TNF associating effect when expressing and cause these necrocytosiss.

(iv) a kind of method is used to handle tumour thing or derivative, when at tumor cells expression, can bring out the expression of IL-8 and causes the death of tumour cell.Kill cell by its " chemotactic activity ", promptly attract granulocyte and other lymphocyte to tumour cell and cause death of neoplastic cells.

In this one side of the present invention, the application of intracellular region (P55-IC) in handling cell of P55-R, its part, analogue and derivative also is provided, can induce the TNF-associating effect; And provide following embodiment:

(i) P55-IC, its part, analogue and derivative are used to handle cell and induce IL-8 genetic expression.

(ii) P55-IC, its part, analogue and derivative are used to handle tumour cell, by inducing IL-8 genetic expression, the kill tumor cell.

Further, at this respect of the present invention, provide a kind of pharmaceutical composition that can bring out the TNF-correlation effect.Comprise P55-IC, its part, analogue and derivative as activeconstituents, and a kind of pharmaceutically acceptable carrier (carrier); And their following embodiment:

(i) a kind of pharmaceutical composition of inducing the TNF-correlation effect comprises that as the recombinant animal virus vector of active ingredient it is encoded to P55-IC, its part and analogue thereof and derivative and can be in conjunction with the protein of cell surface protein to be processed.

(ii) a kind of pharmaceutical composition, when handling tumour cell, it can induce IL-8 to express also kill tumor cell thereupon.

On the other hand, the invention provides a kind of solubility, oligomerization Tumor Necrosis Factor Receptors (TNF-R), comprise at least two kinds from-association fusion rotein, each fusion rotein has: (a) at the one end,-TNF land, it is from the extracellular region of TNF-R, analogue or derivative, and they do not influence it from-association and can be in conjunction with TNF; And (b) at its another end, one from the-district that associates, from: (i) come down to all intracellular regions (P55-IC) of P55TNF-R, promptly from about the 206th amino-acid residue of natural P55TNF-R molecule (P55-R) until the 426th amino-acid residue; The (ii) dead functional domain of P55-IC, from about the 328th amino-acid residue of natural P55-R until about the 426th amino-acid residue; (iii) come down to all intracellular regions (FAS-IC) of FAS/AP01 acceptor; The (iv) dead functional domain of Fas-IC; And (v) any analogue, its part or derivative that can self-association in (i)-(iv), wherein two only can produce from-association at its end (b) from a Rapsyn.(a) that it has is terminal can be in conjunction with at least two kinds of TNF monomers, and each (a) end can be in conjunction with a TNF monomer, reach salt and the functional deriv of said soluble oligomeric TNF-R.

Some embodiment of this respect of the present invention also comprise the combination that all above-mentioned (a) are terminal with (b) terminal, as defined above, for example, a kind of oligomeric TNF-R of solubility comprises as the P55-R extracellular region of extracellular region with as the P55-IC from-association intracellular region.

Further, also provide a kind of processing method of producing soluble oligomeric thing TNF-R of the present invention, having comprised:

(a) make up an expression vector, any component of its encoding fusion protein, its every segment DNA sequence is all from the clone system of extracellular region, analogue or the derivative dna sequence dna of TNF-R; And the clone system that comes the dead functional domain of own coding P55-IC, P55-IC, the dead functional domain of Fas-IC, Fas-IC and their analogues or derivative dna sequence dna, its each end can be interconnected to form the fused protein sequence.And the said carrier of embedding under the adjusting of regulating sequence transcribed and translated to said fusion rotein sequence can;

(b) carrier in (a) is imported proper host cell, said fusion rotein is expressed in this host cell, and

(c) fusion rotein of expressing in the purifying host cell, this fusion rotein before purifying, in the purge process or after the purifying all be can from-associating, and produce the oligomer TNF-R of solubility.

Following content further also is provided: a kind of carrier of the above-mentioned fusion rotein of encoding, it is widely used in aforesaid method of the present invention; The host cell that contains above-mentioned carrier; And a kind of pharmaceutical composition, wherein contain the oligomeric TNF-R of solubility and the mixture of its esters, functional deriv and a kind of aforementioned substances, as activeconstituents, acceptable carrier combines on they and the pathology; Oligomeric TNF-the R of some solubilities and its esters, functional deriv are provided simultaneously, and aforementioned substances mixture, be used for the TNF of supporting short of money at the intravital deleterious effect of Mammals, be used in particular for treating such illness, promptly Yin Neisheng or external source are used and are caused TNF excessive; Or under the another kind of situation, when TNF uses with external source, be used to keep TNF at the intravital long-time advantageous effect of Mammals.

The above-mentioned listed clause according to the present invention is also found, might make a kind of soluble oligomeric thing Fas/AP01 acceptor (Fas-R), is used for the deleterious effect of antagonism FAS aglucon.In view of the above, the present invention provides a kind of oligomerization Fas/AP01 acceptor (Fas-R) of solubility on the other hand, contain at least two kinds from-associated fusion rotein, each fusion rotein has: (a) at its end, it is the Fas ligand-binding domain, this land is the extracellular region from Fas-R, its analogue or derivative, it not can from-associate but can be in conjunction with the Fas aglucon; And (b) at its another end, be from-the district that associates, from: (i) the whole intracellular regions (P55-IC) that come down to P55TNF-R promptly from about the 206th amino-acid residue of natural P55-TNF-R molecule (P55-R) to the section about the 426th amino-acid residue; The (ii) dead functional domain of P55-IC, promptly from about the 328th amino-acid residue of natural P55-R to the section about the 426th amino-acid residue; (iii) come down to whole intracellular regions (Fas-IC) of Fas/AP01 acceptor; The (iv) dead functional domain of Fas-IC; And (v) proteic analogue or derivative described in (i)-(iv), they can produce certainly-association, wherein at least two kinds from-associated albumen only (b) end can from-associate, their end can be in conjunction with at least two Fas aglucon monomers, and each (a) end can be in conjunction with a Fas aglucon monomer; And salt and the functional deriv of the oligomerization Fas-R of solubility.

According to this aspect of the invention, also provide a method of producing soluble oligomeric Fas-R, having comprised:

(a) make up the expression vector that to encode to any component of fusion rotein, the terminal dna sequence dna of each of fusion rotein all from such dna clone is, whole Fas-R promptly encode in fact, the extracellular region of its analogue or derivative, and from such dna clone system, be their whole P55-IC that encode in fact, the dead functional domain of P55-IC, Fas-IC, the dead functional domain of Fas-IC and all aforementioned proteic analogue or derivative, its each end can interconnect and form the fusion rotein encoding sequence, and the fusion rotein encoding sequence is to transcribe and translating the said carrier of embedding under the regulation and control of regulating sequence;

(b) carrier with (a) imports proper host cell, and said expressing fusion protein is in this host cell; And

(c) be expressed in the purifying of the fusion rotein in this host cell, said fused protein before the purge process, among or afterwards all can from-associate and obtain the oligomerization Fas-R of solubility.

Further, also provide a kind of expression vector, it is loaded with the fusion rotein encoding sequence of coding soluble oligomeric Fas-R, and is widely used in said process; A kind of host cell and some pharmaceutical compositions that is loaded with this carrier also is provided, this pharmaceutical composition contains soluble oligomeric Fas-R and its esters or derivative or aforementioned any mixture, as activeconstituents, acceptable carrier combines on they and the pathology.Simultaneously, the mixture of a kind of soluble oligomeric Fas-R and its esters or functional deriv or aforementioned any material also is provided, be used for antagonism Fas aglucon to mammiferous deleterious effect, be used to especially surely handle by Nei Sheng or external source and use excessive some illnesss that form of the Fas aglucon that causes.

With with above-mentioned similar manner about oligomeric TNF-Rs and oligomerization Fas-Rs, also may make some have binding specificity simultaneously to TNF and Fas-R aglucon mixing oligomer.The present invention also provides a kind of mixing oligomeric TNF-R/FAS-R in view of the above, it contains at least two kinds from-associated fusion rotein, wherein a kind of fusion rotein is any from above-mentioned TNF-specificity fusion rotein, another fusion rotein is from above-mentioned FAS-R aglucon specificity fused protein, thereby provide a kind of mixing oligomer, it has at least one TNF-R extracellular region and at least one FAS-R extracellular region, and these extracellular regions rely on and mutual the associating together from-association of each intracellular region of its bonded.These mix the preparation of oligomerization acceptor, as mentioned above, are to prepare oligomer TNF-Rs and oligomer FAS-Rs earlier, then they are mixed together, and selecting according to conventional procedure subsequently can single-minded oligomer in conjunction with FAS-R aglucon and TNF.Another kind is produced the method for mixing the oligomerization acceptor, as mentioned above, be with some carrier cotransfection (co-transfecting) proper host cell, any one TNF specificity fusion rotein of this carrier codified (soluble TNF-Rs) and any one FAS-R aglucon specificity fusion rotein (solubility FAS-Rs) of coding.Purifying then, expressed fusion protein, and it before the purifying, among or afterwards all can self-association and obtain some oligomerization acceptors, select these can be incorporated into the oligomerization acceptor of TNF and FAS-R ligand according to conventional operation then.

Equally, also provide a kind of pharmaceutical composition, comprised the mixing oligomerization acceptor as activeconstituents, their salt or functional deriv, the perhaps mixture of aforementioned substances, acceptable carrier combines on it and a kind of pharmacology.In addition, also provide some to mix oligomer acceptor, their salt or functional deriv, the perhaps mixture of any aforementioned substances, the deleterious effect that is used for interior TNF of antagonism mammalian body and FAS-R aglucon, be used in particular for handling such illness, promptly, Nei Sheng or external source form excessive TNF and FAS-R aglucon because using, perhaps also be used in addition to keep the long advantageous effect of TNF in the mammalian body and/or FAS-R aglucon, for example when they with TNF and/or FAS-R aglucon (with soluble form) during external using.

The present invention also provides other aspect and embodiment, and details are as follows.

Employed following term in should be pointed out that in full: " regulating effect of TNF pair cell " and " regulating effect of Fas aglucon pair cell " is understood to include in vitro and in vivo and handles.

The accompanying drawing summary

Fig. 1 a-c has described coding P55IC and the more protein-bonded cDNA clones' of P75IC-the basic nucleotide sequence of part with chart, and wherein Fig. 1 (a) is clone's 55.11 sequences (SEQ ID NO:9) of coding P55IC-conjugated protein 55.11; Fig. 1 (b) is the clone's 75.3 of coding P75IC-conjugated protein 75.3 a part basic sequence (SEQ ID NO:10 and 11); And Fig. 1 (c) is clone's 75.16 part basic sequences (SEQ ID NO:12 and 13) of the conjugated protein P75.16 of coding P75IC-; All these all such as example 1 description; And Fig. 1 (d) described protein 55.11 amino acid and inferred sequence (SEQ ID NO:14), and albumen 55.11 is to infer from the nucleotide sequence of Fig. 1 (a), also is described in embodiment 1.

Fig. 2 has described Northern blotting detected result, and it shows that 55.11 single-minded mRNAs are the clone that is present in multiple test, as described in example 1.

Fig. 3 A and 3B are the radioautograph detected results, it shows the albumen of the 55.11cDNA that encodes in the external situation that is incorporated into the gst fusion protein matter that contains part P55-IC, and wherein Fig. 3 A has described the situation that overall length 55.11 protein (55.11 total length) are incorporated into various gst fusion proteins; Fig. 3 B has described the situation that the part 55.11 that is blended in the FLAG octapeptide is incorporated into various gst fusion proteins, all is described in embodiment 1.

Fig. 4 is that the human 55.11 proteic deduction aminoacid sequences of expression compare with the chart of the relevant protein sequence of unicellular lower eukaryote, as described in embodiment 1.

Fig. 5 is a Western blotting detected result, promptly with anti--MBP polyclonal antiserum dyeing, this is for the self-association of P55IC is described, when carrying out the SDS-PAGE gel electrophoresis, based on the interaction (swimming lane 1-4) of bacteriogenic chimeric protein P55IC-MBP and P55IC-GST or the regulating and controlling effect between chimeric protein P55IC-MBP and the independent GST, (swimming lane 5-8), and obtain the Western trace.And, before the SDS-PAGE electrophoresis, on the sugared gel of gsh-agar (glue), carry out electrophoresis research to chimeric protein interaction to each other, as described in example 2 above.

Fig. 6 is the phase microscope detected result, explanation is in the expression vector transfection HTtal cell of coding P55IC, the IC cytotoxic effect of total length P55 (right picture), and the restraining effect that this cytotoxic effect is described, i.e. situation (left picture) when handling cell with tsiklomitsin the expression of this carrier is closed, as described in example 2 above.

Fig. 7 has described by P55-R or its intracellular region, or the part intracellular region comprise " dead functional domain " transfection the trigger action that does not rely on aglucon of Hela cell (heLa cell) back cytocidal effect.Wherein

(i) at the Far Left of Fig. 7 coding total length P55-R has been described, the various dna sequence dnas of its intracellular region and part intracellular region, and this sequence is inserted into carrier, is used for transfection Hela cell;

The (ii) left side and intermediary bar graph, be the expression of explanation TNF acceptor in the Hela cell, promptly in every type of acceptor expression in the hela cell shown in Fig. 7 Far Left, left side bar graph is represented the amount (ng/ cell sample) of acceptor, middle bar graph is represented the amount of the acceptor of representing with the radioiodination TNF that is incorporated into transfectional cell, and

(iii) the right bar graph is the viability that the Hela cell of various acceptors is expressed in explanation; Wherein in all bar graphs, some open hurdles are representative cells transfected in the presence of tsiklomitsin, and the sealing hurdle is to represent no tsiklomitsin to have cells transfected down; All are described and all see embodiment 2 herein.

What Fig. 8 illustrated the IL-8 gene does not rely on the aglucon inducing action, this group be expressed in by P55-R or its intracellular region (P55IC) transfection the Hela cell in, wherein Fig. 8 A represents Northern blotting detected result.Wherein RNA extracts personal TNF processing or untreated Hela cell (two row left side swimming lanes are marked with contrast and TNF person respectively), and extract from being encoded P55-R, the carrier transfection of P55-IC the Hela cell or extract from reference protein luciferin enzyme (being marked with all the other each swimming lanes of P55-IC, P55-R and Luc respectively), this Hela cell is in (+) in the presence of the tsiklomitsin or not (-), carries out transfection (so each transfection has two row swimming lanes) respectively; Wherein Fig. 8 B represents to give Yamamoto Methylene Blue ZF dyeing to the 18srRNA in each Hela cell sample shown in the figure A; All above-mentioned all seeing among the embodiment 2.

Fig. 9 (A and B) has described the trigger action that does not rely on aglucon of the cytocidal effect in the Hela cell with diagram.The Hela cell is with P55R or its part, or carries out transfection with FAS-IC, and wherein Fig. 9 has described with P55R or its part and infected result behind the Hela cell, and Fig. 9 B has described with the result behind the FAS-IC cells infected.

Illustrate part P55R or the FAS-IC that is used for transfection in the left side of Fig. 9 A and B, and the right illustrates some experimental results, all these all are described in embodiment 2.

Figure 10 is the basic nucleotide sequence of part (SEQ ID NO:19 and 20) of having described a kind of cDNA clone who is named as " F2 " with graphic, and its coding can be incorporated into a kind of protein of P55IC and FAS-IC, as described in example 3 above.

Figure 11 is the basic nucleotide sequence of part (SEQ ID NO:21-23) of having described a kind of cDNA clone who is named as " F9 " with graphic, and its coding can be incorporated into a kind of protein of P55IC and FAS-IC, as described in embodiment 3.

Figure 12 is the basic nucleotide sequence of part (SEQ ID NO:24) of having described a kind of cDNA clone who is named as " DD11 " with graphic, and its coding can be incorporated into a kind of protein of P55IC, particularly P55DD and FAS-IC, as described in embodiment 3.

The detailed description of invention

The present invention is about being incorporated into some novel proteins of acceptor intracellular region in one aspect, wherein these acceptors belong to the TNF/NGF superfamily, for example TNF-Rs and FAS-R, thereby these protein can be considered as this superfamily receptors for example mediator (mediators) or the conditioning agent of TNF-Rs and FAS-R, and these acceptors are incorporated into FAS-R and cause in the signal production process to have certain effect being incorporated into TNF-Rs and FAS aglucon owing to TNF. These protein refer to be incorporated into the albumen (P55IC) of P55TNF-R intracellular region, for example name here is 55.1,55.3 and some protein (embodiment 1) of 55.11 and according to cDNA clone F2, some protein (embodiment 3) of F9 and DD11 coding; Can be incorporated into the albumen (P75.IC) of P75TNF-R intracellular region, for example naming is some protein (embodiment 1) of 75.3 and 75.16; And some albumen (FAS-IC) that can be incorporated into the FAS-R intracellular region, some protein (embodiment 3) of for example encoding according to cDNA clone F2, F9 and DD11. Protein 55.1 and 55.3 has found to be some parts or the fragment (P55IC) of the intracellular region of P55TNF-R; Other oroteins 55.11,75.3 reach 75.16 representatives had no any report before the present invention protein (75.3,75.16), though perhaps they are had report (55.11, referring to khan etc., 1992), but to its function and other characteristics, particularly can be incorporated into TNF-R, but without any the description (referring to following embodiment 1) of aspect. These clone F2 according to cDNA, some novel proteins of F9 and DD11 coding, " the group database " that the yet albumen of report not yet before the representative, that is their sequence does not deposit DNA or amino acid sequence in (GENEBANK) or " Protein Data Bank " (PROTEIN BANK).

Thereby, the invention relates to the protein by these sequential codings, and the dna sequence dna of these albumen of encoding.

And, the invention still further relates to some dna sequence dnas, their the encode analog with BA and derivatives of these protein, and by analog and the derivative of their codings. The preparation of these analogs and derivative is (such as referring to Sambrook etc. according to the routine operation step, 1989), wherein in these protein DNA sequences of coding, more than one codon disappearance is arranged, increase or replaced by another codon and look for, and obtaining some analogs, these analogs are compared the variation that has an amino acid residue at least with native protein. Welcome analog such as P55IC, P75IC or FAS-IC, the ability that must reservation can be incorporated at least the intracellular region of TNF/ NGF receptor superfamily, the intracellular region of FAS-R or TNF-R for example, perhaps can mediate any other cohesive process or enzymatic activity, such some analogs for example, be that they can be in conjunction with P55IC, P75IC or FAS-IC, but can not produce signal, that is can not be further combined with downstream acceptor (further downstream receptor), protein or other factors, in other words can not certain signal-dependent response of catalysis. Just can produce some analogs with such method, they have so-called dominant-negative effect (dominant-negative effect), that is be incorporated into for example P55IC, P75IC or AFS-IC, or along with in conjunction with and in afterwards the signal production process, this analog all is defective. These analogs are by being combined protein competition in conjunction with can be used to suppress the effect of TNF or the effect of FAS-aglucon with natural IC-. Equally, also can produce so-called dominant-positive-effect analog (dominant-positive analogs), they can be used to strengthen TNF effect or FAS coordination effect. They are compared with natural IC-conjugated protein has identical or better IC-binding ability, and identical or better signal reply performance. Equally, by change the side group of one or more amino acid residues of protein with conventional modification technique, perhaps by gripping hop protein matter in another molecule, such as antibody, enzyme, acceptor etc., also can make some derivatives, as described in prior art.

These novel TNF-R and FAS-R intracellular region are in conjunction with albumen, and for example albumen 55.1,55.3, and 55.11,75.3,75.16 and according to cDNA clone F2, the protein of F9 and DD11 coding (below be called F2, F9 and DD11), many possible purposes are all arranged, for example:

(i) increase time spent of doing of TNF or FAS-R aglucon when needs, when needing TNF or FAS-R aglucon to bring out cytotoxicity when the clinical practices such as antitumor, anti-inflammatory or anti-HIV, they can be used for simulating or strengthening the function of TNF or FAS-R aglucon. Protein in the case, for example can be incorporated into the protein of P55IC, resemble 55.1,55.3, and F2, the protein such as F9 and DD11, and the free P55IC itself (referring to following and embodiment 2) that can strengthen the TNF effect, and P55IC " dead functional domain ", maybe can strengthen the FAS-R coordination effect is the protein F2 of cytotoxic effect, F9 and DD11 and FAS-IC and FAS-DD all can introduce cell according to known conventional method. For example, because these protein are in the born of the same parents, and be introduced in the cell that needs TNF effect or FAS-R coordination effect to be welcome, to need so set up a kind of system that these albumen are introduced in the cell specifically. For reaching this purpose, a kind of method is arranged, by making a kind of recombinant animal virus, a kind of vaccinia virus for example, import following two kinds of genes to its DNA: a kind of is the gene of coding aglucon, this aglucon can be incorporated into the cell surface protein by cell specific expression, the viral gp120 albumen of AIDs (HIV) for example, can selectivity be incorporated into some cell (CD4 lymphocyte and relevant leukaemia), perhaps can selectivity be incorporated into any aglucon of the cell that is loaded with TNF-R or FAS-R, the recombinant virus carrier just can be in conjunction with the cell that is loaded with TNF-R or FAS-R like this; Another kind is encoding novel intracellular region-in conjunction with the gene of albumen or P55IC, P55DD, FAS-IC or FAS-DD albumen. Like this, cell surface will make virus assign tumor cytotoxicity or other cell that is loaded with TNF-R or FAS-R as narrow spectrum target cell in conjunction with the expression of albumen on virus surface, after this just can intracellular region be introduced cell in conjunction with albumen coded sequence or P55IC, P55DD, FAS-IC or FAS-DD coded sequence by virus, and they are in case be expressed in cell, can strengthen TNF effect or FAS-R coordination effect and cause the dead of tumour cell or cause being loaded with the death of the cell of TNF-R or FAS-R. According to routine operation program (such as referring to people such as Sambrook, 1989), can make up this recombinant animal virus. Another kind of may, with novel protein coded sequence or P55IC, P55DD, FAS-IC or FAS-DD with oligonucleotide sequence form transfered cell and make it at cells.

(ii) they can be used to suppress the effect of TNF or FAS-R aglucon. For example repelling (graft-vs-host rejection) because of sepsis body gram, transplant-p-host, or during the tissue that causes of oxyhepatitis damages, need the intracellular signal conductive process of the FAS-R that TNF-R that sealing TNF-brings out or FAS-R aglucon bring out. For example, with antisense (anti-sense) sequence of routine operation method with encoding novel albumen, or the Antisensedigonucleotsequence sequence transfered cell of coding P55IC, P55DD, FAS-IC or FAS-DD, translation and the expression of mRNAs that then can effectively blockade coded protein finally suppresses TNF effect or FAS-R coordination effect.

Such oligonucleotide, the method for available above-mentioned recombinant virus namely carries the second oligonucleotide sequence by virus, and introduces in the cell. Another possibility is to utilize the selectivity antibody of these albumen, and it is active to suppress its intracellular signal conduction. Also have such possibility: these novel proteins have extracellular region and intracellular region, intracellular region can be incorporated into the land of TNF-R or FAS-R, and the antibody that produces can be incorporated into their extracellular region, thereby blockades their the relevant function of TNF or the relevant function of FAS-R aglucon.

Also having the another kind of method that suppresses TNF effect or FAS-R coordination effect, is the ribozyme approach according to latest developments. Nucleic acid is that the RNA molecular energy with catalytic activity cuts the RNA molecule specifically, therefore the selection target hydrolase RNAS that it can be purposive, for example the encode mRNAs of novel protein of the present invention, the mRNA of perhaps encode P55IC, P55DD, FAS-IC or FAS-DD all can be used as target RNAs. This ribozyme can be identified the particular sequence of specific mRNA, and after the mRNA cracking can with its interaction (complementary in conjunction with), the protein expression amount that need to suppress that the result has just reduced (or complete obiteration), minimizing degree (level) is decided by the expression degree of ribozyme in target cell. (for example be loaded with in the cell of TNF-Rs or FAS-R) for ribozyme being introduced in the target cell, can use various suitable carriers (vector), for example plasmid, animal virus (retrovirus) carrier. (described referring to (i), wherein virus has the cDNA sequence of coding purpose ribozyme as the second sequence). And, ribozyme is through making up, just have multiple target substrate (many target nucleus enzyme), the expression that can be used to suppress multiple protein among the present invention with or the protein expressions such as P55IC, P55DD, FAS-IC or FAS-DD (the summary method of relevant ribozyme etc. can be referring to chen etc., 1992; Zhao and pick, 1993; Shore etc., 1993; Joseph and Burke, 1993; And Koizumi etc., 1993).

(iii) they can be used to separate, identify and clone and can be incorporated into some other oroteins relevant with the intracellular signal production process, and these albumen are downstreams (downstream) of TNF-R or FAS-R intracellular region. In the case, the dna sequence dna of these albumen of encoding can be used for the two hybrid systems of yeast, and ((yeast two-hybrid system) (seeing following examples 1), namely these sequences will be used as the protein-bonded albumen coded sequence of intracellular region that " bait " (baits) separates, cloning and identification can be incorporated into these novel TNF-R or FAS-R from cDNA library or genomic library. With the method, just might determine differential protein of the present invention, that is be incorporated into the albumen of P55IC, P75IC or FAS-IC whether to be incorporated into other acceptor of TNF/NGF receptor superfamily, for example, report recently (schwalb etc., 1993; Baen etc., 1993; Crowe etc., 1994), except P55 and P75TNF-Rs, also have other TNF-Rs. Thereby, utilize the two hybrid systems of yeast can test specifically other TNF-Rs or other acceptor that albumen more of the present invention whether can the single-minded TNF/NGF of being incorporated into superfamily. And whether this method also can be used to measure protein more of the present invention can be incorporated into the known receptor that other has functional activity.

(iv) these novel proteins can be used to separate, identify and clone other albumen that belongs to together with it, can be incorporated into relevant acceptor on TNF-R or FAS-R intracellular region or the function, and some protein relevant with the intracellular signal production process. In this uses, can use above-mentioned yeast two-the hybrid system, (wilks etc., the 1989) system that maybe can use a kind of latest developments to get up, namely utilize not tight Southern hybridization (non-stringent southern hybridization), then pcr amplification. In the article of Wilks etc., evaluation and the cloning process of two kinds of generally acknowledged LCKs (kinase) had once been described, according to the known array of these kinases functional areas, i.e. a kind of kinase sequence of imagination, by using not tight Southern hybridization, then pcr amplification. Utilize the novel protein sequence according to the present invention, this method can be used to identify and clone and TNF-R, protein sequence that F AS-R is relevant, or with acceptor (TNF/NGF superfamily receptors) intracellular region in conjunction with the relevant sequence of albumen.

(v) also have another kind of approach to utilize novel protein of the present invention, be about to they be used for affinity chromatography separate and identify other can with protein or the factor of their combinations, for example separate and identify other acceptor relevant for TNF-Rs (TNF/NGF receptor superfamily), or relate to other oroteins or the factor of intracellular signal production process. In this used, novel protein of the present invention can be attached to respectively on the affinity chromatography matrix, and it is contacted with cell extract or isolated protein or the factor, and the said factor is pushed off and has participated in the intracellular signal production process. Along with the carrying out of affinity chromatography, be incorporated into other protein or the factor of novel protein of the present invention, can be by wash-out, separation and evaluation.

(vi) as top pointed, novel protein of the present invention also can be used as immunogene (antigen) and produces its selectivity antibody. These antibody also can be used for from coming from cell extract or producing these novel proteins of purifying their the transformant system. And these antibody can be used for diagnostic purpose, namely for the identification of with TNF or the not normal relevant some diseases of FAS-R aglucon systemic-function, for example hyperactivity or active excessively low TNF or the FAS-R aglucon cytological effect of inducing. Therefore, if these diseases are relevant with the imbalance of novel protein intracellular signal systemic-function, these antibody namely can be used as a kind of important medical diagnosis on disease instrument so.

Should also be noted that separation, evaluation and the sign of novel protein of the present invention, can use any known conventional screening technique. For example the two hybrid methods of yeast will be described in following examples (embodiment 1 and example 3), be used to identify novel protein of the present invention. Equally as top and following as described in, some other method is affinity chromatography for example, the DNA hybrid method, also can be used for separating, identify and characterize novel protein of the present invention, perhaps for separating of, identify and characterize the protein that replenishes, the factor, acceptor etc., these albumen, the factor, acceptor can be incorporated into novel protein of the present invention or be incorporated into some acceptors in the TNF/NGF receptor family.

The antibody of mentioning about context herein, term " antibody " comprises polyclonal antibody, monoclonal antibody (mABs), chimeric antibody, the anti-idiotypic (anti-idiotypic to some antibody, be abbreviated as anti-Id) antibody, they can soluble states or are labeled in conjunction with attitude or with the pieces of being produced by prior art. Such as being labeled synthesize by enzymatic lysis, peptide section or restructuring technology etc.

Polyclonal antibody is from the xenoantibody molecule in the animal blood serum of antigen immune. Monoclonal antibody comprises that namely they have similar epi-position (epitope) binding site in fact to the specific class homologous antibody of some antigen tool. MAbs (monoclonal antibody) can utilize method well known in the art to make. For example referring to Kohler and Milstein, " Nature ", 256:495-497 (1975); United States Patent (USP) NO.4,376,110; The editors such as Ausubel, Harlow and Lan ANTIBODIES: laboratory manual, cold spring harbor laboratory (1988); And the editor such as Coligan, immune general program, Greenes publishing Assoc.and Wiley Interscience N.Y., (1992,1993), the catalogue of these articles is all introduced specification as a reference. These antibody may belong to immune globulin Bai nationality and the subtribe thereof of IgG, IgM, IgE, IgA, GILD. A kind of hybridoma of producing mAB of the present invention can be cultivated or in vivo cultivate in vitro culture or original position. In vivo or original position cultivate and can produce high-titer mAbs, so they are the preferred methods of producing at present this mAbs.

Chimeric antibody be some different pieces derived from the antibody molecule of different animals kind, for example a class variable region is from the antibody molecule of muroid mAb and human immunoglobulin constant region. Chimeric antibody is mainly used to lower immunogenicity and is used for increasing the production yield, and for example mouse mAbs has the higher yields from hybridoma, but higher immunogenicity is arranged in human body, thereby the chimeric mAbs of people/mouse is the chimeric antibody that often uses. The production method of this chimeric antibody is known (Cabilly etc., Proc.Natl Acad.Sci.USA 81:3273-3277 (1984) technically by everybody; Morrison etc., Proc. Natl.Acad.Sci.USA 81:6851-6855 (1984); Boulianne etc., Nature 312:643-646 (1984); Cabilly etc., European Patent Application 125023 (Published November 14,1984); Neuberger etc., Nature 314:268-270 (1985); Taniguchi etc., European Patent Application 171496 (Published February 19,1985); Morrison etc., European Patent Application 173494 (Published March 5,1986); Neuberger etc., PCT Application WO 8601533 (Published March 13,1986); Kudo etc., European Patent Application 184187 (Published June 11,1986); Sahagan etc., J.Immunol.137:1066-1074 (1986); Robinson etc., International Patent Application NO.WO 8702671 (Published May 7,1987); Liu etc., Proc. Natl.ACad.Sci.USA 84:3439-3443 (1987); Sun etc., Proc. Natl.Acad.Sci.USA 84:214-218 (1987); Better etc., Science 240:1041-1043 (1988); And Harlow and Lane, Antibodies:a laboratory Manual, Supta. These articles all as a reference catalogue introduce in the specification.

Anti--Idiotype (Anti-idiotypic is abbreviated as Anti-Id) antibody, refer to identify the idiotype relevant with the antigen-binding site of antibody and determine family (determinants). The preparation of Id antibody can be by realizing as the animal (for example mouse kind) of the mutually of the same race of MAb source and homologous genes type with the MAb immunity. This immune animal is a kind of to the specific antibody of these Idiotype determinant tools (anti--Id antibody) by producing, and identifies or reply these immune antiboidies. See also for example United States Patent (USP) NO.4,699,880, all introduce as a reference herein.

Anti--Id antibody also can be used as a kind of " immunogene " and (immunogen) induces immune response in another animal body, produces so-called resisting-anti--Id (anti-anti-Id) antibody. Should resist-anti--Id may be identical with the originally mAb that brings out anti--Id on epitope. Therefore, utilize the antibody of anti-mAb Idiotype determinant, might identify other clones that express identical specific antibody.

Thereby, the mAbs of anti-IC-conjugated protein of the present invention and analog or derivative, perhaps anti-P55IC, P55DD, FAS-IC, FAS-DD, with and the mAbs of analog or derivative can be used to for example induce anti--Id antibody in the BALB/C mouse body suitable animal. These spleen cells by immunized mice can be used to produce resisting-Id mAbs of anti--Id hybridoma secretion. Further, anti--Id mAbs can be connected in a carrier such as keyhole  keyhole limpet hemocyanin (Keyhole limpet hemocyanin is abbreviated as KLH) and be used for the BALB/C mouse of adding immune. The serum that is produced by these mouse will contain anti--anti--Id antibody, the latter has the originally binding ability of mAb, and originally mAb is to above-mentioned IC-conjugated protein, its analog or derivative, and perhaps the epitope of P55IC, P55DD, FAS-IC or FAS-DD and class thing or derivative has specificity.

Anti--Id mAbs thereby their Idiotype epitope itself is arranged, or " idiotope " is (idiotopes). They structurally are similar to the epitope that will estimate, such as GRB protein-α.

Term " antibody " not only comprised complete antibody molecule but also comprise can conjugated antigen fragment, for example Fab and F (ab ')₂, Fab and F (ab ')₂Fragment, it is the FC fragment that lacks in the complete antibody, pure more transparent quickly than complete antibody capable in circulation, and have and weak non-specificly organize adhesion (non-specitic tissue binding) (Wahl etc., J.Nucl.Med.24:316-325 (1983).

It is worth mentioning that Fab and the F (ab ') of some antibody that use in the present invention according to the method that is used for the complete antibody molecule that discloses herein₂And other fragment, can be used for detection and quantitative analysis IC-in conjunction with albumen or P55IC, P55DD, FAS-IC or FAS-DD. These antibody fragments are to use proteolytic enzyme, for example papayotin (papain) (producing the Fab fragment), or pepsin (pepsin) (production F (ab ')₂Fragment) protein hydrolysate and the typical product that produces.

If an antibody can carry out idiosyncrasy with certain molecule, with this it is incorporated on the antibody, we just say that this antibody can be in conjunction with a molecule. Term (antigen) epi-position (epitope) mean can by antibody in conjunction with, can also be by the fragment of any molecule of antibody recognition. Epi-position (epitopes) or " antigenic determinant " typically refer to some chemical mobility of the surface groups such as amino acid or sugared side chain and have special three-dimensional structure and special charge characteristic.

Antigen is the part of a molecule or a molecule, and it can be by a certain antibody in conjunction with also further inducing animal to produce antibody, and this antibody can be incorporated into the epitope of corresponding antigens. Antigen can have one or more than one epitope. The idiosyncrasy above-mentioned meaning is, antigen is with its corresponding antibody response of high selectivity mode, and do not react with the antibody that is caused by other antigen.

Used all antibody in the present invention, the fragment that comprises these antibody, be used in the sample, quantitatively or qualitative detection IC-in conjunction with albumen or P55IC, P55DD, FAS-IC, FAS-DD, perhaps for detection of expressing the existence of IC-of the present invention in conjunction with the cell of albumen or P55IC, P55DD, FAS-IC, FAS-DD albumen. Detection can be passed through the immunofluorescence art, namely uses FLA (seeing following) in conjunction with finishing with detection techniques such as light microscope, fluidic cell detection technique (flow cytometric) or fluoroscopic examination arts.

Be used for antibody more of the present invention and fragment thereof, can be used on histology, as in immunofluorescence or in the micro-detection technique of Immunoelectron, detect IC-of the present invention in conjunction with albumen or P55IC-P55DD, FAS-IC, FAS-DD with former position detection method. Original position detects and namely organizes sample by cutting from the patient with it, then adds labelled antibody of the present invention and finishes. The best approach that adds antibody (or its fragment) is that antibody (or fragment) with mark is attached to or is superimposed on and organizes on the sample. Whereby, not only may measure IC-in conjunction with the existence of albumen or P55IC, P55DD, FAS-IC, FAS-DD, but also may record it in studied structural distribution. Utilize the present invention, those of ordinary skill can recognize easily that also any (for example decoration method) in the various Histological methods all can be modified to carry out original position and detect.

Being used for IC-of the present invention typically comprises in conjunction with the determination method of albumen or P55IC, P55DD, FAS-IC, FAS-DD, at first can identify in the presence of the antibody of IC-in conjunction with the detectable label of albumen or P 55IC, P55IC, FAS-IC, FAS-DD, culture sample, such as biological fluid, the cell of organizing extract, fresh collection such as lymphocyte or leucocyte, or the cell that in the group training, has cultivated, then detect antibody with any known technology.

Biological sample can be with solid support or carrier such as nitrocellulose, or other solid support or carriers that cell, cell granulations or soluble protein are fixed up are processed. Then these holders or carrier are washed with the buffer solution that is fit to, process with detectable labelled antibody subsequently, according to the present invention, as above-mentioned. Then solid support or carrier are carried out the washing second time with buffer solution, to remove not in conjunction with the antibody that gets on. Be combined in the amount of the label on solid support or the carrier, available commonsense method detects it.

Said " solid support ", " solid phase carrier ", " solid support ", " solid carrier ", " holder " or " carrier ", should extend to any can conjugated antigen or holder or the carrier of antibody. Known holder or carrier comprise glass, polystyrene, polypropylene, polyethylene, glucan, Nilong, amylase, natural and cellulose family modified, polyacrylamide, gabbro, magnetic iron ore. The character of carrier can be solubility on certain program, also can be insoluble. Supporting material, in fact can be any possible node configuration, as long as make the molecule of connection can be incorporated into antigen or antibody. Therefore, the configuration of holder or carrier can be spherical such as pearl, or columnar, for example puts into the inner face of test tube, perhaps is placed on the outside of bar rod. Another situation is, their surface can be flat, such as sheet, the test strip etc. Preferred holder or carrier are the polystyrene colloidal pearls. The personnel that present technique is familiar with will know that other many suitable carriers can be used for binding antibody or antigen, or by determining same carrier with normal experiment.

The combination of the above-mentioned many antibody that provide of the present invention is active, can measure according to known method. People to the art is familiar with can both utilize normal experiment to determine optimal operation and condition determination.

Other operating procedure, such as wash, stir, shake, filtration etc., can take the circumstances into consideration by convention increase and decrease.

According to the present invention, one of detection method of labelled antibody is antibody to be connected with enzyme then be used for enzyme immunoassay (EIA). This kind of enzyme when when suitable substrate contacts, just reacts with this kind of enzyme agent and produces a kind of chemical cracking thing, and this lysate can be detected, and for example measures it with AAS, fluorescence method or visual method. Can be as the enzyme of labelled antibody detection, comprise, but be not limited to malic dehydrogenase, staphylococcal nuclease, δ-5 steroid isomerase, Alcohol Dehydrogenase from Yeast, α-glycerophosphate dehydrogenase, triose-phosphate isomerase, horseradish (horseradish) peroxidase, alkaline phosphatase (ester) enzyme, asparaginase, galactosidase, ribalgilase, urase, catalase, glucoamylase, acetylcholinesterase. Detect available colorimetric method, namely enzyme is used the zymolyte that adds lustre to. Detect and also can relatively finish by observability, be about to the enzyme reaction degree that enzyme acts on substrate and make comparisons to the similar standard that makes.

Detect and also as seen carry out with any other immunoassays. For example utilize radiolabeled various antibody and antibody fragment, might detect RPTP enzyme (R-PTPase) by using radioimmunoassay (radioimmunoassay is abbreviated as RIA). Portion to RIA is described document preferably, can be referring to work, T.S. wait the report of people in " Laboratory Techniques and Biochemistry in Molecular Biology ", North Holland Publishing company, NY (1978), specifically with reference to Chard, T shows and is entitled as " An Introluction to Radioimmune Assay and Related Techniques " chapter, and this paper is the listed in reference catalogue. Utilize r counter or scintillation counter or with autoradiograph, also can detect emitting isotope.

According to the present invention, also may come labelled antibody with fluorescent chemicals. When the antibody with the fluorescent chemicals mark is exposed to lower time of illumination of suitable wavelength, because the generation of fluorescence just can detect its existence. The fluorescence labeling compound that the most generally uses has fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allos phycocyanin (allophycocyanin), ortho-, meta-or p-phthaladehyde and fluorescamine.

Utilize fluorescent radiation metal such as 152E or other lanthanide series, also can make antibody make detectable label. Utilize chelation group such as the diethylene-triamine pentaacetic acid (diethylenetriaminepentaacetic acid, ETPA) of these metals, just these metals can be connected in antibody.

By connecting antibody in chemiluminescence compound, also antibody can be made detectable label. By detecting the light that produces in the chemical reaction process, just can record the antibody of chemiluminescent substance-mark. Useful especially chemiluminescent labeling compound has luminol, different luminol, acridinium ester (theromatic acridinium ester), imidazoles, acridinium salt and oxalate.

Equally, bioluminescent compound also can be used to mark antibody of the present invention. Bioluminescence is a class chemiluminescence phenomenon that comes across in the biosystem, and wherein a kind of catalytic protein mass-energy increases chemiluminescent efficient. The existence of bioluminescent protein matter can record by the appearance that detects light. Be used for the important biomolecule luminophor of mark purpose, fluorescein, luciferase, aequorin (aequorin) are arranged.

Antibody molecule of the present invention is applicable to immune quantitative determination, also is called " two-position " (two-site) or " interlayer " (sandwich) measured. In typical immune quantitative determination, a certain amount of unmarked soluble antibody (or antibody fragment) is incorporated into solid support or carrier, add a certain amount of detectable solubility tags antibody, make might detect and/or quantitative assay at insolubilized antibody, antigen, and mark antibody between formed ternary complex (ternary complex).

Typical and preferred immune quantitative determination method comprises " forward " (forward) determination method, the antibody that will be incorporated into therein on the solid phase at first contacts with the sample of wish test, in order to by forming binary (binary) solid matrix antibody-antigen compound, from sample, extract antigen. Through after the suitable cultivation, solid support or carrier are washed to remove the residue of fluid sample, comprise unreacted antigen (if any), and contact with the labelled antibody that contains unknown quantity (it act as a kind of " reporter molecules ") solution. After second time culture period, allow labelled antibody carry out compound (complex) by unlabelled antibody and the antigen that is incorporated on solid support or the carrier, then solid support or carrier are carried out the washing second time to remove unreacted labelled antibody.

In another kind of " interlayer " determination method, used (reverse) determination method of so-called " simultaneously (simultaneous) " determination method and " oppositely ", " interlayer " determination method and antigen of the present invention all are useful. When the antibody that is incorporated into solid support or solid carrier and labelled antibody join in the sample to be tested simultaneously, " simultaneously determination method " contains an independent incubation step, after cultivation is finished, the labelled antibody that solid support or carrier are washed to remove the residue of fluid sample and compound action does not occur. Then, the labelled antibody that associates according to common " forward " sandwich assay method mensuration and solid support or carrier.

In " oppositely " (reverse) in the determination method, be to adopt progressively additive process, at first labelled antibody is joined in the fluid sample, after suitable culture period, add the unlabelled antibody that is incorporated on solid support or the carrier. After cultivating for the second time, solid phase is washed to remove residue and the unreacted labelled antibody of wanting the test sample product in normal way. Then the labelled antibody of using " simultaneously " determination method and " forward " determination method mensuration and solid support or carrier to associate mutually.

Novel protein of the present invention, in case with any conventional screening technique, for example yeast two-after hybrid method, affinity chromatography and any known technology separate, identify and characterize, just can produce in a large number with conventional recombinant DNA technology (such as referring to Sambrook etc., 1989), namely utilize suitable eucaryon or prokaryotic vector (containing the sequence to protein coding) to transform suitable eucaryon or protokaryon host's cell. Thereby, the invention still further relates to for the production of more such expression vectors of albumen more of the present invention and the host cell that transformed. As mentioned above, these protein comprise that also they have bioactive analog and derivative, and have the carrier of corresponding encoded sequence and for the production of the conversion of these analogs host cell, the derivative of these protein refers to modify the product that produces with protein or its analog that conventional modification method is produced the host cell after transforming.

On the other hand, the present invention also addresses the intracellular region (FAS-IC) of the intracellular region (P55IC) that uses P55TNF-R or FAS-R or their what is called " dead functional domain " (being respectively P55DD or FAS-DD) and strengthens TNF or FAS-R aglucon to the effect of cell as reinforcing agent (agent for enhancing), and to itself effect (seeing embodiment 2). When the somewhere need to be introduced that TNF brings out or during cytotoxic effect that the FAS-R aglucon brings out, for example in the cancer cell or in the HIV-infection cell, utilize above-mentioned (seeing above-mentioned (i)) recombinant animal virus (for example cowpox) approach just P55IC, P55DD, FAS-IC or FAS-DD can be introduced in these cells. Also can use natural P55IC, P55DD, FAS-IC or FAS-DD herein, and have bioactive analog and derivative or fragment, all these materials can prepare as stated above.

Equally, the invention still further relates to the specificity of TNF effect or FAS-R aglucon effect blockade (specific blocking) effect, the activity of namely blockading P55IC, P55DD, FAS-IC or FAS-DD, for example some Antisensedigonucleotsequence sequences can be introduced the expression of blockading P55IC, P55DD, FAS-IC or FAS-DD in these cells.

The invention still further relates to some pharmaceutical compositions, they comprise some coding TNF-R or FAS-R intracellular regions in conjunction with albumen (comprising P55IC, P55DD, FAS-IC and FAS-DD) recombinant animal viral vectors, and these carriers are also encoded and can be guided in conjunction with particular target cell (for example cancer cell) surface protein intracellular region to embed virus surface proteins in some cell in conjunction with protein sequence.

On the other hand, the present invention also at length addresses the effect of intracellular region (P55IC sees example 2) self association of P55TNF acceptor. For example the encode expression of IL-8 gene, this effect normally is incorporated into its receptor-mediated result with TNF, and also can be imitated by the signal activity that P55-IC or its fragment self-association are produced.

IL-8 is a kind of cytokinin (cytokine), it belongs to chemical kinetin (chemokines) subclass, have basic chemotactic activity (chemotactic activity) and proved in the chemotaxis (chemotaxis of granulocytes) of granular cell and the cell relevant with many pathological states and played an important role (referring to such as Endo etc., 1994; Sekido etc., 1993; Harada etc., 1993; Ferrick etc., 1991).

TNF has useful activity, as can be used as that treatment comes tumors destroyed cell and virus infected cell or for increasing the antibacterial activity of granular cell. But as above-mentioned, TNF also has undesirable activity, wishes to blockade in the case these activity, comprises and uses heavy dose of TNF to be used in the situations such as treatment of cancer, antiviral therapy or antibacterial therapy.

Therefore, the material that hope may guide TNF or simulate its useful activity is in need cell or tissue to be processed, and this is useful especially to treatment.

According to the present invention, have now found that P55-R from-association intracellular region (P55-IC) can be not rely on the mode of aglucon, many effects of simulation TNF, for example P55-IC " dead functional domain " can bring out cytocidal effect, and P55-IC can induce IL-8 gene expression. Thereby, might utilize P55-IC to simulate the TNF function in the point location mode, that is induce P55-IC only the cell or tissue of needs treatment to be worked.

One of above-mentioned approach, as mentioned above, dna molecular or its a part of specificity transfection tumor cell or the malignant tissue with coding P55-IC, they not only can be induced these cell or tissue cytocidal effects, and can also by with IL-8 altogether-inducing action (co-induction) strengthens these effects, the result causes having put aside granular cell and other lymphocytes at this cell or tissue, and they will be used for the kill tumor cell or tissue successively. This approach can eliminate heavy dose of TNF of use the side effect of murdering with effect.

Utilize conventional recombinant DNA technology, the various sections (regions) that might prepare P55-IC, and determine which section is relevant to the effect that each TNF-brings out on earth, for example we record " dead functional domain " relevant with cytotoxicity (example 2), and prepared various other and contained the construct of part P55-IC, these parts (with part or all of dead functional domain) are the major reasons that causes other TNF-effects, and the mode that they can not rely on aglucon is used, in case generation self-association, to bring out these effects, for example the induced expression of IL-8.

Should be understood that, induce the sequence of relevant P55-IC to be different from the sequence of inducing relevant P55-IC with cell toxicant with other TNF correlation effect, that is may not comprise or comprise part " dead functional domain " and have sequence motif (motits) from other section of intracellular region, perhaps also may be identical sequence, the different characteristic of this sequence (identical sequence primitive) relates to inducing of different effect.

Thereby, describing in detail as context, the expression vector that contains P55-IC fragment, its analog or derivative can be produced, expression, purifying and its activity tested. Like this, can prepare the multiple P55-IC fragment with one or more TNF-related activity and be used for the treatment of by different way various diseases, such as virus infections, bacterium infection, tumour etc. In all these situations, the increase of specific activity can be by being combined (or being total to-transfection) realization with the P55-IC fragment, the P55-IC fragment is the main cause of inducing IL-8 gene expression, and the chemotactic activity of IL-8 can be used for strengthening the destruction of the cell or tissue that needs are killed.

Like this, need not systematically to use TNF, just might bring out its useful effect by introducing all or part of P55-IC in the cell or tissue of needs treatments.

By one of above-mentioned any method, P55-IC all can be introduced into specifically and wish to kill in the cell or tissue. For example a kind of method is by making up recombinant animal virus, for example from a kind of virus of cowpox, can introduce following two kinds of genes in its DNA: a kind of aglucon of gene code can selectivity be incorporated into cell surface protein, for example can specific binding in some cell (CD₄Lymphocyte and relevant leukaemia) AIDS virus gp120 albumen, perhaps coding can specific binding in the aglucon of the cell that is loaded with T NF-R, such recombinant virus carrier can carry in conjunction with some the cell of TNF-R; Another gene code P55-IC or its fragment. Like this, the protein-bonded expression of the cell surface on virus surface will make virus aiming attack tumour cell or other carry the cell of TNF-R, and P55-IC or its a part of coded sequence will be introduced these cells by virus subsequently. And, just can strengthen TN F effect and cause tumour cell or other to be loaded with the death of TNF-R cell in case in these cells, be expressed, perhaps induce IL-8 and cause these cell deaths. Make up this recombinant animal virus, available routine operation technology (referring to such as Sambrook etc., 1989). Another kind of possibility is with the oligonucleotides form P55-IC sequence or its part to be introduced cell, and this oligonucleotides can be admitted and be expressed in wherein by cell.

Thereby the present invention also is specifically related to some pharmaceutical compositions, above-mentioned this carrier of recombinant animal viral vectors that includes coding P55-IC or its part virus surface proteins of also encoding, this albumen can guide P55-IC in conjunction with special target cell (for example cancer cell) surface protein, or the coded sequence of its fragment embeds these cells.

The present invention also relates to some novel synthetic TNF acceptors on the other hand, it be solubility and can form dimer, also may form more senior poly TNF acceptor molecule, each monomer segment of these acceptors can both be incorporated into a TNF monomer. Spontaneous TNF is a kind of homotrimer, contain three active TNF monomers, each can both be incorporated into a TNF acceptor molecule, and more spontaneous TNF acceptors are monomer, and each only can be in conjunction with a monomer in the TNF homotrimer molecule. Thereby, when TNF is incorporated into some TNF acceptors on the cell surface, just can be incorporated into three acceptor molecules, the result just forms cluster TNF acceptor, and this is considered to the beginning of signal production process, and this process is final to cause viewed TNF effect.

TNF has many useful effects on the one hand, for example it can killing off tumor cells or virus infected cell and the antibacterial activity that increases granular cell, yet TNF also has many adverse effects, such as comprise autoimmunity disease, rheumatoid arthritis, transplant-antagonism-host response (naltrindole), septicaemia shock etc. in many serious diseases, TNF is considered to the main cause that pathological tissue damages. TNF also can cause seriously become thin (cachexia Cachexia), and this is owing to suppressed the activity of adipocyte. And, even need active and when using according to it, for example in the various pernicious or viral disease situations for the treatment of, used TNF dosage is enough height usually, so that in patient body, cause many disadvantageous cytotoxicity side effects, for example to the damage of health tissues.

In all above-mentioned situations, the effect of TNF is disadvantageous, therefore once seeks the effective inhibitor to TNF. Many TNF-blocking agents (TNF-blocking agents) are suggested, the protein that comprises some solubilities, they can in conjunction with TNF, suppress TNF and be incorporated into its acceptor, thereby the cellulotoxic effect (seeing EP308378, EP398327 and EP568925) that suppresses TNF. Yet these TNF are in conjunction with albumen, or soluble TNF acceptor, all are monomers, and each can only be in conjunction with a monomer in the TNF homotrimer. Thereby blockading of TNF function may be not exclusively, and the TNF molecule after each monomeric acceptor body combination still has two TNF monomers and is in free state, can also in conjunction with cell surface TNF acceptor, produce disadvantageous side effect to cell.

Above-mentioned shortcoming when blockading the TNF function in order to overcome, the present invention has developed a method and has been used for making up some soluble oligomeric TNF acceptors, be some fused proteins, they can be in conjunction with at least two TNF monomers in the spontaneous TNF homotrimer molecule. As a result, these soluble oligomeric TNF acceptors in conjunction with albumen or acceptor, just can be incorporated into their TNF aglucon than previously known monomer soluble TNF more consumingly. For example, when soluble TNF acceptor of the present invention is when being in dimeric forms, just might be in conjunction with trimerical two monomers of TNF, thereby cause more completely TNF neutralization. Because the dissociation rate of solvable dimerization acceptor from TNF is lower, therefore this neutralization is more lasting. And this soluble oligomeric acceptor is compared with their monomer counterpart, and molecular weight is larger, and this also has advantage on materia medica, because during their removings in the human body, probably has slower speed.

The development of soluble oligomeric TNF acceptor of the present invention is based on following discovery: the intracellular region of P55-RTNF acceptor can self-association, and further there is a section in discovery in this intracellular region (P55-IC), be called " dead functional domain " (death domain), it also can self-association also can produce in the mode that does not rely on aglucon cytotoxic effect (seeing embodiment 2). Utilize this self-association performance of P55-IC and " dead functional domain " thereof, by conventional recombinant DNA technology, just might make up a kind of fused protein, make it contain whole extracellular regions of TNF acceptor, some acceptors of P75-R or P55-R for example, P55-R preferably, and be blended in the dead functional domain of whole intracellular regions (P55-IC) or P55-IC. So just, can produce a kind of New Fusion product, its end has the TNF land, i.e. the extracellular region of acceptor, and the other end has intracellular region or its dead functional domain, and self-association can occur. Therefore, by the self-association between two (and possibility is more than two) P55-IC or its dead functional domain polymerization occurs, thereby obtain some oligomers that have simultaneously at least two TNF lands (perhaps being at least dimer).

Further, according to the present invention, found once also that the Fas/AP01 acceptor also had self-association, its intracellular region contains self-association " dead functional domain ", and with P55-IC and dead functional domain thereof some homology (homology) (embodiment 2) is arranged.Thereby, might construct soluble oligomeric TNF acceptors more of the present invention by the extracellular region (as mentioned above) of fusion TNF acceptor and intracellular region or " the dead functional domain " of Fas/AP01 acceptor.

In the above two kinds of cases, oligomeric TNF acceptor of the present invention, it all is solubility, in fact only have the two extracellular soluble inner region or its " dead functional domain " of the extracellular soluble outskirt of TNF acceptor and P55-RTNF acceptor or Fas/AP01 acceptor, that is they do not contain transmembrane (transmembranal) district or the insoluble zone of two types of acceptors.

The structure of the above-mentioned oligomeric TNF acceptor of the present invention is specified in the following examples 4.Still need and point out, when making up oligomeric TNF acceptor of the present invention, also can produce a kind of situation, this situation does not still have report before this, it is the extracellular region energy self-association of TNF acceptor, this situation may be disadvantageous, because of this can disturb low oligomerization receptors bind in the plural TNF monomer of homotrimer molecule, perhaps can cause weakening with TNF monomer binding ability.Thereby, in this case, might utilize conventional recombinant DNA technology to modify the extracellular region of TNF acceptor, for example by the one or more amino-acid residues in deletion or the replacement self-association section, to prevent such self-association phenomenon.This modification of the extracellular region of TNF also is a partial content of the present invention, and is named as the analogue or the derivative of the extracellular region of TNF acceptor.Equally, the self-association intracellular region (IC) of P55-R acceptor or Fas/AP01 acceptor or its dead functional domain (DD), be used for oligomeric TNF acceptor of the present invention, they also can be its analogue or derivative, that is can be that P55-IC sequence or it are comprised the segmental modified outcome of dead functional domain (P55DD) or Fas/AP01 intracellular region (FAS-IC) sequence or it are comprised the segmental modified outcome of dead functional domain (FASDD), if these modifications can obtain a kind of self-association product.

Equally, soluble oligomeric TNF acceptor, its analogue or derivative, in case produce and the process purifying, just can utilize the conventional chemical method to make the further salt and the functional deriv of modification so that they to be provided, be used to prepare some pharmaceutical compositions, these pharmaceutical compositions all contain TNF acceptor of the present invention as activeconstituents.

In order to produce soluble oligomeric TNF acceptor of the present invention, the dna sequence dna of coding TNF acceptor extracellular region, be the existing clone who derives from all TNF acceptors, equally also obtain the dna encoding sequence of its intracellular region or dead functional domain, and the dna encoding sequence of the intracellular region of Fas/AP01 acceptor or dead functional domain (seeing embodiment 2 and embodiment 5).With such method, the dna encoding sequence of required extracellular region can be connected in required intracellular region or it comprises the segmental dna encoding sequence of dead functional domain, this fusion product is embedded in (with being connected in) suitable expression vector under the regulation and control of promotor (promoter) and other expression regulation sequence.In case form, expression vector just is introduced into (conversions, transfection etc.) in appropriate host cell, expression vector then, and obtain fusion product of the present invention, promptly some solubilities oneself-association TNF acceptor molecule.Utilize routine operation that these molecules purifying from host cell is come out then, and produce final product, i.e. the oligomeric TNF acceptor of some solubilities.

The better preparation method of coding extracellular region and intracellular region or its a part of fusion product is according to the round pcr route, utilizes required sequence-specific oligonucleotide is made probe.From the clone of all TNF acceptor molecules of encoding, duplicate required sequence.Another kind method also is possible, the required part of for example separating coding extracellular region and intracellular region by restriction endonuclease, by known way they are connected together then, on the endonuclease bamhi end, modify or do not modify, to guarantee the correct fusion that needs part (born of the same parents outer and intracellular region or its some parts) of acceptor.The fusion product intercalation that will obtain is so then gone in the selected expression vector.

With the same manner, the invention still further relates to some soluble oligomeric Fas/AP01 (FAS) acceptors, they contain the extracellular region of Fas/AP01 acceptor and P55-R from-association intracellular region (P55-IC) and dead functional domain (P55DD) thereof, perhaps the Fas/AP01 acceptor from-association intracellular region (FAS-IC) or its dead functional domain (FASDD), or their any analogue or derivative (seeing above-mentioned).The structure of these soluble oligomerics FAS acceptor, be specified in the following examples 5, promptly use FAS acceptor-encoding sequence total length as original material, suitable oligomer primer is used for outside the required born of the same parents and the pcr amplification of intracellular region encoding sequence, then they are coupled together and obtain fusion product, this product intercalation is gone in the suitable expression.

As contextual detailed description, protokaryon or eukaryotic cells carrier and host cell can be used to produce needed soluble oligomeric FAS acceptor, allocate in the pharmaceutical composition with these acceptor purifying with as activeconstituents then.

Above-mentioned soluble oligomeric FAS acceptor of the present invention, plan be used for blockading the effectively aglucon of Fas, also can there be (be similar to TNF, see above-mentioned) with tripolymer in this aglucon, each oligomerization acceptor of the present invention all can be in conjunction with two or may more Fas aglucon, and with this their activity of neutralizing.The Fas aglucon known mainly with cell-surface association, but also can exist with soluble form.Come what may, oligomerization FAS acceptor of the present invention can be incorporated at least two kinds of monomers of this aglucon and with this more effectively in (the monomer FAS acceptor that compares) and the activity of Fas aglucon.The Fas aglucon, activation with the FAS acceptor, involve numerous disease, disease (for example hepatocellular apoptosis (apoptosis) of particularly relevant liver injury, comprise the liver injury relevant with hepatitis, and the autoimmunization symptom, the lymphocyte damage (apoptosis) that is included in the HIV infection human body (is for example seen Ogasawara etc., 1993; Cheng etc., 1994).Therefore soluble oligomeric FAS acceptor of the present invention is intended being used to seal Fas aglucon activity, and the activeconstituents of useful as drug composition, be used for treatment for example with Fas aglucon diseases associated.

Equally, the invention still further relates to the oligomerization acceptor of some solubilities, they have affinity to TNF and FAS-R aglucon, and these oligomerization acceptors are exactly so-called " mixing " TNF-R/FAS-R oligomer acceptor.These oligomer acceptors contain at least one TNF-R extracellular region and at least one FAS-R extracellular region, they can by means of each extracellular region in the oligomerization acceptor with above-mentioned from-one of associating P55IC, P55DD, FASIC or FASDD merge and mutually combine.

These mix the oligomer acceptor and can prepare as follows: any above-mentioned fusion product (a) is provided, they contain the extracellular region (P75TNF-R of TNF-R, or be preferably P55TNF-R) be blended in any one from-association intracellular region P55IC and FASIC or any one from-" dead functional domain " P55DD and FASDD associates, or it is any from-association fragment, any their analogue or derivative, (b) provide any above-mentioned fusion product, they contain the extracellular region of FAS-R and are blended in any one from-association P55IC, FAS-IC, P55DD and FASDD, perhaps any from-association fragment or their any analogue or derivative, and (c) any TNF-specificity fusion product in (a) is mixed with any FAS-R aglucon-specificity fusion product in (b), with provide (routine separate and purification process after) oligomerization (dimer or more high-grade polymer) acceptor, they have the two extracellular region of TNF-R and FAS-R at least, the mutually combining from-association of IC that merges by means of their or DD section.

Be used to prepare the another kind of possibility of above-mentioned mixing oligomerization acceptor, be that appropriate host cell is carried out cotransformation (co-transforming) with above-mentioned expression vector, the specific TNF-fusion product of one of expression vector coding TNF-, another is encoded to FAS-R aglucon-specific FAS-R fusion product.Along with these expression of different fusion products in host cell, mix conventional purifying of oligomerization (TNF-R/FAS-R) acceptor utilization and isolation technique and make.

These purposes of mixing affinity oligomerization acceptor mainly are in being used for and TNF and FAS-R aglucon, use when especially giving birth to overexpression or reaching excessive level in these two takes place after external source is used.Recently evidence is pointed out a kind of possibility, thinks that between FAS-R aglucon (normally combine with cell surface) and TNF-α (it also may be to combine with cell surface) existence acts synergistically on function.Thereby, in some cases, need on the identical point of cell surface, to neutralize simultaneously two kinds of aglucons, that is need a kind of like this TNF that can either stop to be incorporated into its acceptor, can stop the FAS-R aglucon to be incorporated into the mixing affinity acceptor of its acceptor again.

Therefore, these mix the affinity acceptor, in pharmaceutical composition, can be used as a kind of activeconstituents, are used for the treatment of above-mentioned symptom (seeing above-mentioned), and promptly TNF and FAS-R aglucon effect are disadvantageous situations.

Equally, series based on soluble oligomeric TNF-R of the present invention and FAS-R and mixing TNF-R/FAS-R oligomer, might produce the soluble oligomeric acceptor that some are used for other acceptor, perhaps their mixture, particularly any other member's of TNF/NGF superfamily acceptor.In the case, any extracellular region of various acceptors can both be blended in above-mentioned from-association intracellular region or its some section, perhaps being blended in can be from any other intracellular region of-associating superfamily member.

Any recombinant protein of the invention described above can both be expressed in eukaryotic cells (for example yeast, insect or mammalian cell) by suitable expression.Any relevant known technology all can be used.

For example, will utilize technology known in the art to insert in the suitable expression vector (seeing Sambrook etc., 1989) with the dna molecular of the coded protein of above-mentioned any method gained.By the afterbody (tailing) of homopolymer, perhaps, double-stranded cDNA can be connected in the plasmid vector (plasmid vectors) by using synthetic DNA ligase enzyme or blunt ends interconnection technique (blunt-ended ligation) to limit connection.Dna ligase is to be used for connecting dna molecular and will to avoid unwanted connection by using alkaline phosphatase (ester) enzyme to handle.

In order to express the protein that needs, expression vector also should comprise the nucleotide sequence that some are special, and the latter includes the desired protein tool is transcribed dna sequence dna with translational control information, and many genes are expressed.At first, transcribed for making gene, the promotor of being discerned by RNA polymerase (promoters) must come into play, and then polysaccharase is incorporated into this promotor and starts and transcribes.There are many such promotors using their starting efficiency difference (strong or weak promotor).The promotor of prokaryote and eukaryotic cells also is different.

To can be used in promotors more of the present invention both can be composing type, for example phage (bacteriophage) IntThe beta lactamase of promotor, PBR322 BlaThe chloramphenicol acetyl-transferase gene of promotor and PPR325 CATPromotor etc.; Can be again (inducible) of induction type, prokaryote promotor for example, comprise the main right side of phage and left promotor (PL and PR), Trp, RecA, LacZ, LacL, OmpEAnd intestinal bacteria gal promotor, perhaps Trp-lacMix (Glick, B.R. (1987)) such as promotors.Except that using strong promoter to produce a large amount of mRNA, also need use rrna-land to guarantee that mRNA is translated effectively in order to reach in prokaryote high-caliber genetic expression.One example be from start code (codon) suitably localized and with the Shine-Dalgarno sequence (SD sequence) of 16SRNA 3 '-end sequence complementarity.

For the eukaryote host cell, can use the different regulating and controlling sequences of transcribing and translate according to host's character.They can be from viral source, for example adenovirus, bovine papilloma virus, simian virus etc., and wherein conditioning signal and high level (high level) gene of expressing is relevant.The TK promotor of simplexvirus for example, SV40 early promoter, yeast gal4 gene promoter etc.Consider that transcribing the conditioning signal that begun suppresses or activation, need select so that can regulate and control expression of gene it.

The dna molecular that includes coding fusion product of the present invention proteinic nucleotide sequence is inserted into and has in the carrier of transcribing and translating conditioning signal that can carry out attended operation, and these signals can be integrated some required gene orders and enter in the host cell.By the cell of the DNA stable conversion of introducing, can be selected by introducing one or more marker gene (markers).Marker gene can provide nutrition or to the resistibility (biocideresistance) of biocide to auxotrophic host, for example to microbiotic, or the resistance of heavy metal such as copper etc.Selectable marker gene can be directly connected in the dna sequence dna that will express, also can introduce same cell by common-transfection (cotransfection).In order to synthesize protein of the present invention best, also need some additional elements.These key elements can comprise transcripting promoter, enhanser (enhancer).The cDNA expression vector of these key elements of tool comprises Okayama, the carrier that H (1983) is addressed.

In a preferred embodiment, the dna molecular of introducing will be integrated in a plasmid or the virus vector, and this plasmid or virus vector can duplicate in recipient cell independently.Select the important factor of certain plasmid or virus vector to comprise: easiness, the recipient cell that promptly contains carrier is identified easily and separates from carrier-free recipient cell; Whether needed multiple copied number can be between host cell not of the same race " transfer " with it in special host.

Preferred prokaryote carrier comprises some plasmids like this, the plasmid that if can in the intestinal bacteria body, duplicate, and PBR322 for example, COLE1, PSC101, PACYC184 etc. (see Maniatis etc., 1982; Sambrook etc., 1989); Bacillus (Bacillus) plasmid such as PC194, PC221, PT127 etc. (Gryczan, T. (1982)); The streptomyces plasmid comprises PLI101 (Kendall, K.J. etc., (1987)); Streptomyces phage is as φ C31 (Chater, K.F. etc., and Pseudomonas plasmid (John, J.F. etc. in:Sixth Internationalsymposium on Actinomycetales Biology (1986)),, (1986) and Izald, K. (1978)).Preferred eukaryotic cells plasmid comprises BPY, cowpox, SV40,2 microns rings (2-micron circle) etc., perhaps their derivative.These plasmids are known (Botstein, D. etc., (1982) in the present technique field; Broach, J.R.in:The Molecular Biology of the YeastSaccharomyces:Life cycle and Inheritance (1981); Broach, J.R. (1982); Bollon, D.P. etc., (1980); Maniatis, T., in:Cell Biology:A Comprehensive Treatise vol.3, Gene Expression, (1980); And Sambrook etc., (1989)).

In case carrier or the dna sequence dna that contains construct are used for expressing, then DNA construct just can be introduced in the appropriate host cell, uses any appropriate means: conversion, transfection, joint, protoplastis (protoplast) fusion, electric shocking method (electroporation), calcium phosphate precipitation method, direct micro-injection etc.

Being used for host cell of the present invention can be prokaryote, also can eukaryotic cells.Preferred prokaryotic organism host comprises the bacterium class, for example intestinal bacteria, Bacillaceae, streptomyces, Pseudomonas, Salmonella typhimurium (Salmonella typhimurium), serratia marcescens genus (Serratia marcescens) etc.Most preferred prokaryotic organism host is intestinal bacteria.Host bacterium with special role comprises e. coli k12 strain 294 (ATCC3146), intestinal bacteria x1776 (ATCC31537), intestinal bacteria W3110 (F ^-, lambda ^-, prototropy (prototropic) is (ATCC27325)), and other enterobacteria such as Salmonella typhimurium or serratia marcescens and various false monospore kind.Under these conditions, protein does not need glycosylated.The prokaryotic organism host must be complementary with the regulating and controlling sequence in replicon (replicon) and the expression plasmid.

Preferred eukaryote host is a mammalian cell, people, monkey, mouse and Chinese hamster ovary (Chinese hamster ovary for example, be abbreviated as CHO) etc. cell, because they can be translated the back to protein and modify, be included in correct position folding (tolding) or glycosylation and use.Yeast cell also can be translated the back and be modified, and comprises glycosylation.Existing multiple recombinant DNA strategy is to utilize the height of strong promoter sequence and plasmid to duplicate number, and the latter can be used in and produce needed protein in yeast.Yeast can discern the peptide class that some leading sequence justacrines on clone's the mammalian genes product go out to contain leading sequence (that is preceding-peptide class, pre-peptides).

After introducing carrier, host cell is grown in selecting substratum, and this substratum can be used for selecting to contain the cell of carrier.The expression of cloned genes sequence has caused required proteinic production.

Purifying recombinant proteins can carry out according to any of the multiple currently known methods that is used for this purpose, that is any routine operation technology, comprises extraction, precipitation, chromatography, electrophoretic method etc.Proteinic another ideal operation technology of purifying the present invention is to use the affinity chromatography of the monoclonal antibody of anti-TNF acceptor, monoclonal antibody production and being fixed in the gel matrix that is contained in the chromatographic column.The impure prepared product that contains recombinant protein flows out by chromatographic column.Protein is incorporated on the post by specific antibodies, and impurity is removed by pillar.After flushing, protein is because PH changes or ionic strength changes and elute from gel.

Here employed (seeing above-mentioned) term " salt " (Salts) is meant carboxyl salt and add salt by the proteinic amino acid whose acid that currently known methods forms.Carboxyl salt, comprise inorganic salt such as sodium, calcium, and with the salt that organic bases forms, for example, resemble trolamine with amine, the salt that arginine or Methionin form, acid interpolation salt comprises, for example inorganic acid salt and organic acid salt.

Used here " functional deriv " comprised the derivative of preparing from functional group; these functional groups are the known technologies by this area; be as the side chain on the amino-acid residue; or the side chain on the terminal group of N-terminal group or the C-base and existing; and as long as they have kept medicinal acceptability; promptly; injury protein is active and do not authorize toxicity to the component that comprises medicine for they; they will all be contained among the present invention; these derivatives comprise fatty acid ester or amides, and the O-acyl derivative of the N-acyl derivative of free amino group and acyl group fragment (for example alkanoyl or carbocyclic ring aroyl) formation or free hydroxyl class and the formation of acyl group fragment.

Used here " part " refers to any fragment or the part of acceptor, (its intracellular region or extracellular region), or with the proteic part of acceptor intracellular region bonded, if this receptor can keep its bioactive work.

As mentioned above, the present invention relates to various medicinal compositionss, these medicinal compositionss comprise medicinal upward acceptable carrier, and the various activeconstituentss that the present invention mentioned, or its esters, the mixture of functional deriv or above-mentioned substance.Any situation that these components can be used for here being mentioned, for example, the excessive synthetic situation of endogenous TNF resembles sepsis shock disease, emaciation, the transplant reaction active immunity disease to the host, resembles rheumatoid arthritis etc.The medicament administration mode can be by the method for application of received any similar medicament, and also depend on situation to be processed, for example, when being used to suppress the TNF effect, in sepsis shock disease case, they are handled as intravenously, or can be used as local injection (for example in knee) in rheumatic arthritis, or infuse continuously etc.These compositions also can be used for as being used in the TNF poisoning case that excessive TNF causes by external, resemble in cancer therapy or virus disease treatment case.

During as medicament administration, medicinal compositions of the present invention is prepared from by albumen or derivatives thereof and medicinal acceptable carrier, tranquilizer and the mixed with excipients of going up, and is prepared into dosage form, as storing in the bottle with the dry freeze form.The amount of application of active compound depends on application, the disease of treatment and patient's situation.For example, under the rheumatic arthritis inflammation situation, the active compound local injection consumption of basic body weight will lack than the intravenous infusion consumption that sepsis is suffered a shock in the disease case.

To embody others of the present invention more significantly in the following example.

Following unrestricted example and accompanying drawing thereof will illustrate in greater detail the present invention:

Example 1

The clone with separate with ρ 55 and ρ 75TNF acceptor intracellular region bonded protein.

In order to separate and ρ 55 and ρ 75TNF acceptor intracellular region (ρ 55IC and ρ 75IC) interacting proteins, the two hybrid systems (Fields and Song, 1989) of yeast have been used.Briefly, this pair hybrid system is based on the zymic genetic test, by restoring its eukaryotic cell activating transcription factor (activator), comes interior interaction of protein-proteinic body of detection specificity as GAL4.This eukaryotic cell activating transcription factor, GAL4 for example, two zones that separate are arranged: i.e. DNA land and active region, these zones, when expressing and be combined together to form when restoring GAL4 albumen, just can combine with the activation sequence of upstream, then activate the expression of promoter, regulation and control reporter gene, as the expression of LacZ or HIS3 reporter gene, their expression is easy to observe in culturing cell.The inclined to one side sign indicating number gene of interactional candidate albumen matter is entered independently in the expression vector by the clone in this system.In an expression vector, the inclined to one side sign indicating number of a candidate albumen matter sequence is cloned with generation together with the sequence of GAL4 DNA-calmodulin binding domain CaM the same period has GAL4 DNA-in conjunction with the territory hybrid protein; In another carrier, the inclined to one side sign indicating number sequence of the inclined to one side sign indicating number sequence of the second candidate albumen matter and GAL4 active region was cloned by the same period, had the hybrid albumen of GAL4-active region with generation.With two hybrid carrier cotransformations in the yeast host cell system of containing lacZ or HIS3 reporter gene.This report gene is subjected to the regulation and control of upstream GAL4 binding site.Have only those to express the heavy cells in the two kinds of proteic conversion of hybrid places (cotransformation body) interact with each other and can express reporter gene.Under the situation that the lacZ reporter gene exists, the host cell of expressing this gene will become blue look when in the X-gal adding culture.Thereby, the enough facts interact with each other of candidate albumen mass-energy that blue look clone has represented two kinds of clones.

Use this pair hybrid system, intracellular region cloned respectively at ρ 55IC and ρ 75IC enter carrier ρ GBT9 and (carry GAL4 DNA-binding sequence, provide by U.S. CLONTECH, it is described to see below) in, the fusion rotein (intracellular region FAS-IC and the 55IC fragment equally, that have GAL4 DNA-calmodulin binding domain CaM produced, be 55DD, also entered among the ρ GBT9, be used for separating other IC-conjugated protein, as follows the example 3 of face) by the clone.For ρ 55IC and ρ 75IC clone are entered among the ρ GBT9, used coding ρ 55TNF-R (Schall et al., 1990) and ρ 75TNF-R (Smith et al., the clone of full length cDNA sequence 1990), wherein intracellular region (IC) is cut off as follows: cut out ρ 55IIC encode fragment with EcoRI and SalI enzyme, separate the EcoRI-SalI fragment that contains ρ 55IC encoding sequence more according to a conventional method, and with in its embedding ρ GBT9 carrier, this carrier is to be cut in its multiple clone site (MCS) by EcoRI and SalI; Cut out ρ 75IC encode fragment with BspHI and SalI enzyme, separate the BspHI-SalI fragment that contains ρ 75IC encoding sequence more according to a conventional method, and add the Klenow enzyme to produce the fragment that can insert ρ GBT9 carrier, this carrier is cut by Smal and SalI equally.

Then with above-mentioned hybrid (chimeric) carrier and the common transfection HF7C of the PGADGH carrier yeast host cell system that has the GAL4 active region, (carry out respectively, one with P55IC hybrid carrier cotransfection, another and P75IC hybrid carrier cotransfection) the PGADGH carrier also contains simultaneously from the cDNA storehouse of people Hela cell (all above-mentioned carriers, ρ GBT9 with carry the ρ GADGH in Hela cell cDNA storehouse, and yeast system is as the part of the two hybrid systems of MATCHMAKER, #PT1265-1 is from U.S. ClontechLaboratories, and Inc. buys).According to its substratum (His at the shortage Histidine ^-Substratum) energy for growth in is selected the yeast of cotransfection, promptly shows as the growth clone of positive transformant.The yeast clone selected of test is expressed the ability of lacZ gene again, i.e. its LAC Z activity, and this is undertaken by X-gal is added in the substratum, makes X-gal catabolism by the beta-galactosidase enzymes of lacZ genes encoding, generates blue look product.Thereby blue look bacterium colony is represented activatory lacZ gene.The GAL4 activating transcription factor is present among the clone of conversion with activated form, activation to the lacZ gene is necessary, promptly the GAL4 DNA-calmodulin binding domain CaM by one of hybrid carrier of front coding suitably combines with the GAL4 active region of another hybrid vector encoded, is necessary to the activation of Lac gene.This combination only is only possible under two albumen that are blended in each GAL4 zone can stably interact the situation of (combination).Thereby isolated His+ and Lan Se (LACZ+) bacterium colony is such bacterium colony: their carrier of ρ 55IC and coding carrier cotransfections from the protein product of the energy of human body Hela cell and ρ 55IC stable bond that is encoded; Or the carrier of the ρ 75IC that is encoded and coding are from the carrier cotransfection of the protein of the energy of human body Hela cell and ρ 75IC stable bond.

Use ordinary method, separation is from the plasmid DNA of aforementioned His+, LACZ+ yeast clone, and it is squeezed among the e. coli strains HB101 by electric shocking method, then select the transformant of Leu+ and anti-penbritin, these transformant carry the hybrid ρ GADGH carrier that contains AmpR and two kinds of inclined to one side sign indicating number sequences of Leu2.So these transformant are to carry coding to determine the clone that can combine it proteinic sequence with ρ 55IC or ρ 75IC recently.From the intestinal bacteria of these conversions, isolate plasmid DNA then, and test more in the following manner:

(a) as previously mentioned, it is heavily changed into yeast strain HF7 with original intracellular region hybrid plasmid (carrying the hybrid ρ GTB9 of ρ 55IC or ρ 75IC order).In contrast, carry the carrier such as the pACT-lamin (lamin) of irrelevant albumen coded sequence, or independent ρ GBT9 is used for the conjugated protein or protein-bonded coding plasmid of the ρ 75IC-cotransformation with ρ 55IC-.Test the cotransformation yeast then at independent His-substratum or contain growing state in the substratum of different concns 3-aminotriazole at the same time.

(b) yeast host cell of plasmid DNA of addressing in the usefulness (a) and original intracellular region hybrid plasmid and control plasmid cotransformation SFY526 system, and test its LACZ+ activity (it is the efficient that blue look produces that β-gal forms).

Above-mentioned test result shows: when measuring with the color of bacterium colony, at His ^-The colony growth pattern is fully consistent with the active pattern of LACZ in the substratum, and promptly the clone of His+ also is LACZ+.In addition, behind GAL4 DNA land and active region hybrid plasmid transfection SFY526 yeast host, be determined at the LACZ activity in liquid (under the desirable culture condition) culture, SFY526 yeast host amount have will be good than HF-7 yeast host cell the GAL4 activating transcription factor to the inducibility of LACZ.

The results are shown in the following table 1 of above-mentioned cotransfection, therefrom obviously as seen, numerous protein is found and can combines with ρ 55IC or ρ 75IC, that is: be called 55.11 albumen and can combine and be called 75.3 and 75.16 albumen mass-energy with ρ 55IC and combine with ρ 75IC.All these ρ 55-IC-and ρ 75IC-are conjugated protein all to be real human body protein, they are all by cDNA sequence encoding in the people Hela cell cDNA storehouse, and the GAL4 in the two hybrid analysis systems of said here cDNA sequence and above-mentioned yeast in the plasmid ρ GADGH activates a region sequence and merges.

Be enjoyably, also found some fragments of ρ 55IC itself, promptly be called 55.1 and 55.3 albumen, can combine with ρ 55IC.This also will discuss in example 2.

Table 1 is by two hybrid system separated portions cDNA clone feature summaries

DNA-in conjunction with activation-district's colony colour in liquid phase culture sample

The district hybrid	Hybrid		The LacZ activity
				pGBT9-IC55	---	In vain	0.00
pGBT9-IC55	55.1	Blue	0.65
				pGBT9-IC55	55.3	Blue	0.04
---	55.1	In vain	0.00
				---	55.3	In vain	0.00
pACT-Lamin	55.1	In vain	0.00
				pACT-Lamin	55.3	In vain	0.00
pGBT9	55.1	In vain	0.00
				pGBT9	55.3	In vain	0.00
pGBT9-IC55	55.11	Blue	ND
				---	55.11	In vain	ND
pACT-Lamin	55.11	In vain	ND
				pGBT9	55.11	In vain	ND
pGBT9-IC75	75.3	Blue	ND
				pGBT9-IC75	---	In vain	ND
---	75.3	In vain	ND
				pACT-Lamin	75.3	In vain	ND
pGBT9	75.3	In vain	ND
				pGBT9-IC75	75.16	Blue	ND
---	75.16	In vain	ND
				pACT-Lamin	75.16	In vain	ND
pGBT9	75.16	In vain	ND

In above table 1, the plasmid and the hybrid of coding GAL4 DNA-land and GAL4 active region list as follows:

DNA-land hybrid:

ρ GBT9-IC55: the total length intracellular region of ρ 55-TNF-R (ρ 55IC)

ρ ACT-Lamin: irrelevant albumen-lamin

ρ GBT9: carrier itself

ρ GBT9-IC75: the total length intracellular region of ρ 75-TNF-R (ρ 75IC)

The active region hybrid:

55.1 and 55.3 fragments corresponding to ρ 55-TTNF-R intracellular region

55.11 be the new albumen relevant with ρ 55-TNF-R

75.3 with 75.16 is the relevant new albumen of ρ 75-TNF-R

Above-mentioned clone's the following albumen of cDNA coding: new ρ 55IC-and ρ 75IC-protein-bonded 55.11,75.3 and 76.16, check order with conventional dna sequencing method.The partial sequence of all these albumen coded sequences is listed among Fig. 1 a-c, wherein Fig. 1 (a) has listed the sequence cDNA of proteins encoded 55.11, Fig. 1 (b) has listed the cDNA partial sequence of proteins encoded 75.3, and Fig. 1 (c) has been listed as the cDNA partial sequence of proteins encoded 75.16.In Fig. 1 (d), shown the amino-acid sequence of the protein 55.11 of inferring by the nucleotide sequence of Fig. 1 (a).

Yet, what should be mentioned that is: the 55.11 proteic cDNA part orders of encoding were also reported in the research of human brain cDNA sequence by people such as Khan (1992), this research is intended to set up new quick and accurate method, is used for human brain cANA order-checking and draws its physical map and genetic map.But people such as Khan do not provide information to proteic any further feature of 55.11 usefulness cDNA sequence encodings and function, and these functions and other analysis are not the purpose that people such as Khan studies in its research.

55.11 proteic analysis and evaluation

(a) method and material

The clone of (1) 55.11 cDNA

According to analysis revealed: (for example to 55.11 proteic cDNA, the Nothern blotting of face is analyzed as follows), the above-mentioned 55.11cDNA that is cloned by two hybrid screening methods has only represented the partial sequence of 55.11cDNA, contain the fragment between the Nucleotide 925-2863, (seeing Fig. 1 (a)), the fragment between this nucleotide sequence encoding amino acid 309-900 (seeing Fig. 1 (d)).55.11cDNA rest part (fragment (Fig. 1 (d)) between the section between the Nucleotide 1-924 (Fig. 1 (a)) the coded amino acid 1-308 is obtained by ordinary method, promptly clones from human fetus liver cell cDNA storehouse by round pcr.55.11 full length nucleotide sequence (Fig. 1 (a)) is measured from two aspects by dideoxy chain termination

The beta-galactosidase enzymes that (ii) two hybrids are is expressed test

Beta-galactosidase enzymes is expressed test as above-mentioned carrying out.But replaced PGAD-GH (Gal4 active zone) carrier with PVP16 carrier (containing the VP16 active zone) in some test.The numbering of being inserted amino-acid residue in the albumen of fragment coding by cDNA is with reference to the SWiss-Prot database.The mutagenic obtained point mutation (Kunkel, (1994) that obtain deletion mutant and instruct with the PCR method with oligonucleotide.

(iii) Northern analyzes

Adopt routine techniques, with TRI test kit (U.S. Molecular Res CT Inc. product, Cincinnari Oh., U.S.A) isolating total RNA, in formaldehyde/methane amide damping fluid after the sex change, carry out electrophoresis with agarose/formaldehyde gel, again in 10 * SSPE damping fluid trace to Gene ScreenPlus film (du pont company product).Trace point is hybridized (on seeing, the section between the Nucleotide 925-2863) with 55.11 Partial cDNA, makes isotopic labeling with random primer test kit (Berlin, Germany lattice, mannheim biochemical corp product), after the strict flushing, and one week of radioautograph.

(iv) in the Hela cell expression of 55.11cDNA and 55.11 protein binding in the combination of the gsh sulfurtransferase fusion rotein of ρ 55IC

The fusion rotein (GST-ρ 55IC345) of the ρ 55IC of the fusion rotein (GSF-ρ 55IC) of synthesizing glutathion sulfurtransferase (GST) and ρ 55IC and glutathione-S-transferring enzyme and the 345th the following sequence excision of amino acid also is adsorbed onto on gsh-sepharose pearl, as described in following example 2.(with reference to Smith and Corcoran, 1994); Frangioni and Neel, 1993).55.11 the cDMA of the cDNA cDNA of 55.11 total lengths (the 1-2863 Nucleotide promptly), FLAG-55.11 and the cDNA of the plain enzyme of fluorescent be expressed in the Hela cell.FLAG-55.11 is the coding section of 309-900 amino-acid residue in 55.11 albumen, and (55.11 Partial cDNA (Nucleotide 925-2863) are by two hybrid screening clones of system) N end links to each other with the FLAG octapeptide.Expression of Fusion Protein is to be realized by the expression vector (HtTA-1) of tsiklomitsin regulation and control in the Hela cell.The trans-activating factor (seeing following example 2, Gossen and Bujard, 1992) of this Hela cell expressing tsiklomitsin regulation and control.Expressed albumen usefulness ( ³⁵S) methionine(Met) ( ³⁵S) halfcystine (du pont company, Britain Amer-sham company product) carries out the metabolic radiolabeling, follow cracking Hela cell, carry out immunoprecipitation again, then labelled protein is attached to gsh sulfurtransferase fusion rotein, these are all undertaken by the method for following (example 2), but replace 0.1%NonidetP-4 with 0.5% in cell lysis buffer solution.With anti-55.11 the gsh sulfurtransferase fusion rotein that contains amino acid 309-900 section exempt from antiserum(antisera) (dilution in 1: 500), with anti-FLAG octapeptide mouse monoclonal antibody, obtain 55.11 with FLAG-55.11 immunoprecipitation (M ₂Eastman Kodak; 5 μ g/ml cells split Jie's thing).

(b) the combining of 55.11 albumen and ρ 55IC in the transformed yeast

This research is to get 55.11 and ρ 55IC bonded character clear, particularly in these two kinds of albumen with in conjunction with relevant section, for this purpose, adopted above-mentioned pair of hybrid method, going in conjunction with " ravin " as " bait " with " DNA land " construct of the ρ 55IC that has total length and deletion mutantion is that the part 55.11 proteic aminoacid sequences of encoding in the construction (residue 309-900 is as initial isolating product) go to merge " active zone " in GAL4AD and the VP16AD carrier.And then, also made up 55.11 various deletion mutantion strains, and merged with " active zone " of GAC4AD carrier.(as only having 55.11 mutant of 309-680 and 457-900 amino acid fragment).In the SFY526 of transfection yeast cell, detected combining of various " land " construct and various " active zone " constructs.And, measured associativity through two hybrid beta-galactosidase enzymess expression filter-binding assays.Irrelevant Protein S NF1 and SNF4 respectively as " land " and " active zone " construct over against photograph; Gal4 (PGAD-GH) and VP16 (PVP16) empty carrier are as the negative contrast of " active zone " construction, and Gal4 (PGBT9) empty carrier is as the negative contrast of " land " construct.Measurement result is as shown in table 2 below, and symbol in the table " +++" and " ++ " represent to measure the changing conditions (just in conjunction with the result) of Show Color in beginning back 20-60 minute respectively; "-" expression is measured and is begun do not develop the color in back 24 hours (negative test).The place of table empty is represented undeterminate in conjunction with test.

Table 2

55.11 albumen and P55IC's combines in transformed yeast

Listed result can infer from above table 2, the 55.11st, combine with the certain position of P55IC, and this position is different from " dead functional domain " (328-426 amino-acid residue) of P55IC

55.11 the proteic binding ratio of brachymemma P55IC of albumen and lethal zone disappearance (206-328 makes up strain in the table 2) is more effective with combining of the P55IC of not brachymemma.

55.11 also can combine with the albumen construct (construct 206-308) of the further brachymemma of C-terminal and the construct (construct 243-328) of dead functional domain and membrane-proximal region disappearance.But 55.11 albumen do not participate in the N-terminal disappearance to the 266th amino acid whose construct combination (seeing Table 2).These discoveries show, the amino acid region of 55.11 binding site between the 243-308 of P55IC, the amino acid region of the N-terminal of binding site between 243-266.

From " ravin " construct (this construct the contains the Gal4 active zone) 55.11cDNA that derives from the clone transfer to contain the VP16 active zone catch the construct time, that does not reduce by 55.11 albumen and P55IC combines validity (table 2).Thereby, and as if be positioned at 55.11 intramolecularly in conjunction with relevant structure, but as if do not comprise 55.11 with the fusion position of active zone.

Yet, 55.11 with the P55IC protein binding, can be stoped because of the restricted excision of 55.11 PROTEIN C ends (55.11 construct 309-680) or N-terminal (55.11 construct 457-900).(55.11 proteic first amino-acid residues that 309 amino-acid residues are encoded by the Partial cDNA that filters out with two hybrid methods at first).

Observed 55.11 is seemingly specific with combining of P55IC.Because 55.11 not with other protein binding, comprise three receptor proteins (P75-R, Fas/AP01 and CD40) and other albumen of tumour necrosis factor/trk C family, as lamin and cyclin D (data is not delivered).What be worth emphasizing is, in other tumour necrosis factor/trk C albumen of test, also test it and contained the segment of intracellular region, be people's FAS-R (amino-acid residue 175-319), CD40 (amino-acid residue 216-277) and P75-TNF-R (amino-acid residue 287-461) wherein do not have a kind of combine with 55.11 (data is not delivered).

(c) with 55.11cDNA be probe, the RNA of several clones carried out the clone of Northern analysis and total length 55.11cDNA.

Test clone comprises Hela clone, cem cell system, and Jurkat clone and HepG2 clone, they come from human epithelium's cancer cells respectively, acute lymphoblastic T chronic myeloid leukemia, acute T chronic myeloid leukemia and liver cancer cell.The cDNA of initial separation (Nucleotide 925-2863) is as probe, and sample contains 10 μ g RNA/ swimming lanes.The Northern analytical results has wherein reappeared the Northern trace as shown in Figure 2.

As seen from Figure 2, be probe with 55.11cDNA, in several clones, carry out Northern and analyze.Discovery has the single crosses transcript of an about 3kB, and is bigger than the 55.11cDNA (3kB) of initial separation.Utilize and the corresponding Oligonucleolide primers of 55.11 sequences, cloned 5 of the about 1kB of length ' end extension sequence through round pcr (polymerase chain reaction), 5 ' end extension sequence length adds that initial clone's cDNA length summation approaches 55.11 transcript length.3kB cDNA has comprised above-mentioned two portions (sequence of 1kB and the sequence of 2kB), it can be in the Hela of transfection cell effective expression (as follows) and produce the protein of about 84kDa.This shows that 3kB CDNA contains a translation initiation site.

(d) 55.11 albumen and external combination that contains the segmental gsh sulfurtransferase of P55IC fusion rotein.

In order to determine that 55.11 can combine with P55IC really, and the eliminating Yeast protein participates in this process, detected by bacterium and produced gsh sulfurtransferase P55IC fusion rotein and transfection HeLa cell generation, by the external interaction between 3kB 55.11cDNA (55.11 total length) encoded protein.In this research, coding total length 55.11, the cDNA of FLAG-55.11 (merging the N-terminal of FLAG8 peptide by 55.11 309-600 amino-acid residue of initial clone's Partial cDNA coding) and luciferase (comparing) all in the HeLa of transfection cell expression and with [ ³⁵S] methionine(Met) and [ ³⁵S] halfcystine carries out the metabolic radiolabeling.Following albumen and GST merge, the PP55IC of total length (gsh-P55IC fragment) and P55IC fragment (gsh P55IC345), and it by 345 amino-acid residues, removes major part " dead functional domain " formation (seeing Table 2) by the C-terminal of P55IC.Gsh separately in contrast.When 55.11 albumen of total length are used in conjunction with the gsh fusion rotein, anti-55.11 proteic antibody are used for the lysate of immunoprecipitation transfectional cell, or when the FLAG-55.11 fusion rotein was used in conjunction with the gsh fusion rotein, the antibody of anti-FLAG 8 peptides was used for the lysate of immunoprecipitation transfectional cell.Analysis of protein is undertaken by sds polyacrylamide gel electrophoresis (SDS-PAGE, 10% acrylamide), carries out radioautograph subsequently.

Fig. 3 A and B have shown the result of above SDS-PAGE gel autoradiography.Fig. 3 A shows 55.11 albumen (55.11-total length) of bright total length and combining of various gsh fusion roteins, and Fig. 3 B shows combining of bright Flag-55.11 fusion product and various gsh fusion roteins.Rightmost swimming lane is represented the immunoprecipitation result of the cell lysate that contrasts among Fig. 3 A, and these cells are only to use total length 55.11 transfections, and by anti-55.11 antibody (α 55.11Abs) it is carried out immunoprecipitation.The rightmost swimming lane is represented the immunoprecipitation result of the cell lysate that contrasts among Fig. 3 B, and these cells are only to use the FLAG-55.11 transfection, and carry out immunoprecipitation with the antibody (α FLAG-Abs) of anti-FLAG.

Thereby, apparent from the result of Fig. 3 A and B, can in the HeLa cell, express by total length 55.11cDNA encoded protein, and can with contain whole P55IC (gsh-P55IC) or lose the P55IC (fusion rotein of gsh-P55IC345) combine (Fig. 3 A) of the brachymemma of most of lethal zone.55.11 albumen of total length do not combine (contrast) with gsh itself.Similarly, 55.11 Partial cDNA encoded protein matter and FLAG 8 Toplink by initial clone expressed in the HeLa cell form fusion rotein (FLAG-55.11), this fusion rotein can combine with gsh-P55IC and gsh-P55IC345 external, but can not combine (Fig. 3 B) with gsh itself.So above result provides in addition the evidence of (seeing above (b)), prove that 55.11 combine with the upstream region of P55IC " dead functional domain ", that is combine with the zone of striding the film district near P55IC.

In addition, more than research also shows, the result according to the present invention has successfully synthesized anti-55.11 antibody (Fig. 3 A).

(e) human 55.11 albumen and the low amino acid of the associated protein of organism existence that waits infer that sequence compares and 55.11 protein sequence features

55.11 above mentioned as the present invention, that the 55.11cDNA of total length is cloned and checks order (see Fig. 1 (a) in nucleotide sequence) and infers according to the cDNA sequence whole aminoacid sequences (seeing Fig. 1 (d) aminoacid sequence).Database (Gene Bank TM/EMBL Data Bank) key understands that homologue (registration number X80422 and Z311477) is determined in human 55.11cDNA partial sequence (registration number T03659, Z19559 and F091287) and the mouse in the random sequencing of cDNA storehouse.Be present in people's liver cancer HC10 cell culture one and contain 596 proteic cDNA encoding sequences of amino acid whose people, similar to the 55.11cDNA encoding sequence.But liver cancer albumen but lacks and 55.11 corresponding N-terminal fragment (amino acid/11-297), but also is different from 55.11, in the 297-377 and 648-668 amino acid section of 55.11 correspondences.It is bright that database retrieval is also shown, also has the albumen that very high sequence homology is arranged with 55.11 in yeast (Saccharomyces) plant, (Arabidopsisthaliana), worm (Caenorhabditis elegans).55.11 as if albumen have the evolution conservative function.In yeast, known have the dna sequence dna of two primary yeast albumen (opening code-reading frame YHR027C and SEN3) similar to 55.11, and size is also similar with 55.11.Have only by the order-checking of gene clone and could understand YHR027C, and SEN3 is cloned out as a cDNA molecule.55.11 in the site similar to SEN3, also relevant to its site similar in YHR027C.Though evidence shows, 55.11 will surpass the similarity of itself and SEN3 with the similarity of YHR027C.Obtain the information of dna sequence dna from plant (mouseearcress) and worm (Caenorhabditiselegans) albumen, although partial sequence just also clearly illustrates that these albumen are the same with the YHR027C Yeast protein all to have 55.11 similaritys.Up to now, character has been illustrated in four kinds of albumen has only zymic SEN3, and it and 55.11 homology are defined.It is counterpart (the 20S proteasome is the hydrolysis core of 26S the proteasome) [Rechsteiner etc. of the P112 subunit of 20S proteasome incitant in the yeast that SEN3 is identified, 1993.De Martino etc., 1994] (M.R.Culbertson and M.Hock strasser, private communication).

Fig. 4 diagrammatically shown human 55.11 albumen and above-mentioned mention be present in the low comparison that waits the amino acid deduction sequence of associated protein in the organism.The aminoacid sequence that Fig. 4 compares is encoded by following cDNA: 55.11cDNA (seeing Fig. 1 (d)) (SEQ ID NO:14); One opening code-reading frame (YAR027C) is present in yeast (Saocharomyces cerevisiae) the 8th chromosomal clay; (Nucleotide 21253-24234, registration number U10399) (SEQ ID NO:15); The SEN3cDNA (registration number L06321) (SEQ ID NO:16) of coding Yeast protein; Plant (Arabidopsis thaliana) proteic Partial cDNA (registration number T21500) (SEQ ID NO:17); And the proteic Partial cDNA of worm (Caenorhabditiselegans) (registration number D27396) (SEQ ID NO:18).55.11 " KEKE " sequence represent that with solid line AYAGS (x) 8LL sequence dots.PILEUP and PRETTYBOX program with GCG software are in line series arrangement.Being used for increasing the arrangement collinear represents with dash at interval.The feature or the primitive of the various sequences in people's 55.11 aminoacid sequences, as described below: as " KEKE " tumor-necrosis factor glycoproteins in lys614 arrives the Glu632 scope (the beneath black line among Fig. 4), not have to find to contain conservative amino acid sequence motif in 55.11 encoded protein.This " KEKE " sequence is present in the numerous protein, has comprised proteasome subunit and has divided the component ma albumen." KEKE " sequence can promote the association (Realini et al, 1994) of protein complexes, AYAGS (X) ₈The LL sequence has occurred twice in 55.11 albumen, (Fig. 4 is seen in site 479 and 590); This sequence action value is not seen description.

(f) sequence signature in the zone relevant among the P55IC with 55.11 protein binding

As described above, (see (b) and (d), 55.11 protein binding (N-terminal of this binding site is between the 243-266 amino-acid residue) this zone in the zone between the 243-308 amino acid of P55IC is the upstream of " dead functional domain ", and more approach P55-TNT-R stride the film district.55.11 with it in the bonded P55IC zone, proline rich, Serine and threonine residues.Yet this zone is not contained in the RPM1 that occurs in other several cytokine receptors and the primitive (Oneal and Yulee, 1993) of RPM2 proline rich.In the section between the 243-266 amino-acid residue, (this section disappearance can stop P55-R to be attached to 55.11), (see (b) and (d) and table 2), two Serines and two Threonines then can pass through map kinase for the structure of proline residue again, CDC2 and other depend on the kinases of proline(Pro) and become the potential site (Seger and Krebs, 1995) of phosphorylation.The phosphorylation in this site may influence it and 55.11 proteic combinations in the receptor protein.

In view of above-mentioned bonded result of study about albumen 55.11 and it and P55IC, can obtain conclusion, according to the present invention, found a new protein, it can be incorporated into the special area of the upstream of P55IC " dead functional domain ", and this combination can influence the activity of TNF mediation and can not cause necrocytosis.55.11 calmodulin binding domain CaM had before shown relevant with inducing of nitric oxide synthase (Tartagliaet al, 1993), and as if with also relevant to the activation of sphingomyelinase by tumour necrosis factor.(Wiegmam et al，1994)。Thereby 55.11 combine with P55-TNF-R intracellular region (P55IC) and may influence or relate to following process: (i) provide signal for above that address or other TNF effect; (ii) proteic folding or processing (as by 55.11 and 26S proteasome subunits similarity proposed); (iii) active adjusting of P55-TNF-R or expression.

Example 2

The self-association ability of P55TNF acceptor intracellular region (P55IC), cause the ability of necrocytosis and other The intracellular region of feature and active and relevant Fas/AP01 acceptor

As in the above described in the example 1, find that the intracellular region of P55-TNF-R (P55IC) has self-bonded ability, and find that the segment of P55IC is that albumen 55.11 and albumen 55.3 also can combine with P55IC.

Known TNF combines with P55-TNF-R's, can cause the cytocidal effect to the cell that has this receptor.And the antibody self of anti-this acceptor extracellular region also can trigger this effect, and the crosslinked validity of this effect and antibody and acceptor is relevant.

In addition, mutation research [Tartaglia et al (1993); Brakebusch etal., (1992)] show that the P55-R function depends on the integrity of its intracellular region.So also showing the signal enabling of the cytocidal effect of TNF is intracellular region (P55IC) self-association resultant as two or more P55-R, and this be that the congregation of acceptor causes from forming platform.According to the present invention, these results provide further evidence for this opinion.These results show that under the situation of not striding film district or intracellular region, the expression of the intracellular region of P55-R will trigger the death of cell in the cell.These free P55-R intracellular regions show self-associating ability, and this also may be the reason that they have the ability to function that does not rely on TNF.The signal initiation of total length P55-R depends on the stimulation of TNF, and the clear acceptor of this fact table is striden the reflection activity of film district or extracellular region, and its reduces or prevented the generation of self-association.

The self-associative ability of the intracellular region of P55-R (P55IC) is have mercy on extremely accidentally when attempting to clone the effector albumen that acts on mutually with this acceptor fortunately found (seeing above-mentioned example 1).We have used above-mentioned " double cross " technology for this purpose.Except finding that new albumen 55.11 can be with P55IC combines, also found to contain other three clones' of cDNA sequence HeLa cell cDNA s, the part intracellular region of coding P55-R, this shows that P55IC can the oneself associate.Two among these clones is identical, the protein fragments 55.1 that contains insertion fragment (indicating clone 55.1) the coding P55IC of a coded amino acid 328-426, the 3rd then contains long insertion section, fragment between the coded amino acid 277-426 (being denoted as clone 55.3, the protein fragments 55.3 of coding P55IC).

In addition, we have analyzed two kinds of chimeric proteins of bacteriogenic P55IC in external interaction.One of them albumen and maltose binding protein (MBP) merge, and another chimeric protein merges with gsh-sulfurtransferase (GST).These chimeric proteins make up, clone and express with ordinary method.After they were expressed, the oneself who measures P55-R intracellular region (P55IC) by the interaction of measuring above-mentioned bacteriogenic chimeric protein GST-IC55 (Mr-51kD) and MBP-IC55 (Mr-67kD) interacted.The GST-IC55 chimeric protein of equivalent (the sample lane 1-4 among Fig. 5) or separately GST (sample lane 5-8 among Fig. 5) be attached to (Sigma) on gsh-sepharose 4B, then with MBP-IC55 fusion rotein incubation in following wherein a kind of damping fluid of equivalent:

(i) damping fluid I (20mM Tris-HCl, PH7.5,100mM KCl, 2mM CaCl ₂, 2mMMgCl ₂, 5mM DTT, 0.2% Triton X100,0.5mM PMSF, 5% glycerine).The swimming lane 1 among Fig. 5 and the sample of swimming lane 5 are handled with sort buffer.

(ii) contain 5mM EDTA and replace MgCl ₂Damping fluid I.Swimming lane 2 among Fig. 5 and the sample in the swimming lane 6 are handled with this damping fluid.

(iii) contain the damping fluid I that 250mM KCl replaces 100mMKCl, the sample among Fig. 5 in swimming lane 3 and the swimming lane 7 is to handle with such damping fluid.

(iv) damping fluid I contains 400mMKCl and replaces 100mMKCl, and the sample among Fig. 5 in swimming lane 4 and the swimming lane 6 is to handle with such damping fluid.

After 2 hours, sepharose 4B then boils in the SDS-PAGE damping fluid with same damping fluid washing, then by the PAGE electrophoresis at 4 ℃ of rotation incubations.With on the nitrocellulose filter of the Western blot on the glue, the polyclonal antiserum with an anti-MBP dyes then subsequently.Fig. 5 represents the result of this dyeing western engram analysis, and the sample among the swimming lane 1-8 is above-cited.

From Fig. 5, can observe the P55IC-MBP chimeric protein significantly and have nothing to do with divalent cation, and not be subjected to the high salt concentration ionic to influence (0.4MKCl) with combine (the swimming lane 1-4) of P55IC-GST chimeric protein.Can draw the conclusion that P55IC has self-associative ability thus.

In order to assess the action value of P55IC self-association, we attempt to express P55IC in the tenuigenin to TNF cytocidal effect sensitive cells.Consider that P55IC might change or pair cell is poisonous, we select for use a kind of derivable mode to express it.I.e. utilization just grows up recently, by the mammalian expression system (Gossen and Boujard, 1992) of stringent controlled tsiklomitsin regulation and control.The expression of P55IC causes cell mass mortality (Fig. 6, right hurdle).The cell surface that dying cell shows leaks identical with observed phenomenon in the TNF cell killing process.P55IC construct transfectional cell, under the condition that tsiklomitsin exists, the expression of the construct of HD10-3 regulation and control has reduced by 10 ⁵Doubly, but still cause the death of some cell.Although it significantly is lower than viewed result when not having tsiklomitsin to exist.(Fig. 6, the left side).In contrast, with the contrast construct cells transfected that contains the plain enzyme cDNA of fluorescent, do not see dead sign (result does not deliver).

Do not have when striding the expression of receptor of film district or extracellular region, P55IC can trigger cell death.This provides further evidence to show, this district causes relevant with signal.In addition, it shows the other parts that do not have receptor protein, plays direct effect in sort signal causes.Comprise that by studying the influence that suddenlys change to the P55IC function mutant of those researchs shows in the invention, the amino acid section between the amino acid 326-407 of institute residue is a most critical to the function of P55IC.This zone shows sequence similarity significantly with the intracellular region of two other receptor proteins.These two acceptors are respectively to evolve to go up Fas acceptor and the CD40 acceptor relevant with P55TNF-R, and wherein Fas is subjected to physical efficiency trigger cell dead signal (Itoh et al, 1991; Oehm et al, 1992) and CD40 is subjected to physical efficiency to promote cell growth (Stamenkovic et al, 1989).Therefore, as if this regional sequence has constituted a conservative primitive, and plays certain vital role in signal causes.Because the primitive feature dissmilarity of it and known enzymatic activity, thereby it seemingly produces signal in a kind of indirect mode.Be that it may be the stop site of signalase or transmit the proteic stop site that stimulus signal is given signalase.P55IC, Fas acceptor and CD40 can both be stimulated by the antibody of anti-their extracellular regions.And show that this hormesis is relevant with antibody linked ability to acceptor.As if thereby the initiation of signal is because the gathering of extracellular region causes two or more intracellular region results of interaction.In some researchs, also show, this interaction relevant with the startup of receptor signal (Brakebusch et al, 1992), these studies show that, owing to intracellular region sudden change makes acceptor lose function, promptly the function to the normal acceptor of its expression has a kind of " dominant " effect.P55IR produces congregation to TNF reaction, be considered to take place in a kind of passive mode, but be because each TNF molecular energy that occurs with the form of homotrimer in conjunction with two or three receptor protein molecules.Yet discovery of the present invention shows that the generation of this process is slightly different.

The self-associating tendency of P55IC shows that this zone plays activation in its aggregation inducing.As and if then the activation of P55IC is enough to initiating signal, because when it did not rely on other acceptor molecule expression, P55IC can trigger necrocytosis when not having TNF or other external irritant.But when it during as the expression of receptor of total length, the effect of P55-TNF-R unable to get up signal is unless Cai be subjected to TNF and stimulate possible.So someone supposes that when P55-TNF-R was activated, in fact TNF had overcome some and suppressed mechanism, these mechanism stop the self-association of intracellular region, and restraining effect is because P55IC connects with the receptor protein rest part.This inhibiting generation may be owing to stride the orientation effect that film district and zone, extracellular apply intracellular region, or because the association of some other albumen and acceptor maybe may be owing to the receptor protein quantity that allows to be positioned on the cytoplasmic membrane is restricted.It should be noted that this regulatory mechanism is quite effective, according to estimates, only a TNF molecule is attached to a cell, just is enough to cause the death of this cell.

Do not rely on the signal of the spontaneous generation of ligand, can cause occurring extensively chaotic by the process of this receptor regulation and control.The most appropriate known example is the out of control of growth factor receptors.The start signal of spontaneous generation can cause the spontaneous accumulative signal of acceptor as those owing to suddenly change, and plays an important role in the growth out of control of tumour cell.As everyone knows, the TNF effect is induced excessively, is the pathogenesis that causes numerous disease.The free intracellular region (P55ICs) of P55-TNF-R does not rely on the signal initiating power of TNF, may work to this excessive effect.It seems this may, perhaps some virus and cytopathic effect of other pathogenic agent are not the direct cytocidal effect that comes from them, but come from the proteolyzing of the intracellular region of P55-TNF-R, have caused the cytocidal effect of similar TNF.

Reach the cytotoxicity that does not rely on aglucon for further illustrating section relevant among the P55IC with the self-association ability, also, also carried out detailed research below in order to detect the effect whether other relevant member (for example FAS-R) in the TNF/NGF receptor family also has intracellular region that can self-association and do not rely on ligand.

(a) general method and material

(i) detection that beta-galactosidase enzymes is expressed in two hybrid method screenings and the two hybrids

Go out some cDNA with pcr clone and insert fragment, these insert fragment cloning from the full-length cDNA that cloned in the past in our laboratory, or come from the cDNA storehouse of purchase, their coding P55-IC and its mutant, Fas-IC and other albumen (seeing Table 3).With PGBT-9 that contains above-mentioned cDNA and PGAD-GH carrier (containing DNA land (DBD) and active zone (AD) respectively) transformed yeast (SFY526 report (Bartel etc. of system, 1993), beta-galactosidase enzymes expression therein is by liquid testing method (Guarente, 1983) measure, also available filter-binding assay is measured, and the both has produced essential identical result (not delivering).Utilize HF7c yeast report system, recommendation according to manufacturer, (Clontech from the Hela cell cDNA storehouse of the Gal4AD-mark bought, Palo Alto, Ca., the U.S.) with two hybrid sieve methods (Fields andSong, 1989) filter out can with P55-R intracellular region (P55-IC) bonded protein.When isolated clone's the positive was grown in the presence of the 5mM3-aminotriazole by measuring (a) with laxative remedy, transformed yeast was to the prototropy effect of Histidine, (b) expression of beta-galactosidase enzymes, and (c) specificity is tested (with SNF ₄And be blended in interaction between the lamin of Gal4DBD).

The (ii) external self-association of bacterium synthetic P55-IC fusion rotein

The synthetic method by the document narration of glutathione S-transferase (GST) and glutathione S-transferase-P55-IC fusion rotein (GST-P55-IC) is carried out (Frangioni and Neel, 1993; Ausubel etc., 1994).Maltose binding protein (MBP) fusion rotein is to obtain by PMalcRI carrier (New England Biolabs), and on the amylose resin post purifying.MBPP and gst fusion protein Study of Interaction are carried out (5 μ g albumen/20 μ l glue pearls, for the first time incubation is 15 minutes, incubation is 2 hours for the second time, all carries out) successively by gsh-sepharose 4B under 4 ℃ with GST and MBPP fusion rotein incubation.With carry out in the incubation damping fluid below of MBP fusion rotein, promptly it contains: 20mM Tris-HCL, PH7.5,100mM KCl, 2mM CaCl ₂, 2mM MgCl ₂, 5mM dithiothreitol (DTT) (DDT), 0.2% Triton * 100,0.5mM phenmethyl sulfo-fluoro thing, the glycerine of 5% (v/v), or under particular cases, containing 0.4MKCl or 0.5mM EDTA replacement MgCl ₂Above-mentioned damping fluid in carry out incubation.The association of MBP fusion rotein is to analyze by sds page (10% acrylamide) electrophoresis with the associating protein of gsh-sepharose 4B, analyzes through the Western blotting again.The goat-anti rabbit immunoglobulin (Ig) that links to each other with respectful peroxidase with the mouse resisting anteserum (synthesize in this laboratory) of anti-MBP in above-mentioned blotting is analyzed is made probe.

(iii) P55-R and segmental abduction delivering thereof in the Hela cell

The Hela cell of expressing the trans-activator factor of tsiklomitsin regulation and control is grown up by Gossen and Bujard, and (the HtTA-1 clones (Gossen Bujard, 1992)), this cell is grown on the improvement Eagle ' s substratum of Dulbecco, promptly contain 10% foetal calf serum, 100 μ g/ml penicillin, the substratum of 100 μ g/ml Streptomycin sulphates and 0.5mg/ml Xin Meisu.Coding P55-R or its cDNA fragment are inserted the expression vector (pUHD10-3 is so kind as to give by H.Bujard) that fragment is changed over to the tsiklomitsin regulation and control.Again by calcium phosphate precipitation method (Ausubel etc. 1994) with above-mentioned expression vector (5 μ gDNA/6cm flat board) transfectional cell.Under tsiklomitsin (1 μ g/ml) existence or the non-existent situation, the time is measured transfection expression effect of proteic moment in the finger after transfection.With the people P55-ICcDNA transfectional cell in the pUHD10-3 carrier, and set up the cell clone of stable transfection, this is under there is situation in tsiklomitsin, by with the plasmid transfection cDNA of moisture resistance mycin to the HtTA-1 cell, and filter out with Totomycin 200 μ g/ml that resistance clone realizes.Obtain the cDNA expression after removing tsiklomitsin.Generally speaking, tsiklomitsin will be present in the cell growth medium all the time.

(iv) by P55-R and fragment abduction delivering thereof and the mensuration of the similar TNF effect that triggers

The influence of the expression pair cell viability of acceptor that is brought out and TNF is measured by toluylene red absorption process (Wallach, 1984).Inducing of IL-8 genetic expression is to analyze by Northern to measure.RNA TRI test kit (Molecular Research Center, Inc.) separate, and sex change in formaldehyde/methane amide damping fluid, by agarose/formaldehyde gel electrophoresis, trace arrives on " GeneScreen Plus " film (Du Pont) in 10 * SSPE damping fluid then.Above-mentioned these processes all routinely technology carry out.Filter membrane is then with IL-8cDNA probe hybridization (Matsushima waits 1988, Nucleotide No.1-392).(Germany), according to the requirement of manufacturer, strictness is washed for Boehringer Mannheim biochemica, Mannheim to carry out radio-labeling by the random primer box again.Radioautograph 1-2 days.

(the v) mensuration of TNF expression of receptor

Pressing chloramine-t method uses ¹²⁵I mark TNF just can determine the TNF acceptor and containing 1 * 10 by calculating binding capacity with cell ⁶Expression amount in the sample of cell, this method ditto contained (Holtmann and Wallach, 1987), the TNF receptor expression also can be measured by ELISA.Promptly according to the method (Aderka etc., 1991) of measuring soluble TNF acceptor but difference is with RIPA damping fluid (10mM Tris-HCL, PH7.5,150mM NaCl, 1%NP-40,1% deoxycholate salt, 0.1%SDS and 1mM EDTA) decompose cell (70 μ l/10 ⁶Cell) dilution test sample.The solubility P55-R that is purified into from urine is as standard control.

(b) mutation analysis by P55-R intracellular region (P55-IC) detects section relevant with self-association among the P55-IC

As mentioned above, P55-IC can self-association and is triggered the toxic action of pair cell, and some fragment of P55-IC can combine with total length P55-IC.Especially one of them fragment of P55-IC (being called protein fragments 55.1 in above-mentioned example 1) can combine with full-length cDNA, and by its order-checking is found, it contains the amino-acid residue between the 328-426 of P55-TNF-R, and this fragment also is present among the P55-IC.Further also find in (face as follows).Above-mentioned fragment, promptly protein fragments 55.1, can self-association and trigger the toxic action of pair cell.Therefore, this P55-IC fragment is called " dead functional domain " (or title " dead district "), and its section between the 328-426 of people P55-R is likely by the amino-acid residue between the 328-414 and forms.

P55-IC " dead functional domain " has the self-association ability, and this fact is serendipitous.During with the intracellular region bonded albumen of two hybrid technology (see go up example 1) screening and this receptor in the Hela cell cdna library, we find some cDNA, their coded product can be specifically in conjunction with intracellular region-GAL4DBD fusion rotein, some clone (as 55.1 and 55.3) some fragments (marking with an asterisk among the P55-IC, table 3) of P55-R intracellular region of itself just encoding wherein.

The two hybrids of application detect the degrees of specificity of measuring the P55-IC self-association and limit more accurately when being correlated with section, cause following discovery (table 3): (a) self-association of P55-IC is limited to the section in " dead functional domain ", its section of N end between 328-344 amino acid, its C termination is bordering on the 404th amino acid, the upstream of this district C end (the 414th amino acid) of promptly having reported.(b) disappearance of " dead functional domain " nearly membrane portions in upstream will strengthen self-association, and this shows that this section has retarding effect to association.(c) mouse P55-IC energy self-association also can be associated with " the dead functional domain " of people P55-R.(d) three member: Fas/Ap01 (FAS-R), CD40 (Field and the Song in addition of TNF/NGF receptor family, 1989) and P75TNF acceptor (smith etc. 1990), to their studies show that of intracellular region self-association: Fas-IC, it can provide signal for necrocytosis by the sequence motif relevant with P55-R " dead functional domain ", it also has the self-association ability, and with P55-IC to a certain degree association is arranged.Yet it provides growth signal (even it also comprises and " dead functional domain " similar sequence) CD40-IC.In addition, P75-IC does not then have structural similarity with P55-IC, and it can not self-association, does not also combine with P55-IC or Fas-IC.

Table 3

The intracellular region self-association of P55-R and Fas/AP01 in transformed yeast:

Reach-galactoside expression of enzymes method of testing mensuration by two-hybrid

Other albumen

Mouse p55-IC 0.17 0.38 3.0-----2.5----0

Mouse Fas-IC 00 0.14-----0 38.8-0 00

People CD40-IC 00 0-----0 0 0--0

People p75-IC 0--------0-0-0

SNF1 - - - - - - - - - - - - 2.7 0

Lamin (Lamin) 00 0------0---0

pGBT9 0 0 0 - - - - - 0 0 - - - -

Last page table 3 has shown the interactional quantitative test result of Gal4 hybrid construct, Gal4 hybrid construct comprises following albumen: the intracellular region of people P55-R and various deletion mutant thereof (the residue numbering is by (loetscher etc., 1990)) (SEQ ID NO:25); Mouse P55-R intracellular region (zone between the 334-454 amino acid, numbering is by (Goodwin etc. 1991) (SEQ ID NO:26)); Mouse Fas/Ap01 (numbering is by (Watanabe-Fukanaga etc., 1992) for Fas-IC, 166-306); People CD40 (numbering is by (stamenkovic etc., 1989) for CD40-IC, 216-277); People P75TNF acceptor (numbering is by (smith etc., 1990) for P75-IC, 287-461).SNF, with SNF4 as associating over against according to (Field and Song, 1989), lamin is as negative contrast (Bartel etc., 1993).With Gal4DBD construct (pGPT9) encoded protein arranged vertical, horizontal with Gal4AD construct (pGAD-GH) encoded protein.(Clontech Palo Alto, Ca are cloned out in two hybrid screenings U.S.A.) two deletion mutants that asterisk marks, and they are to obtain by the P55-IC work " bait " that is cloned among the pGBT9 in HeLa cell cDNA storehouse.In above-mentioned screening, 4 * 10 ⁶There are 4 to be positively to find that wherein three clones are corresponding to some fragment of people P55-RcDNA (two clones are identical, the zone between the 328-426 amino acid of encoding, another zone between the 277-426 amino acid of encoding) among the cDNA of the individual detection clone.The 4th clones coding one agnoprotein.The expression data of beta-galactosidase enzymes is the testing mean of two independent transformant, and as the product quantity of beta-galactosidase enzymes and list.(activity unit is defined as: enzyme substratum OD ₄₂₀* 10 ³Divided by OD ₆₀₀Product with the reaction times.(OD is the optical density(OD) of yeast culture), the time is minute being unit).Analysis precision is 0.05 unit.In all tests, sub-average 25% (the not test) of the difference between the same sample.

The interactional vitro test of P55-IC glutathione S-transferase (GST) bacterium fusion rotein and P55-IC maltose binding protein (MBP) fusion rotein confirms, and: P55-R can self-association, and in the association process with the female albumen of enzyme irrelevant (on seeing).The increase of salt concn does not influence association, and EDTA does not influence association yet.

In order to assess the action value of " dead functional domain " self-association, we have studied P55-R or its segmental abduction delivering influences the mode of cell for the cytotoxic susceptibility of TNF, analytical results is published in Fig. 7, Fig. 7 has expressed and has used P55-R, in the Hela cell of its intracellular region (P55-IC) or its fragment (including " dead functional domain ") transfection, the trigger action that does not rely on part that cytocidal effect has.

In Fig. 7, represented following content with schematic form: the dna molecular of coding different sorts TNF acceptor, these acceptors are present in the carrier of transfection Hela cell (Fig. 7 leftmost side); Utilize the expression vector of tsiklomitsin regulation and control, moment is expressed various total length P55-R (P55-R), the ability to express (left side and middle segment) and the viability (right side segment) of the Hela cell of P55-IC or its fragment or luciferase in contrast (luc) (every kind of albumen all is described in the leftmost side of Fig. 7).Empty graph piece (left, center, right) is illustrated in the tsiklomitsin (1 μ g/ml) that can suppress to express and has the transfectional cell under the situation; Full segment represents not have the transfectional cell under the tsiklomitsin existence condition.The TNF expression of receptor is 24 hours mensuration after transfection, measuring method ELISA, promptly utilize the antibody (explanation of Fig. 7 left side) of anti-acceptor extracellular region, also measure (in the middle of seeing) proteic cytocidal effect of transfection 48 hours mensuration after transfection simultaneously with combining of cell by measuring radiolabeled TNF.The data of showing is from one in three tests with the identical result of essence, and in these trials, each construct is all tested from bipartite mode." ND " expression. " not detecting ".

From Fig. 7, can obviously find out: utilize and strictness to regulate and control the carrier that transfection cDNA expresses, (expression of this cDNA is by reverse transcription protein factor (Gossen and the Bujard of tsiklomitsin regulation and control, 1992) regulate and control), the moment of transfection cDNA by coding total length acceptor expresses, and P55-R very little raising of expression amount in the Hela cell will cause necrocytosis widely.When only expressing P55-IC, can observe stronger cytotoxicity.In the Hela cell, when only expressing the fragment of P55-IC that contains necessary " dead functional domain " (zone between the 328-426 amino acid), also can observe significant cytotoxicity.On the other hand, if the fragment of P55-IC lack " dead functional domain " or only contain a part of the time, not influence of the viability of their expression pair cell (or expressing luciferase gene, with comparing).Utilize the cell of P55-ICcDNA stable conversion, the cytotoxicity of P55-IC can further be confirmed; These transformants can continue growth during not by abduction delivering at P55-IC, in case but P55-IC express, they will dead (on seeing).

(c) other effect of P55-TNF-R intracellular region

The intracellular region self-association that whether can be contained " dead functional domain " for other activity of studying TNF triggers; We have studied the influence that the expression of the raising expression of total length acceptor (P55-R) and acceptor intracellular region (P55-IC) is transcribed bacteriocidin 8 (IL-8), bacteriocidin 8 (IL-8) now known it can be activated (Matsushima etc., 1988) by TNF.The result is published in Fig. 8, and Fig. 8 has described in the Hela cell, the inducing action that does not rely on aglucon of IL-8 genetic expression, and this Hela cell is the construct that utilizes the tsiklomitsin regulation and control.(also seeing " method and material " and above-mentioned example 1) with P55-R or P55-IC transfection.In the A hurdle of Fig. 8, reproduced the result of Northern blotting analysis (seeing above-mentioned " method and material ") RNA, the RNA (7 μ g/ swimming lane) that analyzes usefulness extracts from Hela cell (HTta-1), with TNF (500 μ g/ml, 4 hours) handle (TNF) or do not handle (contrast), or come from after the transfection 24 hours HTta-1 cell, promptly use P55-IC (' P55-IC ') P55-R (' P55-R ') or luciferase (' luc ') cells transfected (tsiklomitsin exist or non-existent situation under).What the Northern blotting analytical results on Fig. 8 B hurdle was represented is the methylene blue dyeing situation of the 18SrRNA of each sample in the A hurdle.

Thereby, as can be seen from Figure 8, with tsiklomitsin regulation and control, the construction transfection Hela cell of the cDNA of coding P55-R will be induced transcribing of IL-8.And with the coding P55-IC the cDNA transfectional cell, can observe more intensive inducing action.In above-mentioned two examples, to have only after tsiklomitsin is removed from cell growth medium, inducing action just can take place, and this shows, and inducing is the result that expresses of P55-R or P55-IC as transfection and producing.In contrast, the Hela cell is carried out transfection, then transcribing of IL-8 do not influenced with luciferase cDNA.

Therefore, it seems (Fig. 8) from The above results, the expression that only improves P55-R, or the expression that only improves its intracellular region (P55-IC), all be to trigger cytotoxicity or other effect in the mode that does not rely on body (TNF), these effects comprise the reinforcing effect of IL-8 genetic expression in the cell.The triggering of these effects is likely what the self-association owing to P55-R intracellular region (P55-IC) produced.Only as mentioned above, seemingly, in case self-association takes place in P55-IC, its " dead functional domain " will provide signal for inducing of born of the same parents' internal procedure, these born of the same parents' internal procedures will cause the toxic triggering of cell within a cell, and some other effect, induce the signal of IL-8 genetic expression as conduct, be likely because the effect in other district of P55-IC after the self-association, be responsible for TNF inductive effects different in the born of the same parents (as cytotoxicity so be likely the not same district of P55-IC, IL-8 induces), these effects are results that intracellular signal causes after the P55-IC self-association.

P55-IC can induce the triggering of other born of the same parents' internal effect in the mode that does not rely on aglucon (TNF), inducing action as IL-8, this statement of facts, P55-IC, or its specific part can bring such effect as the specificity instrument in the cell or tissue that needs are handled, and these tissues or cell needn't be handled with TNF.Under many pathological conditions (as tumour), handle with TNF, may cause unfavorable side effect when especially high dosage is handled, these side effects are because TNF several born of the same parents' internal effects generations of system induction with receptors bind.The present invention finds that P55-IC can also imitate other specific TNF inductive effect (except that cytotoxicity), as inducing that IL-8 is expressed, this will start a kind of novel method, this method can import P55-IC or its specific fragment in the specific cell or tissue, and can be as the specific desirable born of the same parents' internal effect of signal induction, as inducing of IL-8, thereby overcome common system's side effect in TNF handles.

(d) intracellular region of P55TNF-R and FAS-R (Fas/Ap01) and " dead functional domain " thereof in the Hela cell cytocidal effect do not rely on the aglucon trigger action.

Cytotoxicity about the intracellular region (P55IC and FAS-IC) of P55TNF-R and FAS-R, further set forth now: P55-IC, its " dead functional domain " (P55DD) and FAS-IC in the Hela cell, all have a cytocidal effect do not rely on the aglucon trigger action.In this research, the Hela cell carries out transfection with expression vector.These expression vectors include different constructs: contain total length P55-TNF-R, comprise the segmental construct of P55IC and P55DD; The construct that contains FAS-IC.Therein in the battery of tests, the Hela cell carries out its transfection (details of construct, preparation etc. see on) with construct that contains P55TNF-R and the construct that contains FAS-IC.The result of this research is set forth in Fig. 9 (A and B), wherein is used for the construct of transfection Hela cell to list in the left-hand column with schematic form.In the middle of the result of FAS or TNF expression of receptor lists in schematic form in two hurdles (from a left side several second and third column), the results are shown in the right-hand column of transfectional cell viability.In Fig. 9 A, shown the Hela cell moment expression total length P55-R of transfection, P55-IC or its fragment, or among the result of luciferase in contrast (luc), in these were expressed, what utilized all was the expression vector of tsiklomitsin regulation and control.In Fig. 9 B, shown the Hela cell single expression FAS-IC of transfection or expressed the result of P55-R and FAS-IC simultaneously, utilization also be the expression vector of tsiklomitsin regulation and control.In the diagram result of Fig. 9 A and B, the transfectional cell under tsiklomitsin (the 1 μ g/ml) existence that open piece is represented to suppress to express, the sealing piece represents that there is not the transfectional cell under the situation in the tsiklomitsin that suppresses to express.The TNF expression of receptor is 20 hours mensuration after transfection, a kind of measuring method is ELISA, promptly utilize the antibody (seeing left-hand column) of anti-acceptor extracellular region, use another kind of measuring method simultaneously, promptly measure TNF receptor expression (middle column) by the binding capacity of measuring radiolabeled TNF and cell.The proteic cytocidal effect of transfection is 48 hours mensuration after transfection.The data of delivering is from one in three tests with the identical result of essence, and in these three tests, each construct is all tested with bipartite form.Being called " ND " expression among Fig. 9 A and the B detects.Obviously as seen result from Fig. 9 A and B only is that the expression of P55IC can cause stronger cytotoxicity.When only expressing " dead functional domain ", also produced significant cytotoxicity, in contrast, disappearance " dead functional domain " or only contain some segmental expression of " dead functional domain " segmental P55IC, the viability of pair cell is influence not, the expression of FAS-IC can not cause significant cytotoxicity, but enhancing highly significant the cytotoxicity of P55-R of its expression.

Example 3

Can add albumen with P55TNF-R or FAS-R intracellular region bonded

With method same in the above-mentioned example and technology, other three kinds of albumen have been separated and have been accredited as and can combine with P55IC or FAS-IC.

In Figure 10-12, delivered three kinds of cDNA clones' the basic nucleotide sequence of part with schematic form, these three kinds of cDNA clones are called F2, F9 and DD11.

FAS-IC with mouse makes " bait ", and F2 clone and F9 clone are therefrom isolated in the embryo library of screening mouse.In Figure 10, represented to measure the partial nucleotide sequence of the F2cDNA of sequence with schematic form, represented a part with schematic form among Figure 11 from the nucleotide sequence that contains 1724 bases of the F9cDNA that measures sequence.To analyzing respectively, show by the proteic binding ability of F2 and F9 clones coding:

(a) F2 and people P55IC and P55DD and mouse FAS-IC have intensive to interact, and it and associated protein, people FAS-IC, and with irrelevant (contrast) Protein S NF ₁But very weak with the interaction of lamin.

(b) F9 and people P55-IC and P55DD and mouse FAS-IC have intensive to interact, and very weak with the interaction of people FAS-IC (associated protein) and irrelevant Protein S NF1 and lamin.

(c) F2 and F9 all with people P75IC, pGBT9 (blank carrier) or people CD-40 without any interaction.

In addition, the retrieval from " gene pool " and " protein pool " shows that F2 and F9 have represented two kinds of novel proteins.

Thereby F2 and F9 representative have two kinds of novel proteins of binding specificity to FAS-IC and P55IC.

Personnel selection P55DD makes " bait ", and the DD11 clone is therefrom isolated in screening people Hela library, and Figure 12 has represented a part from the nucleotide sequence that contains 425 bases of the DD11cDNA of known array with the tabulation form.

The DD11 clone approximately contains 800 Nucleotide.Make probe with this clone, find about 1200 bases of total length of its transcription product.The proteic binding ability of DD11 clones coding is analyzed, is shown that DD11 and P55DD (amino acid 326-414) (see figure 9) has intensive to interact, but with this regional deletion mutant, as amino acid 326-404, then do not interact.The FAS-IC of DD11 and people and mouse can interact, and with lamin to a certain degree interaction is arranged also.DD11 fully not with SNF, also do not interact with pGBT9 (empty bait carrier).DD11 is undiscovered in " gene pool " and " protein pool ".Therefore, DD11 be a kind of can with the albumen of P55IC (P55DD) and FAS-IC specific combination.

Example 4

The structure of solubility dimerization TNF acceptor

Based on the discovery in the above-mentioned example 2, the intracellular region of P55-R (P55-IC) and fragment thereof (" dead functional domain "), the intracellular region of Fas/AP01 and fragment thereof (being also referred to as " dead functional domain ") all have the self-association ability.Therefore might make up new TNF acceptor, they will have self-association (gathering) ability and be solubilities.Such TNF acceptor should be a fusion rotein, promptly contains the whole extracellular regions and the P55-R or the whole intracellular regions of Fas/AP01 or the product that its " dead functional domain " merges mutually that contain necessity of necessary P55-R.Thereby what such fusion constructs will lack P55-R strides the film district, so they are soluble.In addition, because intracellular region or its " dead functional domain " have the self-association ability, therefore, these fusion constructs will have the oligomerization ability, and form the dimer (also may be more high-grade polymer) of P55-R at least.As a result, these dimerization TNF acceptor (P55-R) can be in conjunction with at least two TNF monomers of the TNF homotrimer of natural generation, and provide can with its aglucon (homotrimer TNF) bonded soluble TNF acceptor better.

Therefore, need to make up at least four types P55TNF receptor fusion protein, every kind all has the oligomerization ability, and is soluble.

(i) fusion rotein of P55-R extracellular region (EC55) and P55-R intracellular region (p55-IC) formation;

(ii) EC55 and p55-IC " dead functional domain " fusion rotein of (DD55) constituting;

The (iii) fusion rotein that constitutes of EC55 and Fas/Ap01 intracellular region (ICAS); And

(iv) EC55 and ICFAS " dead functional domain " fusion rotein of (DDFAS) constituting.

In above-mentioned each fusion rotein, the monomeric binding ability of TNF is partly produced by EC55, and oligomerization closes ability (or being at least two polymerization abilities) by " tail region " generation, and " tail region " is the p55IC part, or the DD55 part, or the ICFAS part, or the DDAS part.

In order to make up above-mentioned fusion rotein, need to use the routine techniques of recombinant DNA, this technical system is set up well now and (is seen: Sambrook etc., (1989) molecular cloning: laboratory manual, cold spring port experiment press, the cold spring port: New York) in brief, any suitable bacterium, phage or animal virus expression vector (for selecting to insert cloning vector or the plasmid that DNA expresses design) all can be used for inserting the DNA of coding EC55 and one of them " tail region " in one or more stages." tail region " is p55-IC, or DD55, or ICFAS, or DDFAS.The insertion DNA of each fusion rotein of encoding will place under the various expression regulation sequences of cloning vector or plasmid, as promotor, and ribozyme binding site, transcription factor binding site point etc.The selection of expression regulation sequence is depended on the expression vector type of selection and is also just depended on the type (eukaryotic cell or prokaryotic cell prokaryocyte) of host cell, so that fusion rotein of the present invention can be expressed better.Ideal host cell (and carrier) is the eukaryotic cell type, especially mammiferous cell.

Press the dna molecular of above-mentioned each fusion rotein of follow procedure preparation coding, and they are inserted expression vector.

(a) at first, make up used oligonucleotide among the PCR according to ordinary method, these oligonucleotide are expressed as follows:

1) ACC ATG GGC CTC TCC ACC GTG (EC55, positive meaning chain) (SEQ ID NO:1)

2) ACGC GTC GAC TGT GGT GCC TGA GTC CTC (EC55, antisense strand) (SEQ ID NO:2)

3) ACGC GTC GAC CGC TAC CAA CGG TGG AAG (IC55, positive meaning chain) (SEQ ID NO:3)

4) TCA TCT GAG AAG ACT GGG (IC55, antisense strand) (SEQ ID NO:4)

5) ACGC GTC GAC AAG AGA AAG GAA GTA CAG (ICFAS, positive meaning chain) (SEQ IDNO:5)

6) CTA GAC CAA GCT TTG GAT (ICFAS, antisense strand) (SEQ ID NO:6)

7) ACGC GTC GAC CCC GCG ACG CTG TAC GCG (DD55, positive meaning chain) (SEQ ID NO:7)

8) ACGC GTC GAC GAT GTT GAC TTG AGT AAA (DDFAS, positive meaning chain) (SEQ ID NO:8)

B) this laboratory has the plasmid (referring to EP568925 and above-mentioned routine 1-3) that contains total length p55-R and Fas/Ap01 acceptor coding clone, these plasmids are used for carrying out following amplification, thereby produce the dna fragmentation of each fusion rotein of coding, and these dna fragmentations are connected in the above-mentioned expression vector of selecting.

(i) for the dna fragmentation of composite coding as the EC55 of one of 4 fusion rotein integral parts, make primer with above-mentioned oligonucleotide 1 and 2, the plasmid that contains people P55cDNA is carried out pcr amplification (size is about the fragment of 640 base pairs).

(ii) in order to obtain fusion product EC55-IC55, make primer with above-mentioned oligonucleotide 3 and 4, the plasmid that contains people p55cDNA is carried out pcr amplification, obtain the dna fragmentation (about 677 base pairs of size) of a coding IC55, again this fragment is mixed mutually with the EC55 that cuts through the saLI enzyme, then by the mammalian cell expression vector of flush end connection method access under suitable promoter regulation.The direction of insertion of IC55-EC55 is identified by restriction enzyme digestion and sequencing technologies in the carrier.

(iii) in order to obtain fusion product EC55-ICFAS, make primer with oligonucleotide 5 and 6 plasmid that contains the FAScDNA clone is carried out pcr amplification, the dna fragmentation (about 448 base pairs of size) of ICFAS obtains encoding, again this fragment is cut with the SalI enzyme and mix, then insert mammalian cell expression vector under suitable promoter regulation by the flush end connection method with the EC55 that cuts through the salI enzyme.The direction of insertion of EC55-ICFAS is identified by restriction enzyme digestion and sequencing technologies in the carrier.

(iv) in order to obtain fusion product EC55-DD55, make primer with oligonucleotide 7 and 4 people P55cDNA is carried out pcr amplification, obtain a dna fragmentation that has a DD55 encoding sequence, about 314 base pairs of size, this sheet is cut with the SalI enzyme and is mixed with the EC55 that cuts through the SalI enzyme, then is connected into mammalian cell expression vector by the flush end connection method.The direction of insertion of EC55-DD55 is identified by Restriction Enzyme incision technology and sequencing technologies in the carrier.

(V) in order to obtain fusion product EC55-DDFAS, make primer with oligonucleotide 6 and 8 the FAScDNA clone is carried out pcr amplification, obtain a dna fragmentation that contains the DDFAS encoding sequence, about 332 base pairs of size, this fragment is cut with the SalI enzyme and is mixed with the EC55 that cuts through the SalI enzyme, then is connected into mammalian cell expression vector by the flush end connection method.The direction of insertion of EC55-DDFAS is identified by restriction enzyme digestion and sequencing technologies in the carrier.

In case above-mentioned expression vector establishment finishes, they just can be introduced suitable mammalian cell (as Chinese hamster ovary cell (CHO) or monkey-kidney cells (COS)) and express by routine techniques.Expressed fusion protein will be by routine techniques purifying (referring to EP308378, EP398327 and EP568925).Albumen behind the purifying carries out the analysis that oligomerization closes ability (and degree analyzing, promptly whether they form dimer or high-grade polymer more), also they and TNF bonded ability (and binding affinity) is analyzed simultaneously.

Example 5

The structure of solubility dimerization Fas/Ap01 acceptor

According to the same manner in the above-mentioned example 4, can synthesize following four types Fas/Ap01 fusion product, wherein each fusion rotein all has the oligomerization ability and is soluble:

(i) fusion product that constitutes between the intracellular region of Fas/Ap01 extracellular region (ECFAS) and P55-IC;

(ii) ECFAS and P55-IC " dead functional domain " fusion product of constituting between (DD55);

The (iii) fusion product that constitutes between ECFAS and the Fas/Ap01 intracellular region (ICFAS);

(iv) ECFAS and ICFAS " dead functional domain " fusion product of constituting between (DDFAS).

In above-mentioned each fusion rotein, FAS aglucon binding ability is partly produced by ECFAS; And oligomerization closes ability (being two polymerization abilities at least) and is produced by " tail region ", and " tail region " is P55-IC, or DD55, or ICFAS, or in the DDFAS part any one.

Encode the structure of the dna fragmentation of above-mentioned fusion rotein and expression vector thereof with example 4, but here will not prepare the ECFAS fragment and link to each other with different oligonucleotide sequences (delivering) with above-mentioned arbitrary " tail region ".Then, expression vector is introduced proper host cell, and the fusion rotein of expressing is carried out purifying, the oligomerization of analyzing them then closes ability (and degree, promptly whether they form dimer or high-grade polymer more).Also analyzed simultaneously the binding ability (and binding affinity) of they and FAS aglucon.

Example 6

The structure of soluble oligomeric " mixing " TNF/FAS acceptor

The oligomerization acceptor that has " mixing " affinity in order to prepare, i.e. the acceptor that simultaneously TNF and FAS-R aglucon is had affinity, the above-mentioned fusion product of mentioning (example 4 and 5) will be applied to follow procedure:

I) synthetic a kind of fusion product as described in Example 4, the extracellular region that it comprises TNF-R (P75TNF-R or P55TNF-R) are blended in any in P55IC, FAS-IC, these four kinds of albumen of P55DD, FASDD.

Ii) synthetic a kind of fusion product as described in Example 5, the extracellular region that it comprises TNF-R are blended in any in P55IC, P55DD, these four kinds of albumen of FAS-IC, FAS-DD.

Iii) i) in arbitrary fusion product with ii) in arbitrary fusion product mix, obtain a new dimer acceptor (or more high-grade polymer), this receptor has the extracellular region of TNF-R and FAS-R simultaneously.These two kinds of extracellular regions by-IC district or-the DD section links to each other.

In the said procedure, i) fusion product in and ii) in fusion product can synthesize respectively, that is, earlier purifying comes out from the transformant that produces them, again external mixed, thereby obtains blended affinity acceptor.In addition, can in this case, can directly from the host cell of its transfection, obtain the blended affinity receptor with carrier that contains the fusion product encoding sequence and host cell cotransfection.The actual oligomerization of fusion product synthesizes the oligomerization acceptor, can occur in the cell, and also can in the purge process of fusion product, take place, or behind purifying, take place, thus the fusion product that obtains in cell, expressing.In order accurately to select the blended affinity receptor, can use various routine techniquess, for example, use affinity chromatography, in this method,, in the successive chromatographic step, select the acceptor that has two kinds of extracellular regions simultaneously with the antibody of anti-TNF-R and FAS-R extracellular region.

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Sequence table

(1) general information:

(i) applicant:

(A) title: Yeda Research and Development Co., Ltd.

(B) street: P.O.Box 95

(C) city: Rehovot

(E) country: Israel

(F) postcode (ZIP): 76100

(G) phone: 011-972-847-0617

(H) fax: 011-972-847-0733

(A) title: Henry WEINWURZEL

(B) street: Herzl street 43

(C) city: Raanana

(E) country: Israel

(F) postcode (ZIP): 43353

(A) title: David WALLACH

(B) street: 24 Borochov streets

(C) city: Rehovot

(E) country: Israel

(F) postcode (ZIP): 76406

(A) title: Mark BOLDIN

(B) street: Zelenograd 927-53

(C) city: Moscow

(E) country: Russia

(F) postcode (ZIP): 103575

(A) title: Igor METT

(B) street: 60 Levin Epstein streets

(C) city: Rehovot

(E) country: Israel

(F) postcode (ZIP): 76462

(A) title: Eugene VARFOLOMEEV

(B) street: P.O.Box 26

(C) city: Rehovot

(E) country: Israel

(F) postcode (ZIP): 76100

(ii) denomination of invention: the conditioning agent of TNF/NGF superfamily receptors and soluble oligomeric TNF/NGF superfamily receptors

(iii) sequence quantity: 26

(iv) mailing address:

(A) addressee: BROWDY AND NEIMARK

(B) street: 419 Seventh streets, N.W., Suite 300

(C) city: Washington

(D) continent: D.C.

(E) country: USA

(F)ZIP：20004

(v) computer-reader form:

(A) media types: floppy disk

(B) computer: IBM PC compatibility

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release #1.0, Version #1.30 (EPO)

(v) current application materials:

Application number: PCT/US95/05854

(vi) application materials formerly:

(A) application number: IL109,632

(B) applying date: 11-MAY-1994

(vi) application materials formerly:

(A) application number: IL111,125

(B) applying date: 02-OCT-1994

(viii) lawyer/proxy's information:

(A) title: BROWDY, Roger L.

(B) number of registration: 25,618

(C) reference/file number: WALLACH=15 PCT

(ix) telecommunication information:

(A) phone: 202-628-5197

(B) fax: 202-737-3528

(C) telegram: 248633

(2) SEQ ID NO:1 information:

(i) sequence signature:

(A) length: 21bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:1:

ACCATGGGCC TCTCCACCGT G 21

(2) information SEQ ID NO:2:

(i) sequence signature:

(A) length: 28bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:2:

ACGCGTCGAC TGTGGTGCCT GAGTCCTC 28

(2) information SEQ ID NO:3:

(i) sequence signature:

(A) length: 28bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:3:

ACGCGTCGAC CGCTACCAAC GGTGGAAG 28

(2) information SEQ ID NO:4:

(i) sequence signature:

(A) length: 18bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:4:

TCATCTGAGA AGACTGGG 18

(2) information SEQ ID NO:5:

(i) sequence signature:

(A) length: 28bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:5:

ACGCGTCGAC AAGAGAAAGG AAGTACAG 28

(2) information SEQ ID NO:6:

(i) sequence signature:

(A) length: 18bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:6:

CTAGACCAAG CTTTGGAT 18

(2) information SEQ ID NO:7:

(i) sequence signature:

(A) length: 28bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:7:

ACGCGTCGAC CCCGCGACGC TGTACGCC 28

(2) information SEQ ID NO:8:

(i) sequence signature:

(A) length: 28bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:8:

ACGCGTCGAC GATGTTGACT TGAGTAAA 28

(2) information SEQ ID NO:9:

(i) sequence signature:

(A) length: 2866bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:9:

ATTCGGGTGC AGCCCCAGCA GTCTCCAGCG GCGGCCCCCG GCGGCACGGA CGAGAAGCCG 60

AGCGGCAAGG AGCGGCGGGA TGCCGGGGAC AAGGACAAAG AACAGGAGCT GTCTGAAGAG 120

GATAAACAGC TTCAAGATGA ACTGGAGATG CTCGTGGAAC GACTAGGGGA GAAGGATACA 180

TCCCTGTATC GACCAGCGCT GGAGGAATTG CGAAGGCAGA TTCGTTCTTC TACAACTTCC 240

ATGACTTCAG TGCCCAAGCC TCTCAAATTT CTGCGTCCAC ACTATGGCAA ACTGAAGGAA 300

ATCTATGAGA ACATGGCCCC TGGGGAGAAT AAGCGTTTTG CTGCTGACAT CATCTCCGTT 360

TTGGCCATGA CCATGAGTGG GGAGCGTGAG TGCCTCAAGT ATCGGCTAGT GGGCTCCCAG 420

GAGGAATTGG CATCATGGGG TCATGAGTAT GTCAGGCATC TGGCAGGAGA AGTGGCTAAG 480

GAGTGGCAGG AGCTGGATGA CGCAGAGAAG GTCCAGCGGG AGCCTCTGCT CACTCTGGTG 540

AAGGAAATCG TCCCCTATAA CATGGCCCAC AATGCAGAGC ATGAGGCTTG CGACCTGCTT 600

ATGGAAATTG AGCAGGTGGA CATGCTGGAG AAGGACATTG ATGAAAATGC ATATGCAAAG 660

GTCTGCCTTT ATCTCACCAG TTGTGTGAAT TACGTGCCTG AGCCTGAGAA CTCAGCCCTA 720

CTGCGTTGTG CCCTGGGTGT GTTCCGAAAG TTTACCCGCT TCCCTGAAGC TCTGAGATTG 780

GCATTGATGC TCAATGACAT GGAGTTGGTA GAAGACATCT TCACCTCCTG CAAGGATGTG 840

GTAGTACAGA AACAGATGGC ATTCATGCTA GGCCGGCATG GGGTGTTCCT GGAGCTGAGT 900

GAAGATGTCG AGGAGTATGA GGACCTGACA GAGATCATGT CCAATGTACA GCTCAACAGC 960

AACTTCTTGG CCTTAGCTCG GGAGCTGGAC ATCATGGAGC CCAAGGTGCC TGATGACATC 1020

TACAAAACCC ACCTAGAGAA CAACAGGTTT GGGGGCAGTG GCTCTCAGGT GGACTCTGCC 1080

CGCATGAACC TGGCCTCCTC TTTTGTGAAT GGCTTYGTGA ATGCAGCTTT TGGCCAAGAC 1140

AAGCTGCTAA CAGATGATGG CAACAAATGG CTTTACAAGA ACAAGGACCA CGGAATGTTG 1200

AGTGCAGCTG CATCTCTTGG GATGATTCTG CTGTGGGATG TGGATGGTGG CCTCACCCAG 1260

ATTGACAAGT ACCTGTACTC CTCTGAGGAC TACATTAAGT CAGGAGCTCT TCTTGCCTGT 1320

GGCATAGTGA ACTCTGGGGT CCGGAATGAG TGTGACCCTG CTCTGGCACT GCTCTCAGAC 1380

TATGTTCTCC ACAACAGCAA CACCATGAGA CTTGGTTCCA TCTTTGGGCT AGGCTTGGCT 1440

TATGCTGGCT CAAATCGTGA AGATGTCCTA ACACTGCTGC TGCCTGTGAT GGGAGATTCA 1500

AAGTCCAGCA TGGAGGTGGC AGGTGTCACA GCTTTAGCCT GTGGAATGAT AGCAGTAGGG 1560

TCCTGCAATG GAGATGTAAC TTCCACTATC CTTCAGACCA TCATGGAGAA GTCAGAGACT 1620

GAGCTCAAGG ATACTTATGC TCGTTGGCTT CCTCTTGGAC TGGGTCTCAA CCACCTGGGG 1680

AAGGGTGAGG CCATCGAGGC AATCCTGGCT GCACTGGAGG TTGTGTCAGA GCCATTCCGC 1740

AGTTTTGCCA ACACACTGGT GGATGTGTGT GCATATGCAG GCTCTGGGAA TGTGCTGAAG 1800

GTGCAGCAGC TGCTCCACAT TTGTAGCGAA CACTTTGACT CCAAAGAGAA GGAGGAAGAC 1860

AAAGACAAGA AGGAAAAGAA AGACAAGGAC AAGAAGGAAG CCCCTGCTGA CATGGGAGCA 1920

CATCAGGGAG TGGCTGTTCT GGGGATTGCC CTTATTGCTA TGGGGGAGGA GATTGGTGCA 1980

GAGATGGCAT TACGAACCTT TGGCCACTTG CTGAGATATG GGGAGCCTAC ACTCCGGAGG 2040

GCTGTACCTT TAGCACTGGC CCTCATCTCT GTTTCAAATC CACGACTCAA CATCCTGGAT 2100

ACCCTAAGCA AATTCTCTCA TGATGCTGAT CCAGAAGTTT CCTATAACTC CATTTTTGCC 2160

ATGGGCATGG TGGGCAGTGG TACCAATAAT GCCCGTCTGG CTGCAATGCT GCGCCAGTTA 2220

GCTCAATATC ATGCCAAGGA CCCAAACAAC CTCTTCATGG TGCGCTTGGC ACAGGGCCTG 2280

ACACATTTAG GGAAGGGCAC CCTTACCCTC TGCCCCTACC ACAGCGACCG GCAGCTTATG 2340

AGCCAGGTGG CCGTGGCTGG ACTGCTCACT GTGCTTGTCT CTTTCCTGGA TGTTCGAAAC 2400

ATTATTCTAG GCAAATCACA CTATGTATTG TATGGGCTGG TGGCTGCCAT GCAGCCCCGA 2460

ATGCTGGTTA CGTTTGATGA GGAGCTGCGG CCATTGCCAG TGTCTGTCCG TGTGGGCCAG 2520

GCAGTGGATG TGGTGGGCCA GGCTGGCAAG CCGAAGACTA TCACAGGGTT CCAGACGCAT 2580

ACAACCCCAG TGTTGTTGGC CCACGGGGAA CGGGCAGAAT TGGCCACTGA GGAGTTTCTT 2640

CCTGTTACCC CCATTCTGGA AGGTTTTGTT ATCTTCGGAA GAACCCCAAT TATGATCTCT 2700

AAGTGACCAC CAGGGGCTCT GAACTGCAGC TGATGTTATC AGCAGGACAT GCATCCTGCT 2760

GCCAAGGGTG GACACGGCTG CAGACTTCTG GGGGAATTGT CGCCTCCTGC TCTTTTGTTA 2820

CTGAGTGAGA TAAGGTTGTT CAATAAAGAC TTTTATCCCC AAGGTC 2866

(2) information SEQ ID NO:10:

(i) sequence signature:

(A) length: 676bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:10:

GAATTCGGCA CGAGCGGCAC GAGGACAGAG TGAGACTCTG TCTCTTAAAA TAATAATAAA 60

AATAAAAATA AAATGTGGGG CCGGGCAAGG TGGCTCATGC CTGTAATCCC AGCACCTTGG 120

GAGGCTGAGG CAGGAGGATT GCCTAAGCCC AGGAGTTTGA CATCAGCCTG GGCAACATGG 180

TGAAACCCCA TCTCTACAAA AAATGCAAAA ATTAGCCAGG TGTGGTGGGT GTGCTCCTAT 240

AGTCTCAGCT ACTCAGGAGG CTGAGGTAGA GGGGATCACC TGAGCCCAGG AAGTTTGGAG 300

GCTATAGTGA GCTGAAGACC CGCACCATTG CACGCCAGCC TGGAGCAAGA GACNCTGTCT 360

CCACATAAAT AAATAAATAA ATAAAAGTGG GGAACTTCTG TGTTAAGTCA GAAGGCACCA 420

CACAATTTGN ATAGCCANCA ACCATATTCA ATACCCAATC TCTTTATTGC AATATAAGTA 480

TTTGTAAACC CCTACACAAA TATTCCCAAG AATAAGTTGG AATATAAATT ACTATATCAA 540

TCANCCAATA AAAATAAACA CATACAGTAT TTATTTCCTG TTGCTCCATA TAAAGCTTTG 600

CTATTTCAAT ATAAAGCTTA CCTAGTATGG TCATTTGAGC CTGAGCAGAG AATATGCCCA 660

AGCTCGTGCC GAATTC 676

(2) information SEQ ID NO:11:

(i) sequence signature:

(A) length: 1153bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:11:

GGCGNTCTGA CTCTCTACTG AACCAAGACT GAATCAGAGA GACTCGAGTG CNCTTATTTG 60

ATTAANCCCA AATTATTGAA ACCTNTGATT TTTTCTGGAG GNGGATGATA AAGATGTGAA 120

AGTGTGATGA ACAGTGTGTA TCCCTACTCT TGATCCTGGA ACCAGACAAG CAAGAAGCTT 180

TGATTGAAAG CCTATGTGAA AAGCTGGTCA AATTTCGCGA AGGTGAACGC CCGTCTCTGA 240

GACTGCAGTT GTTAAGCAAC CTTTTCCACG GGATGGATAA GAATACTCCT GTAAGATACA 300

CAGTGTATTG CAGCCTTATT AAAGTGGCAG CATCTTGTGG GGCCATCCAG TACATCCCAA 360

CTGAGCTGGA TCAAGTTAGA AAATGGATTT CTGACTGGAA TCTCACCACT GAAAAAAAGC 420

ACACCCTTTT AAGACTACTT TATGAGGCAC TTGTGGATTG TAAGAAGAGT GATGCTGCTT 480

CAAAAGTCAT GGTGGAATTG CTCGGAAGTT ACACAGAGGA CAATGCTTCC CAGGCTCGAG 540

TTGATGCCCA CAGGTGTATT GTACGAGCAT TGAAAGATCC AAATGCATTT CTTTGTGACC 600

ACCTTCTTAC TTTAAAACCA GTCAAGTTTG TGGAAGGCGA GCTTATTCAT GATCTTTTAA 660

CCATTTGTGT GAGTGCTAAA TTGGCATCAT ATGTCAAGTT TTATCAGAAT AATAAAGACT 720

TCATTGATTC ACTTGGCCTG TTACATGAAC AGAATATGGC AAAAATGAGA CTACTTACTT 780

TTATGGGAAT GGCAGTAGAA AATAAGGAAA TTTCTTTTGA CACAATGCAG CAAGAACTTC 840

AGATTGGAGC TGATGATGTT GAAGCATTTG TTATTGACGC CGTAAGAACT AAAATGGTCT 900

ACTGCAAAAT TGATCAGACC CAGAGAAAAG TAGTTGTCAG TCATAGCACA CATCGGACAT 960

TTGGAAAACA GCAGTGGCAA CAACTGTATG ACACACTTAA TGCCTGGAAA CAAAATCTGA 1020

ACAAAGTGAA AAACAGCCTT TTGAGTCTTT CTGATACCTG AGTTTTTATG CTTATAATTT 1080

TTGTTCTTTG AAAAAAAAGC CCTAAATCAT AGTAAAACAT TATAAACTAA AAAAAAAAAA 1140

AAAAAAACTC GAG 1153

(2) information SEQ ID NO:12:

(i) sequence signature:

(A) length: 220bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:12:

GTCCGGTTTA CTTTAACTTA GTTTTGCATA GTTCTAGTGC ACGTGAAATT GAAAAGTTAT 60

TTCCCTTTAG CTGTGTTATT ATAGAGCAGA AATTCTGTTT TTAAAAATTA GCCTAAGATA 120

TACTTGTTTT TGTAAAGAAA AATATTTAAT GCTTGAACAA AATAAATTGG AGTTGGAGTA 180

GAATGTAGTT TGAGGAAATT TGCAGCTTCC AATGCCTCTG 220

(2) information SEQ ID NO:13:

(i) sequence signature:

(A) length: 220bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:13:

CAGAGGCAT TGGAAGCTGCA AATTTCCTCA AACTACATTC TACTCCAACT CCAATTTATT 60

TTGTTCAAGC ATTAAATATT TTTCTTTACA AAAACAAGTA TATCTTAGGC TAATTTTTAA 120

AAACAGAATT TCTGCTCTAT AATAACACAG CTAAAGGGAA ATAACTTTTC AATTTCACGT 180

GCACTAGAAC TATGCAAAAC TAAGTTAAAG TAAACCGGAC 220

(2) information SEQ ID NO:14:

(i) sequence signature:

(A) length: 900AA

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:14:

Arg Val Gln Pro Gln Gln Ser Pro Ala Ala Ala Pro Gly Gly Thr Asp

1 5 10 15

Glu Lys Pro Ser Gly Lys Glu Arg Arg Asp Ala Gly Asp Lys Asp Lys

20 25 30

Glu Gln Glu Leu Ser Glu Glu Asp Lys Gln Leu Gln Asp Glu Leu Glu

35 40 45

Met Leu Val Glu Arg Leu Gly Glu Lys Asp Thr Ser Leu Tyr Arg Pro

50 55 60

Ala Leu Glu Glu Leu Arg Arg Gln Ile Arg Ser Ser Thr Thr Ser Met

65 70 75 80

Thr Ser Val Pro Lys Pro Leu Lys Phe Leu Arg Pro His Tyr Gly Lys

85 90 95

Leu Lys Glu Ile Tyr Glu Asn Met Ala Pro Gly Glu Asn Lys Arg Phe

100 105 110

Ala Ala Asp Ile Ile Ser Val Leu Ala Met Thr Met Ser Gly Glu Arg

115 120 125

Glu Cys Leu Lys Tyr Arg Leu Val Gly Ser Gln Glu Glu Leu Ala Ser

130 135 140

Trp Gly His Glu Tyr Val Arg His Leu Ala Gly Glu Val Ala Lys Glu

145 150 155 160

Trp Gln Glu Leu Asp Asp Ala Glu Lys Val Gln Arg Glu Pro Leu Leu

165 170 175

Thr Leu Val Lys Glu Ile Val Pro Tyr Asn Met Ala His Asn Ala Glu

180 185 190

His Glu Ala Cys Asp Leu Leu Met Glu Ile Glu Gln Val Asp Met Leu

195 200 205

Glu Lys Asp Ile Asp Glu Asn Ala Tyr Ala Lys Val Cys Leu Tyr Leu

210 215 220

Thr Ser Cys Val Asn Tyr Val Pro Glu Pro Glu Asn Ser Ala Leu Leu

225 230 235 240

Arg Cys Ala Leu Gly Val Phe Arg Lys Phe Thr Arg Phe Pro Glu Ala

245 250 255

Leu Arg Leu Ala Leu Met Leu Asn Asp Met Glu Leu Val Glu Asp Ile

260 265 270

Phe Thr Ser Cys Lys Asp Val Val Val Gln Lys Gln Met Ala Phe Met

275 280 285

Leu Gly Arg His Gly Val Phe Leu Glu Leu Ser Glu Asp Val Glu Glu

290 295 300

Tyr Glu Asp Leu Thr Glu Ile Met Ser Asn Val Gln Leu Asn Ser Asn

305 310 315 320

Phe Leu Ala Leu Ala Arg Glu Leu Asp Ile Met Glu Pro Lys Val Pro

325 330 335

Asp Asp Ile Tyr Lys Thr His Leu Glu Asn Asn Arg Phe Gly Gly Ser

340 345 350

Gly Ser Gln Val Asp Ser Ala Arg Met Asn Leu Ala Ser Ser Phe Val

355 360 365

Asn Gly Phe Val Asn Ala Ala Phe Gly Gln Asp Lys Leu Leu Thr Asp

370 375 380

Asp Gly Asn Lys Trp Leu Tyr Lys Asn Lys Asp His Gly Met Leu Ser

385 390 395 400

Ala Ala Ala Ser Leu Gly Met Ile Leu Leu Trp Asp Val Asp Gly Gly

405 410 415

Leu Thr Gln Ile Asp Lys Tyr Leu Tyr Ser Ser Glu Asp Tyr Ile Lys

420 425 430

Ser Gly Ala Leu Leu Ala Cys Gly Ile Val Asn Ser Gly Val Arg Asn

435 440 445

Glu Cys Asp Pro Ala Leu Ala Leu Leu Ser Asp Tyr Val Leu His Asn

450 455 460

Ser Asn Thr Met Arg Leu Gly Ser Ile Phe Gly Leu Gly Leu Ala Tyr

465 470 475 480

Ala Gly Ser Asn Arg Glu Asp Val Leu Thr Leu Leu Leu Pro Val Met

485 490 495

Gly Asp Ser Lys Ser Ser Met Glu Val Ala Gly Val Thr Ala Leu Ala

500 505 510

Cys Gly Met Ile Ala Val Gly Ser Cys Asn Gly Asp Val Thr Ser Thr

515 520 525

Ile Leu Gln Thr Ile Met Glu Lys Ser Glu Thr Glu Leu Lys Asp Thr

530 535 540

Tyr Ala Arg Trp Leu Pro Leu Gly Leu Gly Leu Asn His Leu Gly Lys

545 550 555 560

Gly Glu Ala Ile Glu Ala Ile Leu Ala Ala Leu Glu Val Val Ser Glu

565 570 575

Pro Phe Arg Ser Phe Ala Asn Thr Leu Val Asp Val Cys Ala Tyr Ala

580 585 590

Gly Ser Gly Asn Val Leu Lys Val Gln Gln Leu Leu His Ile Cys Ser

595 600 605

Glu His Phe Asp Ser Lys Glu Lys Glu Glu Asp Lys Asp Lys Lys Glu

610 615 620

Lys Lys Asp Lys Asp Lys Lys Glu Ala Pro Ala Asp Met Gly Ala His

625 630 635 640

Gln Gly Val Ala Val Leu Gly Ile Ala Leu Ile Ala Met Gly Glu Glu

645 650 655

Ile Gly Ala Glu Met Ala Leu Arg Thr Phe Gly His Leu Leu Arg Tyr

660 665 670

Gly Glu Pro Thr Leu Arg Arg Ala Val Pro Leu Ala Leu Ala Leu Ile

675 680 685

Ser Val Ser Asn Pro Arg Leu Asn Ile Leu Asp Thr Leu Ser Lys Phe

690 695 700

Ser His Asp Ala Asp Pro Glu Val Ser Tyr Asn Ser Ile Phe Ala Met

705 710 715 720

Gly Met Val Gly Ser Gly Thr Asn Asn Ala Arg Leu Ala Ala Met Leu

725 730 735

Arg Gln Leu Ala Gln Tyr His Ala Lys Asp Pro Asn Asn Leu Phe Met

740 745 750

Val Arg Leu Ala Gln Gly Leu Thr His Leu Gly Lys Gly Thr Leu Thr

755 760 765

Leu Cys Pro Tyr His Ser Asp Arg Gln Leu Met Ser Gln Val Ala Val

770 775 780

Ala Gly Leu Leu Thr Val Leu Val Ser Phe Leu Asp Val Arg Asn Ile

785 790 795 800

Ile Leu Gly Lys Ser His Tyr Val Leu Tyr Gly Leu Val Ala Ala Met

805 810 815

Gln Pro Arg Met Leu Val Thr Phe Asp Glu Glu Leu Arg Pro Leu Pro

820 825 830

Val Ser Val Arg Val Gly Gln Ala Val Asp Val Val Gly Gln Ala Gly

835 840 845

Lys Pro Lys Thr Ile Thr Gly Phe Gln Thr His Thr Thr Pro Val Leu

850 855 860

Leu Ala His Gly Glu Arg Ala Glu Leu Ala Thr Glu Glu Phe Leu Pro

865 870 875 880

Val Thr Pro Ile Leu Glu Gly Phe Val Ile Phe Gly Arg Thr Pro Ile

885 890 895

Met Ile Ser Lys

900

(2) information SEQ ID NO:15:

(i) sequence signature:

(A) length: 995AA

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:15:

Lys Lys Met Val Asp Glu Ser Asp Lys Lys Gln Gln Thr Ile Asp Glu

1 5 10 15

Gln Ser Gln Ile Ser Pro Glu Lys Gln Thr Pro Asn Lys Lys Asp Lys

20 25 30

Lys Lys Glu Glu Glu Glu Gln Leu Ser Glu Glu Asp Ala Lys Leu Lys

35 40 45

Thr Asp Leu Glu Leu Leu Val Glu Arg Leu Lys Glu Asp Asp Ser Ser

50 55 60

Leu Tyr Glu Ala Ser Leu Asn Ala Leu Lys Glu Ser Ile Lys Asn Ser

65 70 75 80

Thr Ser Ser Met Thr Ala Val Pro Lys Pro Leu Lys Phe Leu Arg Pro

85 90 95

Thr Tyr Pro Asp Leu Cys Ser Ile Tyr Asp Lys Trp Thr Asp Pro Asn

100 105 110

Leu Lys Ser Ser Leu Ala Asp Val Leu Ser Ile Leu Ala Met Thr Tyr

115 120 125

Ser Glu Asn Gly Lys His Asp Ser Leu Arg Tyr Arg Leu Leu Ser Asp

130 135 140

Val Ser Asp Phe Glu Gly Trp Gly His Glu Tyr Ile Arg His Leu Ala

145 150 155 160

Leu Glu Ile Gly Glu Val Tyr Asn Asp Gln Val Glu Lys Asp Ala Glu

165 170 175

Asp Glu Thr Ser Ser Asp Gly Ser Lys Ser Asp Gly Ser Ala Ala Thr

180 185 190

Ser Gly Phe Glu Phe Ser Lys Glu Asp Thr Leu Arg Leu Cys Leu Asp

195 200 205

Ile Val Pro Tyr Phe Leu Lys His Asn Gly Glu Glu Asp Ala Val Asp

210 215 220

Leu Leu Leu Glu Ile Glu Ser Ile Asp Lys Leu Pro Gln Phe Val Asp

225 230 235 240

Glu Asn Thr Phe Gln Arg Val Cys Gln Tyr Met Val Ala Cys Val Pro

245 250 255

Leu Leu Pro Pro Pro Glu Asp Val Ala Phe Leu Lys Thr Ala Tyr Ser

260 265 270

Ile Tyr Leu Ser Gln Asn Glu Leu Thr Asp Ala Ile Ala Leu Ala Val

275 280 285

Arg Leu Gly Glu Glu Asp Met Ile Arg Ser Val Phe Asp Ala Thr Ser

290 295 300

Asp Pro Val Met His Lys Gln Leu Ala Tyr Ile Leu Ala Ala Gln Lys

305 310 315 320

Thr Ser Phe Glu Tyr Glu Gly Val Gln Asp Ile Ile Gly Asn Gly Lys

325 330 335

Leu Ser Glu His Phe Leu Tyr Leu Ala Lys Glu Leu Asn Leu Thr Gly

340 345 350

Pro Lys Val Pro Glu Asp Ile Tyr Lys Ser His Leu Asp Asn Ser Lys

355 360 365

Ser Val Phe Ser Ser Ala Gly Leu Asp Ser Ala Gln Gln Asn Leu Ala

370 375 380

Ser Ser Phe Val Asn Gly Phe Leu Asn Leu Gly Tyr Cys Asn Asp Lys

385 390 395 400

Leu Ile Val Asp Asn Asp Asn Trp Val Tyr Lys Thr Lys Gly Asp Gly

405 410 415

Met Thr Ser Ala Val Ala Ser Ile Gly Ser Ile Tyr Gln Trp Asn Leu

420 425 430

Asp Gly Leu Gln Gln Leu Asp Lys Tyr Leu Tyr Val Asp Glu Pro Glu

435 440 445

Val Lys Ala Gly Ala Leu Leu Gly Ile Gly Ile Ser Ala Ser Gly Val

450 455 460

His Asp Gly Glu Val Glu Pro Ala Leu Leu Leu Leu Gln Asp Tyr Val

465 470 475 480

Thr Asn Pro Asp Thr Lys Ile Ser Ser Ala Ala Ile Leu Gly Leu Gly

485 490 495

Ile Ala Phe Ala Gly Ser Lys Asn Asp Glu Val Leu Gly Leu Leu Leu

500 505 510

Pro Ile Ala Ala Ser Thr Asp Leu Pro Ile Glu Thr Ala Ala Met Ala

515 520 525

Ser Leu Ala Leu Ala His Val Phe Val Gly Thr Cys Asn Gly Asp Ile

530 535 540

Thr Thr Ser Ile Met Asp Asn Phe Leu Glu Arg Thr Ala Ile Glu Leu

545 550 555 560

Lys Thr Asp Trp Val Arg Phe Leu Ala Leu Ala Leu Gly Ile Leu Tyr

565 570 575

Met Gly Gln Gly Glu Gln Val Asp Asp Val Leu Glu Thr Ile Ser Ala

580 585 590

Ile Glu His Pro Met Thr Ser Ala Ile Glu Val Leu Val Gly Ser Cys

595 600 605

Ala Tyr Thr Gly Thr Gly Asp Val Leu Leu Ile Gln Asp Leu Leu His

610 615 620

Arg Leu Thr Pro Lys Asn Val Lys Gly Glu Glu Asp Ala Asp Glu Glu

625 630 635 640

Glu Thr Ala Glu Gly Gln Thr Asn Ser Ile Ser Asp Phe Leu Gly Glu

645 650 655

Gln Val Asn Glu Pro Thr Lys Asn Glu Glu Ala Glu Ile Glu Val Asp

660 665 670

Glu Met Glu Val Asp Ala Glu Gly Glu Glu Val Glu Val Lys Ala Glu

675 680 685

Ile Thr Glu Lys Lys Asn Gly Glu Ser Leu Glu Gly Glu Glu Ile Lys

690 695 700

Ser Glu Glu Lys Lys Gly Lys Ser Ser Asp Lys Asp Ala Thr Thr Asp

705 710 715 720

Gly Lys Asn Asp Asp Glu Glu Glu Glu Lys Glu Ala Gly Ile Val Asp

725 730 735

Glu Leu Ala Tyr Ala Val Leu Gly Ile Ala Leu Ile Ala Leu Gly Glu

740 745 750

Asp Ile Gly Lys Glu Met Ser Leu Arg His Phe Gly His Leu Met His

755 760 765

Tyr Gly Asn Glu His Ile Arg Arg Met Val Pro Leu Ala Met Gly Ile

770 775 780

Val Ser Val Ser Asp Pro Gln Met Lys Val Phe Asp Thr Leu Thr Arg

785 790 795 800

Phe Ser His Asp Ala Asp Leu Glu Val Ser Met Asn Ser Ile Phe Ala

805 810 815

Met Gly Leu Cys Gly Ala Gly Thr Asn Asn Ala Arg Leu Ala Gln Leu

820 825 830

Leu Arg Gln Leu Ala Ser Tyr Tyr Ser Arg Glu Gln Asp Ala Leu Phe

835 840 845

Ile Thr Arg Leu Ala Gln Gly Leu Leu His Leu Gly Lys Gly Thr Met

850 855 860

Thr Met Asp Val Phe Asn Asp Ala His Val Leu Asn Lys Val Thr Leu

865 870 875 880

Ala Ser Ile Leu Thr Thr Ala Val Gly Leu Val Ser Pro Ser Phe Met

885 890 895

Leu Lys His His Gln Leu Phe Tyr Met Leu Asn Ala Gly Ile Arg Pro

900 905 910

Lys Phe Ile Leu Ala Leu Asn Asp Glu Gly Glu Pro Ile Lys Val Asn

915 920 925

Val Arg Val Gly Gln Ala Val Glu Thr Val Gly Gln Ala Gly Arg Pro

930 935 940

Lys Lys Ile Thr Gly Trp Ile Thr Gln Ser Thr Pro Val Leu Leu Asn

945 950 955 960

His Gly Glu Arg Ala Glu Leu Glu Thr Asp Glu Tyr Ile Ser Tyr Thr

965 970 975

Ser His Ile Glu Gly Val Val Ile Leu Lys Lys Asn Pro Asp Tyr Arg

980 985 990

Glu Glu Glu

995

(2) information SEQ ID NO:16:

(i) sequence signature:

(A) length: 945AA

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:16:

Met Ser Leu Thr Thr Ala Ala Pro Leu Leu Ala Leu Leu Arg Glu Asn

1 5 10 15

Gln Asp Ser Val Lys Thr Tyr Ala Leu Glu Ser Ile Asn Asn Val Val

20 25 30

Asp Gln Leu Trp Ser Glu Ile Ser Asn Glu Leu Pro Asp Ile Glu Ala

35 40 45

Leu Tyr Asp Asp Asp Thr Phe Ser Asp Arg Glu Met Ala Ala Leu Ile

50 55 60

Ala Ser Lys Val Tyr Tyr Asn Leu Gly Glu Tyr Glu Ser Ala Val Lys

65 70 75 80

Tyr Ala Leu Ala Ala Lys Asp Arg Phe Asp Ile Asp Glu Lys Ser Gln

85 90 95

Phe Val Glu Thr Ile Val Ser Lys Ser Ile Glu Met Tyr Val Gln Glu

100 105 110

Ala Ser Lys Gln Tyr Thr Lys Asp Glu Gln Phe Tyr Thr Lys Asp Ile

115 120 125

Ile Asp Pro Lys Leu Thr Ser Ile Phe Glu Arg Met Ile Glu Lys Cys

130 135 140

Leu Lys Ala Ser Glu Leu Lys Leu Ala Leu Gly Ile Ala Leu Glu Gly

145 150 155 160

Tyr Arg Leu Asp Ile Ile Glu Ser Ala Leu Lys Ser Lys Leu Asp Gln

165 170 175

Asp Ser Thr Ser Glu Asn Val Lys Ile Ile Asn Tyr Leu Leu Thr Leu

180 185 190

Ala Ile Thr Thr Val Thr Asn Ser Lys Phe Arg Ser Ser Ile Leu Arg

195 200 205

Lys Ser Phe Asp Phe Leu Met Asn Met Pro Asn Cys Asp Tyr Leu Thr

210 215 220

Leu Asn Lys Val Val Val Asn Leu Asn Asp Ala Gly Leu Ala Leu Gln

225 230 235 240

Leu Phe Lys Lys Leu Lys Glu Glu Asn Asp Glu Gly Leu Ser Ala Gln

245 250 255

Ile Ala Phe Asp Leu Val Ser Ser Ala Ser Gln Gln Leu Leu Glu Ile

260 265 270

Leu Val Thr Glu Leu Thr Ala Gln Gly Tyr Asp Pro Ala Leu Leu Asn

275 280 285

Ile Leu Ser Gly Leu Pro Thr Cys Asp Tyr Tyr Asn Thr Phe Leu Leu

290 295 300

Asn Asn Lys Asn Ile Asp Ile Gly Leu Leu Asn Lys Ser Lys Ser Ser

305 310 315 320

Leu Asp Gly Lys Phe Ser Leu Phe His Thr Ala Val Arg Leu Ala Asn

325 330 335

Gly Phe Met His Ala Gly Thr Thr Asp Asn Ser Phe Ile Lys Ala Asn

340 345 350

Leu Pro Trp Leu Gly Lys Ala Gln Asn Trp Ala Lys Phe Thr Ala Thr

355 360 365

Ala Ser Leu Gly Val Ile His Lys Gly Asn Leu Leu Glu Gly Lys Lys

370 375 380

Val Met Ala Pro Tyr Leu Pro Gly Ser Arg Ala Ser Ser Arg Phe Ile

385 390 395 400

Lys Gly Gly Ser Leu Tyr Gly Leu Gly Leu Ile Tyr Ala Gly Phe Gly

405 410 415

Arg Asp Thr Thr Asp Tyr Leu Lys Asn Ile Ile Val Glu Asn Ser Gly

420 425 430

Thr Ser Gly Asp Glu Asp Val Asp Val Leu Leu His Gly Ala Ser Leu

435 440 445

Gly Ile Gly Leu Ala Ala Met Gly Ser Ala Asn Ile Glu Val Tyr Glu

450 455 460

Ala Leu Lys Glu Val Leu Tyr Asn Asp Ser Ala Thr Ser Gly Glu Ala

465 470 475 480

Ala Ala Leu Gly Met Gly Leu Cys Met Leu Gly Thr Gly Lys Pro Glu

485 490 495

Ala Ile His Asp Met Phe Thr Tyr Ser Gln Glu Thr Gln His Gly Asn

500 505 510

Ile Thr Arg Gly Leu Ala Val Gly Leu Ala Leu Ile Asn Tyr Gly Arg

515 520 525

Gln Glu Leu Ala Asp Asp Leu Ile Thr Lys Met Leu Ala Ser Asp Glu

530 535 540

Ser Leu Leu Arg Tyr Gly Gly Ala Phe Thr Ile Ala Leu Ala Tyr Ala

545 550 555 560

Gly Thr Gly Asn Asn Ser Ala Val Lys Arg Leu Leu His Val Ala Val

565 570 575

Ser Asp Ser Asn Asp Asp Val Arg Arg Ala Ala Val Ile Ala Leu Gly

580 585 590

Phe Val Leu Leu Arg Asp Tyr Thr Thr Val Pro Arg Ile Val Gln Leu

595 600 605

Leu Ser Lys Ser His Asn Ala His Val Arg Cys Gly Thr Ala Phe Ala

610 615 620

Leu Gly Ile Ala Cys Ala Gly Lys Gly Leu Gln Ser Ala Ile Asp Val

625 630 635 640

Leu Asp Pro Leu Thr Lys Asp Pro Val Asp Phe Val Arg Gln Ala Ala

645 650 655

Met Ile Ala Leu Ser Met Ile Leu Ile Gln Gln Thr Glu Lys Leu Asn

660 665 670

Pro Gln Val Ala Asp Ile Asn Lys Asn Phe Leu Ser Val Ile Thr Asn

675 680 685

Lys His Gln Glu Gly Leu Ala Lys Phe Gly Ala Cys Val Ala Gln Gly

690 695 700

Ile Met Asn Ala Gly Gly Arg Asn Val Thr Ile Gln Leu Glu Asn Ala

705 710 715 720

Asp Thr Gly Thr Leu Asp Thr Lys Ser Val Val Gly Leu Val Met Phe

725 730 735

Ser Gln Phe Trp Tyr Trp Phe Pro Leu Ala His Phe Leu Ser Leu Ser

740 745 750

Phe Thr Pro Thr Thr Val Ile Gly Ile Arg Gly Ser Asp Gln Ala Ile

755 760 765

Pro Lys Phe Gln Met Asn Cys Tyr Ala Lys Glu Asp Ala Phe Ser Tyr

770 775 780

Pro Arg Met Tyr Glu Glu Ala Ser Gly Lys Glu Val Glu Lys Val Ala

785 790 795 800

Thr Ala Val Leu Ser Thr Thr Ala Arg Ala Lys Ala Arg Ala Lys Lys

805 810 815

Thr Lys Lys Glu Lys Gly Pro Asn Glu Glu Glu Lys Lys Lys Glu His

820 825 830

Glu Glu Lys Glu Lys Glu Arg Glu Thr Asn Lys Lys Gly Ile Lys Glu

835 840 845

Thr Lys Glu Asn Asp Glu Glu Phe Tyr Lys Asn Lys Tyr Ser Ser Lys

850 855 860

Pro Tyr Lys Val Asp Asn Met Thr Arg Ile Leu Pro Gln Gln Ser Arg

865 870 875 880

Tyr Ile Ser Phe Ile Lys Asp Asp Arg Phe Val Pro Val Arg Lys Phe

885 890 895

Lys Gly Asn Asn Gly Val Val Val Leu Arg Asp Arg Glu Pro Lys Glu

900 905 910

Pro Val Ala Leu Ile Glu Thr Val Arg Gln Met Lys Asp Val Asn Ala

915 920 925

Pro Leu Pro Thr Pro Phe Lys Val Asp Asp Asn Val Asp Phe Pro Ser

930 935 940

Ala

945

(2) information SEQ ID NO:17:

(i) sequence signature:

(A) length: 142AA

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:17:

Trp Xaa Ile Arg Ser Asp Glu Arg Val Leu Gln Tyr Gly Glu Gln Asn

1 5 10 15

Ile Arg Arg Ala Val Pro Leu Ala Leu Gly Leu Leu Cys Ile Ser Asn

20 25 30

Pro Lys Val Thr Val Met Asp Thr Leu Ser Arg Leu Ser His Asp Arg

35 40 45

Phe Arg Ser Cys Asn Gly Ser Asn Tyr Leu Pro Trp Ile Asp Arg Arg

50 55 60

Trp Asn Gln Gln Cys Lys Asp Ser Trp His Ala Lys Ser Leu Gln Leu

65 70 75 80

Leu Leu Gln Gly Cys Pro Xaa Phe Phe Ser Val Cys Ala Ser Leu Lys

85 90 95

Gly Phe Xaa His Met Gly Lys Gly Leu Leu Thr Leu Asn Pro Phe His

100 105 110

Ser Glu Arg Ala Xaa Phe Leu Xaa Xaa Asn Pro Asp Phe Pro Trp Val

115 120 125

Gly Xaa Asn Phe Leu Gln Xaa Xaa Xaa Phe Xaa Ile Glu Thr

130 135 140

(2) information SEQ ID NO:18:

(i) sequence signature:

(A) length: 97AA

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:18:

Met Gln Pro Arg Met Leu Thr Thr Leu Val Glu Asp Glu Met Lys Pro

1 5 10 15

Gly Ser Leu Lys Gln Leu Asn Val Ser Val Arg Val Gly Gln Pro Val

20 25 30

Asp Val Val Ala Gln Ala Gly Lys Pro Lys Thr Ile Thr Gly Phe Gln

35 40 45

Thr His Thr Thr Pro Val Leu Leu Ala His Gly Glu Arg Ala Glu Leu

50 55 60

Ala Asn Asp Glu Tyr Leu Ser Val Thr Pro His Leu Glu Gly Leu Val

65 70 75 80

Ile Leu Lys Lys Asn Pro Asp Tyr Gln Pro Val Val Val Ser Thr Lys

85 90 95

Lys

(2) information SEQ ID NO:19:

(i) sequence signature:

(A) length: 390bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:19:

ATCAGTGTCA CTACGGATAG TGATGACACT CACAGGAGGG CTGGGGGTAT CTGGAATGAT 60

GATTGGCTGA TGCTGGTCTT GGACAGGAAC TAGGGAATTA TAAGAAGATG TGGTACGAAG 120

AGGACTACTC CCANCCAGAG AATAAACTTG AGAAGGCAGG ACTTCCAGAG AGGATTTGGA 180

TGAAACTGGA GCAGACTGCT TATTCTACTT TGAAGGGAGG GAACTAGACT GTTGTTGTCT 240

GACAACATGG GCAACACCAA CATTCAGAGG CTGAGCAGTN GCCAAGGNCA CATGGTTGGT 300

CAGCAAAGAT GGCTGCTGCA TAATAGTGCT GTACTGGTCG NCATGAGAGT GGGCATTCCC 360

CAGTCAGCTA GCTGGTGGGC TGCTCCCCAT 390

(2) information SEQ ID NO:20:

(i) sequence signature:

(A) length: 385bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:20:

CCTCTCAGTT ATCTCTGTTG GAGTAGTCCT CTTCGTACCA CATCTTCTTA TAATTCCCTA 60

GTTCCTGTCC AAGACCAGCA TCAGCCAATC ATCATTCCAG ATACCCCCAG CCCTCCTGTG 120

AGTGTCATCA CTATCCGTAG TGACACTGAT GAAGAAGAGG ACAACAAATA CAAGCCCAAT 180

AGCTCGAGCC TGAAGGCGAG GTCTAATGTC ATCAGTTATG TCACTGTCAA TGATTCTCCA 240

GACTCTGACT CCTCCCTGAG CAGCCCACAT TCCACAGCCA CTCTGAGTGC TCTGCGGGGC 300

AACAGTGGGA CCCTTCTGGA GGGACCTGGC AGACCTGCAG CAGATGGCAT TGGCACCCGT 360

ACTATCATTG TACCTGAGCG GCCGC 385

(2) information SEQ ID NO:21:

(i) sequence signature:

(A) length: 444bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:21:

GGGAGCCTGT GCACCCCGAT GTCACCATGA AGCCACTGCC CTTCTATGAA GTCTATGGGG 60

AGCTCATCCG ACCCACCACC CTTGCGTCCA CCTCCAGCCA GAGGTTCGAG GAAGCCCACT 120

TCACCTTCGC GCTCACTCCC CAGCAGCTGC AGCAGATTCT CACGTCCAGG GAGGTTATGC 180

CAGGAGCCAA GTGTGATTAC ACCATACAAG TGCAGCTCAG ATTCTGTCTC TGTGAGACCA 240

GCTGCCCTCA GGAGGACTAT TTCCCCCCTA ACCTCTTTGT TAAGGTTAAT GGGAAACTCT 300

GCCCCCTGCC GGGTTACCTC CCTCCAACCC AAGAATGGAG CTGAGCCCAA GAGGCCCAGC 360

CGTCCGATCA ACATCACACC CTTGGCTCGA CTCTCAGCCA CTGTCCCCAA CACCATCGTA 420

GTTAATTGGG TCATCTTGAA GTTT 444

(2) information SEQ ID NO:22:

(i) sequence signature:

(A) length: 888bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:22:

TCCAACACCA TCGTAGATAA ATTGGTCATC TGAGTTTGGA CCGGAATTAC TCCTTGTCCG 60

TGTACCCTGG TGAGGCAATT GACTGCAGGG ACCCTTCTAC ACAAACTCAG AGCCAAGGGG 120

ATCCGGAATC CAGACCATTC CCGGGCACTG ATCAAGGAGA AACTGACTGC TGACCCCGAC 180

AGTGAAGTGG CTACTACAAG TCTCCGGGTG TCACTCATGT GCCCGCTAGG GAAGATGCGC 240

CTGACTGTCC CGTGTCGTGC CCTCACCTGT GCCCATCTGC AGAGTTTCGA TGCTGCCCTT 300

TATCTACAGA TGAATGAGAA GAAGCCGACA TGGACGTGTC CTGTGCGTGA CAAGAAGGCT 360

CCCTATGAGT CGCTGATTAT TGATGGTTTA TTCATGGAAA TTCTTAATTC CTGTTCGGAT 420

TGTGATGAGA TCCAGTTCAT GGAAGATGGA TCCTGGTGTC CGATGAAACC CAAGAAGGAG 480

GCATCAGAGG TTTGCCCCCC GCCAGGGTAT GGGCTGGATG GTCTCCAGTA CAGCGCAGTC 540

CAGGAGGGAA TTCAGCCAGA GAGTAAGAAG AGGGTCGAAG TCATTGACTT GACCATCGAA 600

AGCTCATCAG ATGAGGAGGA TTTGCCCCCC ACCAAGAAGC ACTGCCCTGT CACCTCAGCG 660

GTCATTCCAG CCCTTCCTGG AAGCAAAGGA GCCCTGACCT CTGGTCACCA GCCATCCTCG 720

GTGCTGCGGA GCCCTGCAAT GGGCACACTG GGCAGTGACT TCCTGTCTAG TCTCCCGCTA 780

CATGAGTACC CACCTGCCTT CCCACTGGGG GTTGACATCC AAGGTTTAGA TTTTATTTTC 840

TTTTCTTCAG ACTGAGAGTC AGAATTACGG GCCTTCAGTT ATCATTCG 888

(2) information SEQ ID NO:23:

(i) sequence signature:

(A) length: 392bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:23:

CCACTTCCTG GCCCACTGCC CCCAAACTGG GGACTCTCAC CGCAAGCTCC AACTCCAGCG 60

CCCCCTCCTG GTCGTGTCAG CAGCATTGTG GCTCCTGGGA GCTCCTTGAG GGAAGGGCAT 120

GGAGGACCCC TGCCTTCAGG TCCCTCTTTG ACTGGCTGTC GGTCAGACGT CATTTCCTTG 180

GACTGAGCTT TTTGGATTAT GAAATCAATC TCCATTGGCC CCAGCACTGA GCAGATCACG 240

TTGTGGGTTC CGAACCCCTG GCTGCTCTGA TCCCTCAGGG GTCATTGGCC AAAGGCCAGG 300

CCAGAGCTTC ATGGATACCT GCTTTTGGCC TTATCGCTGC CTAACAGGCC AGTACTCACA 360

GGGTTAACAT TTAACCTTTT TATGGTGGCC CG 392

(2) information SEQ ID NO:24:

(i) sequence signature:

(A) length: 425bp

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:24:

AATTCGGCAC GAGGTTGTGC TGTGGGGAAG GGAGAAGGAT TTGTAAACCC CGGAGCGAGG 60

TTCTGCTTAC CCGAGGCCGC TGCTGTGCGG AGACCCCCGG GTGAAGCCAC CGTCATCATG 120

TCTGACCAGG AGGCAAAACC TTCCAACTGA GGACTTGGGG GATAAGAAGG AAGGTGAATA 180

TATTAAACTC AAAGTCATTG GACAGGATAG CAGTGAGATT CACTTCAAAG TGAAAATGAC 240

AACACATCTC AAGAAACTCA AAGAATCATA CTGTCAAAGA CAGGGTGTTC CAATGAATTC 300

ACTCAGGTTT CTCTTTGAGG GTCAGAGAAT TGCTGATAAT CATACTCCAA AAGAACTGGG 360

AATGGAGAAG AAAGATTGTG ATTTGAAGTT TTATCAGGAA CAAACGGGGG GTCATTCAAC 420

AGCTT 425

(2) information SEQ ID NO:25:

(i) sequence signature:

(A) length: 455AA

(B) type: amino acid

(C) chain: strand

(D) topology linearity

(ii) molecule type: polypeptide

(xi) sequence description: SEQ ID NO:25:

mglstvpdll lplvllellv giypsgvigl vphlgdrekr dsvcpqgkyi hpqnnsicct 60

kchkgtylyn dcpgpgqdtd crecesgsft asenhlrhcl scskcrkemg qveissctvd 120

rdtvcgcrkn qyrhywsenl fqcfncslcl ngtvhlscqe kqntvctcha gfflrenecv 180

scsnckksle ctklclpqie nvkgtedsgt tvllplviff glcllsllfi glmyryqrwk 240

sklysivcgk stpekegele gtttkplapn psfsptpgft ptlgfspvps stftssstyt 300

pgdcpnfaap rrevappyqg adpilatala sdpipnplqk wedsahkpqs ldtddpatly 360

avvenvpplr wkefvrrlgl sdheidrlel qngrclreaq ysmlatwrrr tprreatlel 420

lgrvlrdmdl lgcledieea lcgpaalppa psllr 455

(2) information SEQ ID NO:26:

(i) sequence signature:

(A) length: 454AA

(B) type: amino acid

(c) chain: strand

(D) topology linearity

(ii) molecule type: polypeptide

(xi) sequence description: SEQ ID NO:26:

mglptvpgll lslvllallm gihpsgvtgl vpslgdrekr dslcpqgkyv hsknnsicct 60

kchkgtylvs dcpspgrdtv crecekgtft asqnylrqcl scktcrkems qveispcqad 120

kdtvcgcken qfqrylseth fqcvdcspcf ngtvtipcke tqntvcncha gfflresecv 180

pcshckknee cmklclpppl anvtnpqdsg tavllplvil lglcllsfif islmcryprw 240

rpevysiicr dpvpvkeeka gkpltpapsp afsptsgfnp tlgfstpgfs spvsstpisp 300

ifgpsnwhfm ppvsevvptq gadpllyesl csvpaptsvq kwedsahpqr pdnadlaily 360

avvdgvppar wkefmrfmgl seheierlem qngrclreaq ysmleawrrr tprhedtlev 420

vglvlskmnl agclenilea lrnpapsstt rlpr 454

Claims (21)

1. isolated DNA molecule, a kind of polypeptide that can be incorporated into the intracellular region of p55 TNF acceptor of encoding,

This polypeptide is

(i) polypeptide of the residue 206-426 of SEQ ID NO:25,

The (ii) polypeptide of the residue 334-454 of SEQ ID NO:26, perhaps

The (iii) polypeptide of SEQ ID NO:14.

2. carrier, foreign DNA wherein is the described dna molecular of claim 1.

3. eucaryon that has transformed or the protokaryon chief cell that contracts contains the described carrier of claim 2.

4. a production can be incorporated into the method for polypeptide of the intracellular region of p55 TNF acceptor, this method comprises: cultivate the described host cell that has transformed of claim 3 be fit to the condition that expression product expresses from described cell under, in case of necessity, carry out the posttranslational modification of described expression product so that obtain said polypeptide, and in the substratum of the said cell that has transformed or in the cell extract of the said cell that has transformed, extract said polypeptide.

5. isolated DNA molecule, its coding are selected from the protein 55.1 of this paper definition, i.e. the residue 328-426 of SEQ ID NO:25,55.3, i.e. the residue 277-426 and 55.1 of SEQ ID NO:25, the i.e. protein of SEQ ID NO:14.

6. the described isolated DNA molecule of claim 5, its coded protein 55.1, this protein have the aminoacid sequence corresponding to the amino-acid residue 328-426 of SEQ ID NO:25.

7. the described isolated DNA molecule of claim 5, its coded protein 55.3, this protein have the aminoacid sequence corresponding to the amino-acid residue 277-residue 426 of SEQ ID NO:25.

8. the described isolated DNA molecule of claim 5, its coded protein 55.11, this protein has the aminoacid sequence of SEQ ID NO:14.

9. carrier, foreign DNA wherein is the described dna molecular of claim 5.

10. eucaryon that has transformed or the protokaryon chief cell that contracts contains the described carrier of claim 9.

11. a production can be incorporated into the method for polypeptide of the intracellular region of p55 TNF acceptor, this method comprises: cultivate the described host cell that has transformed of claim 10 be fit to the condition that expression product expresses from described cell under, in case of necessity, carry out the posttranslational modification of described expression product so that obtain said polypeptide, and in the substratum of the said cell that has transformed or in the cell extract of the said cell that has transformed, extract said polypeptide.

12. the described isolated DNA molecule of claim 1 is the sequence of SEQ ID NO:9.

13. a peptide species, by claim 1, any one described dna molecule encode among the 5-7 and 12, described polypeptide can be incorporated into the intracellular region of p55 TNF acceptor.

14. antibody has specificity for the described polypeptide of claim 13.

15. be used to handle the purposes of the medicine of cell with the described Antibody Preparation of claim 14, said processing is in said cell with described medicinal application, wherein when the IC-of said cell conjugated protein is exposed to born of the same parents' outside surface, then described medicine is just prepared as born of the same parents and is used outward, and when said IC-conjugated protein be in the born of the same parents time, then described medicine is just prepared as using in the born of the same parents.

16. one or more polypeptide according to claim 13 preparations are used to handle the purposes of the medicine of cell, described polypeptide can be incorporated into the intracellular region of TNF-R and the activity of regulating TNF-R, and it is by described medicine is introduced in the said cell to be applicable to the form of introducing in its born of the same parents that wherein said cell is handled.

17. use the described isolated DNA molecule of claim 1 to be prepared into the purposes of the medicine that is used to handle cell with the suitable carrier format that is loaded with this sequence, it is to import in the said cell by the mode that said medicine is expressed in said cell with said sequence that wherein said cell is handled.

18. one or more polypeptide according to claim 13 are used for preparing the purposes that is used for inducing cell or organizes the medicine of the relevant effect of TNF-, wherein said inducing is by said medicine is introduced in the said cell with the form that is suitable for introducing in its born of the same parents.

19. use the described isolated DNA molecule of claim 1 to be prepared into the purposes of the medicine that is used for the relevant effect of inducing cell or tissue TNF-with the suitable carrier format that is loaded with this sequence, wherein said induce be by with said medicine so that the mode that said sequence is expressed in described cell import in the said cell.

20. a medicinal compositions that is used to regulate TNF-R part pair cell effect comprises the described peptide species of claim 13 as activeconstituents.

21. a medicinal compositions that is used to regulate TNF-R part pair cell effect, the recombinant animal virus vector that comprises the described peptide species of coding claim 13 is as activeconstituents.

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