CN1235910C - Target-specific cytotoxin chimeric with short-peptide harmone - Google Patents

Abstract

The present invention relates to chimeric toxin which comprises gonadotropin-releasing hormone and releasing hormone for promoting melanin, uses short peptide hormone as a guiding agent, and uses recombination pseudomonas exotoxin as a cytotoxicity agent, a preparation method thereof, and the application for remedial gland reactivity or non-reactivity tumors. The present invention is characterized in that the guiding part of the chimeric toxin is connected in series by two molecules.

Description

The target-specific cytotoxin agent chimeric with the small peptide hormone

The present invention generally relates to the fused protein with target cell SC.More particularly, the present invention relates to the structure of the reorganization ETA (rPE) that merges with the small peptide parahormone, and said reconstitution cell toxicity fusion rotein is suppressing tumour and melanomatous generation and the developmental application that steroid hormone stimulates as antineoplastic agent.

Traditional tumor therapeuticing method is to use the intravital tumour cell of chemicals direct killing.Yet most of antitumor drugs also kill and wound normal cell in killing tumor cells.In order to improve the selectivity of antitumor drug to tumour cell, since the later stage seventies 20th century, people just attempt with some antitumor drug or lacked originally target optionally toxin (toxin that comprises bacterium or plant origin) chemical coupling to suitable guide molecule or claim on the identification molecule, with the antineoplastic agent with target-specific of preparation hybridization.Along with the development of Protocols in Molecular Biology, and then set up the method for preparing such fused protein with the DNA recombinant technology.

In the generation and growth course of tumour, many tumour cells are overexpression growth factor receptor protein matter in its surface all, for example EGF-R ELISA (EGF-R) and transforming growth factor-alpha acceptor (TGF α-R).Therefore, some cytotoxic agent (as Pseudomonas exotoxin (PE), diphtheria toxin, Toxins,exo-, cholera, staphylococcus intracellular toxin and Ricin etc.) can be connected on EGF, the TGF-α or bFGF molecule as directed agents, have the hybrid molecule of tumour cell guide function and cytotoxic activity with generation concurrently.These hybrid molecules are guided whole molecule target cell into and are killed and wounded target cell by its toxin moiety target tumour cell guidance capability by it.

More and the more deep cytotoxic agent of research is ETA (PE) (referring to United States Patent (USP) 4,545,985) at present.The single chain polypeptide that PE is made up of 613 amino acid.The X-ray crystallography research of PE molecule and mutation analysis are shown, the PE molecule comprise three with produce the relevant structural region of cytotoxicity: responsible and sensitive cells bonded N-terminal cell receptor land (I district); Be responsible for middle the transposition district (II district) of lps molecule transposition in cytosol: and the responsible inactivating proteins C-terminal enzymatic activity district (III district) that also finally causes necrocytosis.Wherein the I district comprises mediated cell bonded Ia district (amino acid/11-252) and at present not the Ib district of clear and definite its function (amino acid 365-399) as yet.Can use biological chemistry or recombinant DNA technology to modify the PE molecule, so that the PE fragment of the various modifications of one or more aminoacid deletion or replacement to be arranged in the preparation PE molecule, for example, generally will leave out Ia district in the PE molecule, only contain enzymatic and transposition district, the PE-A protein that molecular weight is about 40KDa is called PE40.After having now found that the Ia and most of Ib district (amino acid 365-380) of deletion PE, lps molecule still keeps its SC, but has reduced non-specific toxicity (for example referring to Hwang et al., Cell 48:129-136,1987; United States Patent (USP) 4,892,827 and European patent 0261671).

PE described by many prior art documents and various somatomedin, antibody, hormone or CD4 merge, optionally lead and kill and wound the have different epicyte proteins hybridization method of protein of target cell of (acceptor or antigen) to produce (referring to Pastan and Fitz.G., Science 254:1173-1177,1991).For example, people such as Chaudhary (PNAS USA 84:4538-4542,1987) have described the crossbred fused protein that forms between PE40 and TGF α (TGF α-PE40).The clinical practice of TGF α-PE40 depends on its combination and kills and wounds the ability of the cell with EGF-R ELISA.People such as Murphy (PNAS USA 83:8258-8262,1986) have described gene constructed, the expression and the melanoma selecting cell toxicity thereof of diphtheria toxin-α melanotropin (MSH) fused protein.United States Patent (USP) 5,428,143 disclose the hybridization protein of the cell that is used for selective killing HIV infection and have encoded that this hybridizes the structure of proteinic mosaic gene.Wherein said hybridization protein is made up of with the cytotoxic protein matter (PE40) that can kill the HIV infection the people CD4 that contains the HIV binding site.

As the prior art more relevant with the present invention, international monopoly WO93/15751 discloses gonadotropin-releasing hormone (GnRH) peptide directly has been coupled to the chimeric protein molecule of making on the Pseudomonas exotoxin molecule.It is said that such chimeric molecule that comes into operation can cause carrying in the pituitary gland destruction of the cell of GnRH acceptor, and with the reduction of sex hormone secretion, thereby the propagation that is expected to use it for the animal sterilization and suppresses the steroid hormone related neoplasms.In addition, International Patent Application WO 97/15325 has been described a kind of immunogenic carrier system that contains with the chemically combined GnRH of false unit cell extracellular toxin, can bring out the anti-GnRH antibody of generation high density with it in animal body as vaccine, thereby can be used for controlling behavior and the reactive tumour of treatment steroid hormone of becoming pregnant, reduce the reproductive hormone driving.

An object of the present invention is to provide a kind of chimeric toxin that forms by small peptide parahormone and the fusion of reorganization Pseudomonas exotoxin, be characterised in that wherein said small peptide parahormone part is as directed agents, can with its surface on have the reactive or non-reacted tumour cell specific combination of periphery gonadal hormone of corresponding hormone receptor, and after internalization was in these tumour cells, said Pseudomonas exotoxin part can be killed and wounded these tumour target cells effectively as cytotoxic agent.

This purpose preferred embodiment according to the present invention, wherein said small peptide class trop(h)ic hormone are that gonadotropin-releasing hormone (GnRH) and melanochrome stimulate hormone (MSH).

This purpose preferred embodiment according to the present invention, the false unit cell extracellular toxin of wherein said reorganization is PE40.

This purpose preferred embodiment according to the present invention, wherein said GnRH and MSH molecule are to be connected to the position that is equivalent to the Ia district in the PE molecule with two molecule series connection forms.

Another object of the present invention provides above-mentioned GnRH-PE40 or the MSH-PE40 chimeric toxin that contains the cytotoxicity significant quantity, and the pharmaceutical composition of one or more pharmaceutically acceptable carriers or vehicle.

A further object of the present invention relates to the application of aforementioned pharmaceutical compositions in therapeutic glandular hormone reactivity or non-reacted tumour.

Fig. 1 a demonstration is used to express the construction of recombinant plasmid of GnRH-PE40.

Fig. 1 b demonstration is used to express the construction of recombinant plasmid of 2 * GnRH-PE40.

The influence of the protein synthesis of the GnRH-PE40 of Fig. 2 display part purifying reactive or non-reacted tissue-derived tumour cell and some healthy tissues primary cell culture to some sexual hormoue.■: mammary cancer MCF-7 cell; ●: liver cancer HepG-2 cell; ▲: colorectal carcinoma Caco2 cell; ※: cervical cancer Hela cell; Zero: the calf testis cell; △: mouse chest cell; : Turnover of Mouse Peritoneal Macrophages; ⊙ human cervical cancer 1 cell.

The GnRH-PE40 (●) of Fig. 3 display part purifying and 2 * GnRH-PE40 (zero) are right ¹²⁵I-GnRH and Hela cytolemma bonded competition binding ability.

Fig. 4 demonstration is used to express the construction of recombinant plasmid of 2 * MSH-PE40.

The MSH-PE40 of Fig. 5 display part purifying or 2 * MSH-PE40 are respectively to the inhibition of the protein synthesis of melanin tumour b16 cell (● or zero) and former normal mouse melanocyte of being commissioned to train foster (▲ or △).Numerical value is with in the presence of different concns MSH-PE40 or 2 * MSH-PE40 (X-coordinate), and the specific chimeric toxin protein suppresses percentage (ordinate zou) expression that the malignant cell protein synthesis accounts for optimum cellular control unit.

Term used herein " directed agents ", referring to can be only and the lip-deep acceptor of target cell to be killed and wounded Or the molecule of antigen-specific combination or part. " directed agents " is also referred to as " identification molecule " or " part knot sometimes Mixture ". The example of such identification molecule is antibody or its fragment that can identify target cell, can with target cell on Growth factor, lymphokine, cell factor, antigen and the hormone etc. of molecular specific combination. According to this Bright preferred embodiment, said directed agents is hormone, better is little molecule small peptide parahormone, and Well gonadotropin-releasing hormone (GnRH) and short melanocyte (MSH).

Term as used herein " GnRH " refer to can with its surface on have the cell of steroid hormone receptor In conjunction with, and when coming into operation in mammalian hosts with high dose, can cause natural that anti-GnRH antibody produces GnRH and analog thereof or derivative.

Natural GnRH is also referred to as luteinizing hormone releasing hormone (LHRH or LRH), and it is to have the lower decapeptide molecule that shows amino acid sequence:

pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2

Used amino acid symbol is the general trigram dummy suffix notation in this area in the formula, and pGlu wherein Represent pyroglutamic acid. Comprise Lys applicable to GnRH analog of the present invention or derivative⁶-GnRH、Cys ⁶-Terminal GnRH, the GnRH of brachymemma that extend of two GnRH, C of the end modified nonapeptide of GnRH, N, series connection The GnRH that the terminal pGlu of fragment and C is replaced by Gln. Result of study shows, in the molecule the 3rd～6 Amino acid is that to keep the GnRH activity necessary, and Ser⁴And Tyr⁵Then in receptors bind, play key Effect. Show that on evidence gonad cell particularly GC surface has the GnRH acceptor. Proved little The expression of receptor on mouse testicular cell (Legdig cell) surface is relevant with the hormone in vivo level (Harwood, J.P.et al., Endocrinology 107:407-413,1980). In addition, also has human hair Have on the existing rat endometrium the very high LHRH acceptor of affinity (Cao Yongqing etc., Chinese science B collects 1: 32-37,1984). Although at present the outer mechanism of action of the hypophysis of GnRH is still imperfectly understood, can affirm Be the knot of specific receptor on the steroid hormone linked groups such as GnRH and sexual gland or endometrium or the cell Closing is an important step of the outer effect of GnRH performance hypophysis. And shown, with target cell membrane and cell The LHRH receptors bind that exists on the endocrine granules basically be do not have species specificity (Zhang Chongli etc., in State's applied physiology magazine 5 (1): 87-93,1988). We are to the GnRH-PE40 by the inventive method preparation The analysis of biological activity of fused protein proves that clearly GnRH-PE40 has and comprises the Hela cell Tumour cell in interior multiple sexual organ source has killing activity, and to the normal cell in Various Tissues source Then there is not this specific killing activity (referring to embodiment 2).

Term used herein " MSH " refer to can with its surface on have the cell-specific of MSH acceptor In conjunction with natural MSH and analog or derivative. Natural MSH has lower 13 of the amino acid sequence that shows Peptide molecule:

Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2

Structure-functional study to MSH finds that continuous four the amino acid His-Phe-Arg-Trp in the molecule are absolutely necessary for keeping its biologic activity.Said MSH analogue or derivative are any one or a plurality of amino acid whose disappearance, displacement or the adding forms that does not change the MSH in this active centre basically.In fused protein of the present invention, MSH can combine with the cell-specific that overexpression MSH acceptor is gone up on its surface.Show that on evidence though melanocyte and melanoma cell surface all have the MSH acceptor, the density of melanoma cell surface MSH acceptor increases 40～200% than normal melanocyte.(referring to Donectien, P.D.et al., Arch.Dermatol.Res.284 (7): 424-426,1992; Ghamen, G.E.et al., Int J.Cancer 45 (2): 248-255,1988).

Term " ID used herein ₅₀" be meant and suppress the concentration that the target cell protein synthesis reaches 50% required chimeric toxin of control group.Use standard among the present invention ³The H leucine mixes method and detects " ID ₅₀" value.

The invention further relates to the method with recombinant DNA technology production and small-molecular peptides parahormone link coupled target-specific, cytotoxicity reorganization PE fused protein, this method comprises: (1) provides and carries the PE40 expression carrier; (2) nucleotide sequence of the coding small molecule peptide hormone that is connected in series of synthetic two molecules, and it is cloned in the expression vector of linearizing step (1); (3) expression vector with step (2) transforms appropriate host cell; (4) be suitable for expressing cultivate under the condition of the said fused protein of forming by small-molecular peptides parahormone and Pseudomonas exotoxin said by transformed host cells; (5) from cell culture, reclaim and the said fused protein of purifying.

The PE molecule that is used to make up fused protein of the present invention can be the natural PE molecule that has lacked the Ia district, but also can and better be the PE40 molecule through modifying.For example, can be 1～4 halfcystine (Cys wherein through the PE40 molecule of modifying like this ²⁶⁵, Cys ²⁸⁷, Cys ³⁷²And Cys ³⁷⁹) PE40 deleted or that replaced by other amino acid such as L-Ala is (referring to European patent 0383599 and United States Patent (USP) 5,621,078), has amino acid 286～364 and 381～613 (aminoterminal 1～28 amino acids in the disappearance II district), and the reorganization PE40 molecule that 287 halfcystine is replaced by Serine is (referring to United States Patent (USP) 5,821,238), and can between the amino acid 600-613 of PE molecule C-terminal, insert first identification molecule respectively, and connect second identification at the N-terminal of PE molecule and divide the reorganization Pseudomonas exotoxin of giving (referring to United States Patent (USP) 5,705,163).In addition, can use site-specific mutagenesis technology well known to those skilled in the art or other technologies further to modify the PE molecule, to change it according to the application-specific purpose.Certainly, the prerequisite of these modifications is that the functional performance that is provided by the PE molecule is not provided in fact.

Owing to discern the molecular weight all less relatively (10～13 peptide) of the small-molecular peptides parahormone of molecule, can use general dna synthesizer on the 1000A solid phase carrier, to synthesize the nucleotide sequence of single or these hormones of the placed in-line coding of two molecules or its varient as directed agents in the antitumor chimeric toxin of the present invention or title.For the ease of being connected, can introducing suitable endonuclease (as NdeI) restriction enzyme site at 5 ' and/or 3 ' end of synthetic hormone gene sequence, and cause the sticky end that is suitable for connecting with free or the PE encoding sequence that is carried on the specific recombinant vectors.Can be according to recombinant technology well known by persons skilled in the art (for example referring to Sambrook et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harborlaboratory, 1989), the gene of these synthetic small peptide parahormones of clones coding (or with its cDNA or genomic dna form), and be connected in the carrier of the linearizing PE40 of carrying as pVC8-PE40 (Chandhary etal. at the position that is equivalent to deleted Ia district or the suitable restriction enzyme site place in the III district, PNAS USA 84:4538-454,1987) or among the PET-17b-PE40 (Novagen Co.).The recombinant vectors system that so obtains can be transformed into then in prokaryotic cell prokaryocyte such as the Bacillus coli cells to produce GnRH-PE40 or MSH-PE40 fused protein.In the DNA reorganization operation, generally use the required gene fragment of the pcr amplification technology amplification of standard and cause suitable restriction enzyme site.Use the Restriction Enzyme cutting method to identify the exactness of sequence closure and possible sudden change, at last with people's such as Sanger dideoxy chain termination carry out sequencing with further confirmation it.

Certainly, also can with known chemical process two parts be combined by suitable chemical coupling group.For example can use SPDP (N-succinimide-3-2-pyridine dithio)-third sulphur ester, glutaraldehyde, maleimide aminobenzoyl-N-succinic diamide ester, imino-sulfane etc. to carry the isodigeranyl functional cross-link agent of functional groups as " joint " that connect GnRH or MSH and PE40 molecule.

What should particularly point out here is, because with respect to cytotoxicity part PE40, the molecular weight of targeting part GnRH or MSH is much smaller, so form new secondary space conformation probably by the PE40 part in the fused protein of both generations, cause PE40 part to the curling of GnRH or MSH, influence the degree of the receptors bind of fused protein.Under the cysteine residues in the PE molecule not being replaced/deletes the situation of modifying, this possibility is bigger especially.In addition, as previously mentioned, because GnRH and MSH are mammalian pituitary excretory one-level hormone, and corresponding Rd is lower on the tumor cell surface of relevant peripheral tissues, think increase these guide molecules and receptor in target cell combine probability and combination stability, best with placed in-line two the hormone molecules of head-tail as the targeting part in the chimeric toxin.

Small-molecular peptides parahormone as ligand binding agent can be inserted in the PE40 I district, preferably be equivalent on the position in Ia district of natural PE molecule (referring to Heimbrook et al., PNAS USA 87:4697-4701,1990).But also can be inserted on the position of PE molecule C-terminal amino acid 605～613.

Can and comprise at intestinal bacteria or other prokaryotic cell prokaryocytes, yeast and express the fused protein that comprises PE molecule and small-molecular peptides parahormone in the various higher eucaryotic cells of COS, CHO.The recombinant protein plasmagene will operably be connected on the suitable expression control sequenc, as be connected to the T that is suitable for using in intestinal bacteria ₇, on trp or λ promotor, ribosome bind site and the transcription termination signal.Can use known method for transformation, as the electroporation that is suitable for the calcium chloride facture of prokaryotic cell prokaryocyte or is suitable for mammalian cell with recombinant plasmid transformed of the present invention in the host cell of selecting.Can select the positive cell that transformed based on the antibiotics resistance that contained antibiotics resistance gene on the plasmid is given.In case expressed required fused protein, can separate and this fused protein of purifying according to methods known in the art.For example, can be from fermenting culture centrifugal collection somatic cells and with N,O-Diacetylmuramidase or Guanidinium hydrochloride cracking it, ultracentrifugation and dialysis repeatedly in low phosphorus hydrochlorate (about 20mM) solution then.In mixture, add urea then and forgive intravital protein with dissolved cell.After recentrifuge is removed insolubles, successively through ion exchange chromatography (IEC) and required GnRH-PE40 or the MSH-PE40 reorganization hybridization protein of volume-exclusion chromatography (SEC) purifying.In addition, also can use saltout, methods such as affinity chromatography and the gel electrophoresis of preparation property carry out purifying (referring to R.Scopes, Protein Purificafion, Springer-Verlag, N, Y., 1982).

In general, use polyacrylamide gel with SDS-PAGE electrophoretic method (Laemmli, Nature227:680-689,1970) analyze each column chromatography wash-out part, and use anti-PE antiserum(antisera) of multi-clone rabbit and Vectastain test kit (Vector Labs, Buvlingame Calif.) monitors it with immunoblotting.The recombinant fusion protein purified product is carried out protein synthesis inhibition test (Prior et al., Cell 64:1017-1029,1991), to detect the cytotoxicity (ID of fused protein ₅₀).For the GnRH-PE40 fused protein, then mainly detect its external killing activity, and use the different target cells that comprise tumour or normal cell ³The leucine of the H mark method of mixing detects fusion rotein to the isocellular protein synthesis restraining effect of HeLa, Swiss 3T3.In addition, can use the wheat germ extract that is rich in elongation factor 2 to detect the ADP-ribosylation activity of protein example by Colliev and the described method of Kondel (J.Biol.Chem.246:1496-1503,1971).

Can be with GnRH-PE40 of the present invention or MSH-PE40 fused protein as the primary activity composition, and add one or more pharmaceutically acceptable carrier or vehicle, make the pharmaceutical composition that is suitable for clinical application.Said carrier or vehicle comprise but be not only limited to phosphate buffered saline (PBS), physiological saline, etc. ooze glucose solution, dextran, dextran etc.According to the difference of the disease of being treated, can add in pharmaceutical composition of the present invention that one or more and fused protein of the present invention have that auxiliary or synergistic other are natural, the active compound of synthetic or reorganization.In addition, can in pharmaceutical composition of the present invention, add and be selected from human serum albumin, low molecular weight peptide, glycine or Methionin and metallic cation (as Zn ²⁺, Mn ²⁺, Mg ²⁺And Ca ²⁺) protein protectant, and the stablizer that is selected from polyoxyethylene glycol, carboxymethyl cellulose, polyglycine, gsh.

Can be by the outer approach of conventional route of administration, the particularly gi tract pharmaceutical composition of the present invention that comes into operation, for example by administration in intravenously, intraperitoneal, intramuscular, intracutaneous, the subcutaneous or mucous membrane.The effective dosage ranges of pharmaceutical composition of the present invention can be from several nanogram(ng)s to tens milligram/kg body weight/sky, but at the concrete dosage of each given patient will be according to age of the character of disease to be treated or pathological state and severity, patient, body weight, factors such as the response capacity of medicine and administering mode are decided.

What should particularly point out is, although the more deep mechanism of action is still indeterminate, our laboratory has proved promptly as far back as 1994 that GnRH-PE40 protein of the present invention all has tangible specific combination activity and cytotoxicity to comprising tumor cell lines such as colorectal carcinoma HT-29 cell, ovarian cancer OVCAR3 cell, adenocarcinoma of cervix HeLa cell and liver cancer HepG-2 cell.

Therefore as seen, GnRH-PE40 protein of the present invention not only has obvious external lethal effect to the reactive tumour of the so-called steroid hormone that is subjected to hypothalamic pituitary gonadal axis control, and the adenocarcinoma cell of certain non-sexual gland organ origin such as S-180 sarcoma cell and myeloma cell are also had similar identical specificity in conjunction with, internalization and cytotoxic activity.Simultaneously, prove clearly also that MSH-PE40 fusion rotein confrontation melanoma cell of the present invention has external significantly and the interior killing activity of body.

Below further illustrate the present invention by embodiment, but it will be appreciated by those of skill in the art that these embodiment do not constitute the await the reply restriction of claim scope to the present invention.

The preparation of embodiment 1:GnRH-PE40 fused protein

A. the structure of recombinant expression plasmid and evaluation

A. the amplification of initial plasmid DNA and linearizing: be in T with carrying ₇Plasmid pVC (the Chaudhary et al. of amino acid/11～3 of the coding PE molecule under promotor and SD sequence and the terminator control and the nucleotide sequence of amino acid 253～613, PNAS USA 84:4538-4542,1987) transformed competence colibacillus intestinal bacteria HB101 cell, and will be incubated in the LB substratum that contains penbritin (50 μ g/ml), by cell transformed with amplified plasmid dna.After cultivation is finished, smudge cells, centrifugal collection plasmid also extracts plasmid DNA with phenol/chloroform.With NdeI plasmid DNA is cut into linearity, and handles so that its terminal dephosphorylation with calf intestinal alkaline phosphatase.

B. contain the preparation of the oligonucleotide of GnRH gene:, use the A and the B chain oligonucleotide sequence (36bp) of the synthetic coding GnRH shown in SEQ ID NO.1 of dna synthesizer (Applied Biosystems Inc.AB1394 type) according to the aminoacid sequence of people GnRH.At T ₄Polynucleotide kinase (Promega) exists makes it terminal phosphateization down respectively.Mixing A, B chain and annealing back preserves standby down in-20 ℃.

C. the structure of recombinant plasmid pVC8-GnRH-PE40 and screening: at T ₄Dna ligase exists down, and according to about 1: the molar ratio of 2-4 links together the double-stranded GnRH nucleotide coding sequence of linearizing and terminal dephosphorylized pVC8-PE40 and the processing of process terminal phosphate.Because 3 ' end leaves the AT protuberance when NdeI digestion pVC8-PE40, and the synthetic GnRH of institute encoding sequence A, B chain have the TA base at 5 ' end respectively, thereby are easy to realize under the situation of only using single enzyme to cut the GnRH gene order of modifying and correct connection of the pVC8 plasmid vector (pVC8-PE40) that carries PE40 after the annealing.Figure Ia and Ib have shown the structure of recombinant plasmid pVC8-GnRH-PE40 and pVC8-2 * GnRH-PE40 respectively.

Use resulting ligation mixture transformed into escherichia coli HB101 then.The inoculation that will be transformed is selected the amicillin resistance bacterium colony in containing on penbritin (50 μ g/ml) the LB agar plate after the cultivation, and on nitrocellulose filter with radiolabeled probe (γ- ³²P-GnRH) (A chain) in situ hybridization (65 ℃ of prehybridizations 2 hours; Hybridized 12 hours for 55 ℃).From in situ hybridization male transformant, extract in a small amount plasmid DNA, once more with above-mentioned probe with identical method carry out dot hybridization (usefulness synthetic GnRH gene B chain is as positive control, and with empty pVC8 carrier sequence as negative control.

Picked at random hybridization male plasmid is cut identification method and sepharose (2%) electrophoretic method is identified with enzyme.Select correctly to be connected with the recombinant plasmid of single GnRH encoding sequence and it named to be pVC8-GnRH-PE40.And the recombinant plasmid that head-tail is connected in series with two GnRH encoding sequences named be pVC8-2 * GnRH-PE40.

In general, as in ligation, increasing the molar ratio of GnRH encoding sequence, should wherein be connected in series with the recombinant plasmid of a plurality of GnRH encoding sequences in theory to pVC8-PE40.After the SDS-PAGE electrophoretic analysis, only select with forward be connected with that single (GnRH) or two are connected in series (recombinant plasmid of 2 * GnRH) nucleotide coding sequences carries out dna sequence analysis.The nucleotide sequence that has shown measured GnRH-PE40 and 2 * GnRH-PE40 hybrid gene among SEQ ID NO:2 and the SEQ ID NO:3 respectively.SEQ ID NO:4 then shows the aminoacid sequence of the fused protein of being inferred by 2 * GnRH-PE40.

The expression of B.GnRH-PE40 fused protein and the purifying of product:

To (contain T with carrying the recombinant plasmid pVC8-PE40-GnRH or the pVC8-2 * GnRH-PE40 of GnRH-PE40 hybrid gene and not containing the e. coli bl21 (DE3) that the initial plasmid pVC8-PE40 (in contrast) of GnRH gene order transforms respectively ₇Rna polymerase gene) cultivate in the LB substratum that contains penbritin (50 μ g/ml) (Studier, F.W.andMoffatt, B.A., J.Mol, Biol, 189:113-130,1986).Work as A ₆₀₀Reach at about 0.8～1.2 o'clock and add 1mM isopropylthio-(IPTG) (final concentration 1mM/L), 37 ℃ are continued to cultivate 3 hours, to induce the expression of purpose product.Centrifugal collecting cell and will be suspended in lysis buffer (50mMTris-HCl behind the cell precipitation thing multigelation then, pH7.5,5mM EDTA and 0.2mg/ml N,O-Diacetylmuramidase) in, the centrifugal supernatant (soluble fractions) of removing, and with resuspended thing ultrasonic (3 * 20 seconds) processing, with smudge cells.Recentrifuge (25,000g, 20 minutes) collecting precipitation thing (insoluble part) back adds ice-cold sex change damping fluid (100mM Tris-HCl, pH7.5,1mM EDTA 15mM 3-mercaptoethanol, 7M Guanidinium hydrochloride).Stir recentrifuge after 2 hours under the room temperature, and the gained protein soln is diluted to refolding damping fluid (the 50mM Tris-HCl of 200 times of volumes, pH7.8,1mMEDTA, 0.1M NaCl and 10mM 3-mercaptoethanol) in, placed 24 hours for 4 ℃, with (PBS) (pH7.4) the thoroughly dialysis (changing liquid 4 times) in the 10mM phosphate buffered saline (PBS) of folding again protein soln, centrifugal then (20,000g, 30 minutes), supernatant is the GnRH-PE40 crude extract.

The clarifying supernatant liquor that as above obtains is added on the DEAE-Sepharose Fast Flow post of crossing with above-mentioned refolding damping fluid balance (Pharmacia), with TE damping fluid (the 20mM Tris-HCl that contains 0.05～0.5M NaCl, pH8.0,1mM EDTA) stepwise elution, and collect protein active peak part.After the Amicon ultra-fine filter that the YM-30 film is equipped with in use concentrates, make the 2.6 * 100cm Sephacryl S-200 post (Pharmacia) of enriched material by crossing with above-mentioned refolding damping fluid balance, and with the TE damping fluid that contains 0.15M NaCl (20mM TrisHCl, pH8.0,1mM EDTA) wash-out.Collect active peak part and cross 2.0 * 20cm Q-Sepharose post (Pharmacia) once more, with collecting protein peak value (A behind the NaCl gradient elution ₂₈₀) part and thoroughly dialysis in 20mM PBS, the dialysis back stores for future use under-20 ℃.Lipidated protein＞96% of purifying like this.

According to the method for manufacturer recommendation, use Vectastain ABC test kit (Vector LaboratoriesInc.) and the anti-PE antiserum(antisera) of multi-clone rabbit that the GnRH-PE40 protein of purifying is identified.

The target-specific of embodiment 2:GnRH-PE40 fused protein and biologic activity analysis

A. protein synthesis inhibition test: detect the verify restraining effect of protein synthesis of the tumour selected or normal cell system of GnRH-PE40 fusion rotein of the present invention according to the described method of people such as Prior (Cell 64:1017-1023,1991).

The target cell of using in the experiment comprises the normal cell culture of former generation such as calf testis cell, mouse chest cell, mammary gland cell and peritoneal macrophage of knurls such as colorectal carcinoma HT-29 cell, ovarian cancer OVCAR cell, adenocarcinoma of cervix Hela cell and liver cancer HepG-2 cell system and pre-preparation.

With every hole 2 * 10 ⁴The density of individual cell with various tested cell inoculations in 96 hole microtest plates, at 5%CO ₂The following 37 ℃ of insulations of environment add the GnRH-PE40 of different concns and the equivalent PE40 of sample in contrast in each hole after 24 hours, final volume is 200 μ l/ holes.All all are diluted in 0.2% bovine serum albumin-phosphate buffered saline (PBS) (BSA-PBS) by test agent.37 ℃ of insulations add after 24 hours [ ³H] (Amershanm, Corp.) (5 μ Ci/ holes are diluted among the BSA-PBS) continues insulation 12 hours for the leucine of mark.-70 ℃ freezing and melt fast after with cell harvesting to glass fiber filter, detect the radioactivity be incorporated in the cell protein with the β counter then.Do not contact toxin protein and only add the results are shown among Fig. 2 that the percentage incorporation of the control cells of BSA-PBS represents to account for.

From result shown in Figure 2 as can be seen, the partially purified GnRH-PE40 fusion rotein of the present invention (the peak value part behind the DEAE-Sepharose post) can kill the tumour cell (ID in the reactive organ mammary gland of steroid hormone, uterine cervix source in dosage dependence mode ₅₀Value is about 15-40 μ g/ml).And find that toxin has also showed significant cytotoxicity (ID to colorectal carcinoma and liver cancer cell ₅₀Value is about 55-80 μ g/ml).

In addition, can find out further that from Fig. 2 GnRH-PE40 of the present invention does not then show detectable cytotoxic activity for the primary cell culture in the normal sexual gland related tissue (calf testis cell and human cervical cancer 1 cell) that derives from healthy donors, Lymphoid tissue (mouse chest cell) and epithelium (rat skin or fibrocyte) source.

The specificity of B.GnRH-PE40 is in conjunction with test

Basically according to Qayum, the described method of people such as A. (Br.J.Cancer 62:96-99,1990) is carried out GnRH-PE40 pair and is combined it with Hela cell cytosol membrane portions ¹²⁵The specificity competition of I-GnRH and replacement research.

The Hela cell monolayer of cultivating is separated to contains 10mM Tris-HCI, pH7.5,1mM EDTA, with the preparation cell homogenates, and centrifugal (500g, 15 minutes) remove big fragment in the damping fluid of 1mMDTT and 1mg/ml bovine serum albumin.With 25,000g obtained the endochylema membrane portions in centrifugal 30 minutes with 4 ℃ of supernatant parts, and it is suspended in the cold above-mentioned damping fluid (0.5mg protein/ml).Final volume with every hole 100 μ l is added on this endochylema film suspension (100 μ g/ hole) in each hole of 24 well culture plates.Respectively add 5 μ M (0.25 μ Ci) then ¹²⁵I-GnRH and 37 ℃ of temperature were bathed 1 hour.Add unlabelled GnRH (0.01-1000 μ M) or GnRH-PE40 of the present invention or 2 * GnRH-PE40 chimeric toxin (0.1-500mM) after 1 hour more respectively and continue insulation 24 hours.The insulation back is washed sample and is detected the bonded part with the γ calculating instrument with above-mentioned damping fluid.The result as shown in Figure 3.Partially purified 2 * the GnRH-PE40 of zero representative among Fig. 3, ● represent partially purified GnRH-PE40.The numerical value that X-coordinate provides is the concentration of competition thing 2 * GnRH-PE40 or GnRH-PE40.Ordinate zou is the B/BO value, and wherein B is in the presence of different concns competition thing, with Hela cytolemma bonded ¹²⁵The amount of I-GnRH (cpm); BO is not for when competing thing and exist and Hela cytolemma bonded ¹²⁵The amount of I-GnRH (cpm).

From data shown in Figure 3 as can be seen, adding partially purified GnRH-PE40 chimeric toxin with cumulative concentration can replace and Hela cytolemma bonded gradually along with the increase of GnRH-PE40 concentration ¹²⁵I-GnRH.And as if the chimeric toxin that the chimeric toxin that merges with two molecule GnRH series connection and toxin is connected with unit molecule GnRH has stronger receptor in target cell binding ability.

Though be not limited to theory, but we infer that this higher receptor-specific affinity of 2 * GnRH-PE40 may be not have the space extensibility of the guide molecule of halfcystine owing to prolonged this, and then have increased guide molecule and associated receptor or the antigenic chance that combines.In addition, as if the prompting of this experimental result of the present inventionly is more suitable for being developed to the antineoplastic agent that can use with placed in-line two molecule GnRH end to end as the chimeric toxin of targeting part in clinical practice.

The preparation of embodiment 3:MSH-PE40 fused protein

A. make up in the recombinant expression plasmid and identify

Basically make up the recombinant expression vector of carrier's melanotropin (MSH) gene order according to the method for the structure pVC8-GnRH-PE40 described in the embodiment 1.

Briefly, at first by known ordinary method (referring to Sambrook et al, MolecularCloning:A Laboratory Mamual; Cold Spring Harbor Laboratory, 1989) be in T with carrying ₇PE40 gene under the promotor control, and contain T ₇The plasmid pET17b-PE40 transformed into escherichia coli HB101 competent cell of transcription terminator and phage replication starting point, wherein said PE gene also contain an OmpA signal sequence (Chaudhary et al., PNAS USA 85:2939-2943,1988).The HB101 cell inoculation that will be transformed is containing on the LB agar plate of penbritin then, with a large amount of preparation pET17b-PE40 plasmid DNA.

With NdeI plasmid DNA is cut into linearity, and uses the small intestine alkaline phosphatase treatment, so that its sticky end dephosphorylation.

According to two complementary oligonucleotide chains (SEQID NO:5) of the known amino acid sequence composite coding MSH of people MSH, complementary annealed MSH double-stranded DNA two ends are the sticky end of NdeI restriction enzyme site respectively.After terminal phosphateization, the synthetic oligonucleotide fragment is inserted among the linearizing pET17b-PE40.

In order to obtain the MSH fragment that two MSH encoding sequences are connected in series mutually, can be respectively in the presence of the T4 dna ligase, according to the ligation condition of standard with linearizing pET17b-PE40 and double-stranded MSH fragment by 1: the 2-4 molar ratio mixes, to finish ligation (total reaction volume 50 μ l).

With resulting ligation mixture transformed into escherichia coli JM105 competent cell.Use above-mentioned oligonucleotide as probe, on nitrocellulose filter, carry out in situ hybridization with the amicillin resistance bacterium colony that is transformed.After under stringent condition, washing cell, extract plasmid DNA from the medium and small amount of positive bacterium colony.With NdeI, EcoRI and XbaI recombinant plasmid dna is carried out enzyme and cut evaluation.

To enzyme cut identify that correct plasmid DNA is carried out pcr amplification after, (AppliedBiosystems Inc.) carries out sequencing with the automated DNA sequenator.The complete sequence analysis result is shown in SEQ ID NO:6 and SEQ ID NO:7.The MSH gene order is linked the recombinant plasmid of being named respectively on the pET17b-PE40 carrier to pET17b-MSH-PE40 or pET17b-2 * MSH-PE40 with single copy or placed in-line two copy by correct direction.Fig. 4 has shown the structure of pET17b-2 * MSH-PE40.

By recombinant plasmid pET17b-MSH-PE40 or the pET17b-2 * MSH-PE40 difference transformed into escherichia coli BL21 (DE3) of standard method with as above preparation.Will be after containing on the LB agar plate of penbritin 37 ℃ of incubated overnight by cell transformed, select positive bacteria and drop into capable cell proliferation and cultivate.Work as OD ₆₀₀Add IPTG during for 0.6-0.8, with the expression (37 ℃, 3 hours) of induced protein product.

Owing to contain an intestinal bacteria secretory signal sequence (OmpA) among the E.Coli.BL21 (DE3) as this research, therefore there is the chimeric toxin of being expressed more than 80% can be secreted into (Chaudhary etal. in the cell pericentral siphon approximately, PNAS, USA 85:2939-2943,1988).Prepare the pericentral siphon part according to the described method of people such as Chaudhary (ibid for document), and this pericentral siphon partly carried out SDS-PAGE electrophoresis and Western engram analysis (using anti-PE antiserum(antisera) of multi-clone rabbit and anti-MSH antiserum(antisera)), with the existence and the molecular size thereof of MSH-PE40 and 2 * MSH-PE40 chimeric toxin in the tentative confirmation cell pericentral siphon.The aminoacid sequence of 2 * MSH-PE40 is shown in SEQ ID NO:8.

Can use Mono cationic exchange coloum and the required fused protein of Sephacry S-200 HR post (Pharmacia) purifying that is connected in the FPLC system (Pharmacia) successively according to the method for the purifying GnRH-PE40 described in the Embodiment B.With each wash-out part of SDS-polyacrylamide gel electrophoresis (Laemmli, Nature227:680-685,1970) analytical column chromatography, and use the anti-PE antiserum(antisera) of polyclone to identify it with immunoblotting.

The target-specific of embodiment 4:MSH-PE40 fused protein and biologic activity analysis

A. basically according to the method described in the embodiment 2, use murine melanoma B16 clone as target cell, and with the normal mouse melanocyte in contrast, partially purified MSH-PE40 of the present invention and 2 * MSH-PE40 chimeric toxin are carried out target-specific and cytotoxicity analysis.Test-results as shown in Figure 5.

From result shown in Figure 5 as can be seen, partially purified MSH-PE40 of the present invention and 2 * MSH-PE40 can suppress to have on its surface the murine melanoma B16 cell of MSH acceptor in dosage dependence mode, and performance has tangible cytotoxic activity (ID ₅₀Be about 80 μ g/ml), but can detected cytotoxicity to having that the source takes place in homologue normal mouse melanocyte then do not have.On the other hand, MSH-PE40 more of the present invention and 2 * MSH-PE40 chimeric toxin to the cytotoxicity of B16 cell as can be seen, with two molecule MSH series connection back and lps molecule chimeric obtain fused protein as if performance obtain fusion rotein confrontation target cell stronger cytotoxicity arranged than being connected with unit molecule MSH.

Sequence table

(1) general information

(I) applicant: Univ. of Farming and Stockbreeding, PLA

(II) denomination of invention: with the target-specific cytotoxin agent of small peptide hormone fusion

(III) sequence number: 8

(IV) address:

(A) contact person: Zhu Ping

(B) street: No. 175, Xi'an main road

(C) city: Changchun

(D) country: the People's Republic of China (PRC)

(E) postcode: 130062

(V) computer-reader form:

(A) amboceptor type: 3.5 inches floppy disks

(B) computer: IBM PC

(C) operating system: WINDOWS95

(D) software: WORD97

(VI) telecommunication information:

(A) phone: 86-0431-7962109

(B) fax: 86-0431-7973911-66692

(2) information of SEQ ID No:1

(I) sequence signature:

(A) length: 36 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: cDNA

(III) sequence description: SEQ ID No:1

TATGCAGCAC?TGGTCCTATG?GACTGCGCCC?TGGACA

(2) information of SEQ ID No:2

(I) sequence signature:

(A) length: 1134 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: cDNA

(III) sequence description: SEQ ID NO:2

ATGCAGCACT?GGTCCTATGG?ACTGCGCCCT?GGACATATGG?CCGAAGAGGG?CGGCAGCCTG

GCCGCGCTGA?CCGCGCACCA?GGCTTGCCAC?CTGCCGCTGG?AGACTTTCAC?CCGTCATCGC

CAGCCGCGCG?GCTGGGAACA?ACTGGAGCAG?TGCGGCTATC?CGGTGCAGCG?GCTGGTCGCC

CTCTACCTGG?CGGCGCGGCT?GTCGTGGAAC?CAGGTCGACC?AGGTGATCCG?CAACGCCCTG

GCCAGCCCCG?GCAGCGGCGG?CGACCTGGGC?GAAGCGATCC?GCGAGCAGCC?GGAG

CGTCTGGCCC?TGACCCTGGC?CGCCGCCGAG?AGCGAGCGCT?TCGTCCGGCA?GGGC

AACGACGAGG?CCGGCGCGGC?CAACGCCGAC?GTGGTGAGCC?TGACCTGCCC?GGTCGCCGCC

GGTGAATGCG?CGGGCCCGGC?GGACAGCGGC?GACGCCCTGC?TGGAGCGCAA?CTATCCCACT

GGCGCGGAGT?TCCTCGGCGA?CGGCGGCGAC?GTCAGCTTCA?GCACCCGCGG?CACGCAGAAC

TGGACGGTGG?AGCGGCTGCT?CCAGGCGCAC?CGCCAACTGG?AGGAGCGCGG?CTATGTGTTC

GTCGGCTACC?ACGGCACCTT?CCTCGAAGCG?GCGCAAAGCA?TCGTCTTCGG?CGGGGTGCGC

GCGCGCAGCC?AGGACCTCGA?CGCGATCTGG?CGCGGTTTCT?ATATCGCCGG?CGATCCGGCG

CTGGCCTACG?GCTACGCCCA?GGACCAGGAA?CCCGACGCAC?GCGGCCGGAT?CCGCAACGGT

GCCCTGCTGC?GGGTCTATGT?GCCGCGCTCG?AGCCTGCCGG?GCTTCTACCG?CACCAGCCTG

ACCCTGGCCG?CGCCGGAGGC?GGCGGGCGAG?GTCGAACGGC?TGATCGGCCA?TCCGCTGCCG

CTGCGCCTGG?ACGCCATCAC?CGGCCCCGAG?GAGGAAGGCG?GGCGCCTGGA?GACCATTCTC

GGCTGGCCGC?TGGCCGAGCG?CACCGTGGTG?ATTCCCTCGG?CGATCCCCAC?CGACCCGCGC

AACGTCGGCG?GCGACCTCGA?CCCGTCCAGC?ATCCCCGACA?AGGAACAGGC?GATCAGCGCC

CTGCCGGACT?ACGCCAGCCA?GCCCGGCAAA?CCGCCGCGCG?AGGACCTGAA?GTAA

(2) information of SEQ ID NO:3:

(I) sequence signature:

(A) length: 1179 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: cDNA

(III) sequence description: SEQ ID NO:3

ATGCAGCACT?GGTCCTATGG?ACTGCGCCCT?GGACATATGC?AGCACTGGTC?CTATGGACTG

CGCCCTGGAC?ATATGGCCGA?AGAGGGCGGC?AGCCTGGCCG?CGCTGACCGC?GCACCAGGCT

TGCCACCTGC?CGCTGGAGAC?TTTCACCCGT?CATCGCCAGC?CGCGCGGCTG?GGAACAACTG

GAGCAGTGCG?GCTATCCGGT?GCAGCGGCTG?GTCGCCCTCT?ACCTGGCGGC?GCGGCTGTCG

TGGAACCAGG?TCGACCAGGT?GATCCGCAAC?GCCCTGGCCA?GCCCCGGCAG?CGGCGGCGAC

CTGGGCGAAG?CGATCCGCGA?GCAGCCGGAG?CAGGCCCGTC?TGGCCCTGAC?CCTGGCCGCC

GCCGAGAGCG?AGCGCTTCGT?CCGGCAGGGC?ACCGGCAACG?ACGAGGCCGG?CGCGGCCAAC

GCCGACGTGG?TGAGCCTGAC?CTGCCCGGTC?GCCGCCGGTG?AATGCGCGGG?CCCGGCGGAC

AGCGGCGACG?CCCTGCTGGA?GCGCAACTAT?CCCACTGGCG?CGGAGTTCCT?CGGCGACGGC

GGCGACGTCA?GCTTCAGCAC?CCGCGGCACG?CAGAACTGGA?CGGTGGAGCG?GCTGCTCCAG

GCGCACCGCC?AACTGGAGGA?GCGCGGCTAT?GTGTTCGTCG?GCTACCACGG?CACCTTCCTC

GAAGCGGCGC?AAAGCATCGT?CTTCGGCGGG?GTGCGCGCGC?GCAGCCAGGA?CCTCGACGCG

ATCTGGCGCG?GTTTCTATAT?CGCCGGCGAT?CCGGCGCTGG?CCTACGGCTA?CGCCCAGGAC

CAGGAACCCG?ACGCACGCGG?CCGGATCCGC?AACGGTGCCC?TGCTGCGGGT?CTATGTGCCG

CGCTCGAGCC?TGCCGGGCTT?CTACCGCACC?AGCCTGACCC?TGGCCGCGCC?GGAGGCGGCG

GGCGAGGTCG?AACGGCTGAT?CGGCCATCCG?CTGCCGCTGC?GCCTGGACGC?CATCACCGGC

CCCGAGGAGG?AAGGCGGGCG?CCTGGAGACC?ATTCTCGGCT?GGCCGCTGGC?CGAGCGCACC

GTGGTGATTC?CCTCGGCGAT?CCCCACCGAC?CCGCGCAACG?TCGGCGGCGA?CCTCGACCCG

TCCAGCATCC?CCGACAAGGA?ACAGGCGATC?AGCGCCCTGC?CGGACTACGC?CAGCCAGCCC

GGCAAACCGC?CGCGCGAGGA?CCTGAAGTAA

(2) information of SEQ ID NO:4

(I) sequence signature:

(A) length: 393 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: protein

(III) sequence description: SEQ ID NO:4

Met-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-His-Met-Glu-His-Trp-Ser-Tyr-Gly-

Leu-Arg-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-

Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-

Glu-Gln-Leu-Gly-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-

Ala-Arg-Leu-Ser-Trp-Asp-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-

Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Gln-Gln-Ala-Arg-Leu-

Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-

Asp-Gln-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-

Gly-Glu-Cys-Ala?Gly-Pro-Ala-Asp-Gly-Asp-Ala-Len-Len-Glu-Arg-Asn-Tyr-Pro-Thr-

Gly-Ala-Glu-Phe-Len-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-

Asn-Trp-Thr-Val-Gln-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Gln-Gln-Arg-Gly-Tyr-

Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Len-Gln-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-

Gly-Arg-Ala-Arg-Ser-Gln-Gln-Asp-Leu-Asp-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-

Ala-Gly-Asp-Pro-Ala-Len-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Gln-Pro-Asp-Ala-Arg-

Gly-Arg-Ile-Arg-Asp-Gly-Ala-Len-Len-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Len-Pro-

Gly-Phe-Tyr-Arg-Thr-Ser-Len-Thr-Len-Ala-Ala-Pro-Gln-Ala-Ala-Gly-Gln-Val-Gln-

Arg-Len-Ile-Gly-His-Pro-Len-Pro-Len-Arg-Len-Asp-Ala-Ile-Thr-Gly-Pro-Gln-Gln-

Gly-Gly-Arg-Len-Gln-Thr-Ile-Len-Gly-Trp-Pro-Len-Ala-Gln-Arg-Thr-Val-Val-Ile-

Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Len-Asp-Pro-Ser-Ser-

Ile-Pro-Asp-Lys-Gln-Gln-Ala-Ile-Ser-Ala-Len-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-

Lys-Pro-Arg-Gln-Asp-Len-Lys-end.

(2) information of SEQ ID NO:5

(I) sequence signature:

(A) length: 45 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: cDNA

(III) sequence description: T ATG AGT TAT AGT ATG GAG CAC TTC AGG TGG GGA AAG CCA GTA CA

(2) information of SEQ ID NO:6

(I) sequence signature:

(A) length: 1143 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: cDNA

(III) sequence description:

ATGAGTTATA?GTATGGAGCA?CTTCAGGTGG?GGAAAGCCAG?TACATATGGC?CGAAGAGGGC

GGCAGCCTGG?CCGCGCTGAC?CGCGCACCAG?GCTTGCCACC?TGCCGCTGGA?GACTTTCACC

CGTCATCGCC?AGCCGCGCGG?CTGGGAACAA?CTGGAGCAGT?GCGGCTATCC?GGTGCAGCGG

CTGGTCGCCC?TCTACCTGGC?GGCGCGGCTG?TCGTGGAACC?AGGTCGACCA?GGTGATCCGC

AACGCCCTGG?CCAGCCCCGG?CAGCGGCGGC?GACCTGGGCG?AAGCGATCCG?CGAGCAGCCG

GAGCAGGCCC?GTCTGGCCCT?GACCCTGGCC?GCCGCCGAGA?GCGAGCGCTT?CGTCCGGCAG

GGCACCGGCA?ACGACGAGGC?CGGCGCGGCC?AACGCCGACG?TGGTGAGCCT?GACCTGCCCG

GTCGCCGCCG?GTGAATGCGC?GGGCCCGGCG?GACAGCGGCG?ACGCCCTGCT?GGAGCGCAAC

TATCCCACTG?GCGCGGAGTT?CCTCGGCGAC?GGCGGCGACG?TCAGCTTCAG?CACCCGCGGC

ACGCAGAACT?GGACGGTGGA?GCGGCTGCTC?CAGGCGCACC?GCCAACTGGA?GGAGCGCGGC

TATGTGTTCG?TCGGCTACCA?CGGCACCTTC?CTCGAAGCGG?CGCAAAGCAT?CGTCTTCGGC

GGGGTGCGCG?CGCGCAGCCA?GGACCTCGAC?GCGATCTGGC?GCGGTTTCTA?TATCGCCGGC

GATCCGGCGC?TGGCCTACGG?CTACGCCCAG?GACCAGGAAC?CCGACGCACG?CGGCCGGATC

CGCAACGGTG?CCCTGCTGCG?GGTCTATGTG?CCGCGCTCGA?GCCTGCCGGG?CTTCTACCGC

ACCAGCCTGA?CCCTGGCCGC?GCCGGAGGCG?GCGGGCGAGG?TCGAACGGCT?GATCGGCCAT

CCGCTGCCGC?TGCGCCTGGA?CGCCATCACC?GGCCCCGAGG?AGGAAGGCGG?GCGCCTGGAG

ACCATTCTCG?GCTGGCCGCT?GGCCGAGCGC?ACCGTGGTGA?TTCCCTCGGC?GATCCCCACC

GACCCGCGCA?ACGTCGGCGG?CGACCTCGAC?CCGTCCAGCA?TCCCCGACAA?GGAACAGGCG

ATCAGCGCCC?TGCCGGACTA?CGCCAGCCAG?CCCGGCAAAC?CGCCGCGCGA?GGACCTGAAG

TAA

(2) information of SEQ ID NO:7:

(I) sequence signature:

(A) length: 1188 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: cDNA

(III) sequence description:

ATGAGTTATA?GTATGGAGCA?CTTCAGGTGG?GGAAAGCCAG?TACATATGAG?TTATAGTATG

GAGCACTTCA?GGTGGGGAAA?GCCAGTACAT?ATGGCCGAAG?AGGGCGGCAG?CCTGGCCGCG

CTGACCGCGC?ACCAGGCTTG?CCACCTGCCG?CTGGAGACTT?TCACCCGTCA?TCGCCAGCCG

CGCGGCTGGG?AACAACTGGA?GCAGTGCGGC?TATCCGGTGC?AGCGGCTGGT?CGCCCTCTAC

CTGGCGGCGC?GGCTGTCGTG?GAACCAGGTC?GACCAGGTGA?TCCGCAACGC?CCTGGCCAGC

CCCGGCAGCG?GCGGCGACCT?GGGCGAAGCG?ATCCGCGAGC?AGCCGGAGCA?GGCCCGTCTG

GCCCTGACCC?TGGCCGCCGC?CGAGAGCGAG?CGCTTCGTCC?GGCAGGGCAC?CGGCAACGAC

GAGGCCGGCG?CGGCCAACGC?CGACGTGGTG?AGCCTGACCT?GCCCGGTCGC?CGCCGGTGAA

TGCGCGGGCC?CGGCGGACAG?CGGCGACGCC?CTGCTGGAGC?GCAACTATCC?CACTGGCGCG

GAGTTCCTCG?GCGAGGGCGG?CGACGTCAGC?TTCAGCACCC?GCGGCACGCA?GAACTGGACG

GTGGAGCGGC?TGCTCCAGGC?GCACCGCCAA?CTGGAGGAGC?GCGGCTATGT?GTTCGTCGGC

TACCACGGCA?CCTTCCTCGA?AGCGGCGCAA?AGCATCGTCT?TCGGCGGGGT?GCGCGCGCGC

AGCCAGGACC?TCGACGCGAT?CTGGCGCGGT?TTCTATATCG?CCGGCGATCC?GGCGCTGGCC

TACGGCTACG?CCCAGGACCA?GGAACCCGAC?GCACGCGGCC?GGATCCGCAA?CGGTGCCCTG

CTGCGGGTCT?ATGTGCCGCG?CTCGAGCCTG?CCGGGCTTCT?ACCGCACCAG?CCTGACCCTG

GCCGCGCCGG?AGGCGGCGGG?CGAGGTCGAA?CGGCTGATCG?GCCATCCGCT?GCCGCTGCGC

CTGGACGCCA?TCACCGGCCC?CGAGGAGGAA?GGCGGGCGCC?TGGAGACCAT?TCTCGGCTGG

CCGCTGGCCG?AGCGCACCGT?GGTGATTCCC?TCGGCGATCC?CCACCGACCC?GCGCAACGTC

GGCGGCGACC?TCGACCCGTC?CAGCATCCCC?GACAAGGAAC?AGGCGATCAG?CGCCCTGCCG

GACTACGCCA?GCCAGCCCGG?CAAACCGCCG?CGCGAGGACC?TGAAGTAA

(2) information of SEQ ID NO:8:

(I) sequence signature:

(A) length: 396 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: protein

(III) sequence description:

Met-Ser-Tyr-Ser-Met-Gln-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-His-Met-Ser-Tyr-Ser-

Met-Gln-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-

Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-

Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Gly-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-

Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asp-Gln-Val-Asp-Gln-Val-Ile-Arg-

Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-

Pro-Gln-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-

Arg-Gln-Gly-Thr-Gly-Asn-Asp-Gln-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-

Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Gly-Asp-Ala-Len-Len-

Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Len-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-

Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Gln-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-

Leu-Gln-Gln-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Len-Gln-Ala-Ala-

Gln-Ser-Ile-Val-Phe-Gly-Gly-Arg-Ala-Arg-Ser-Gln-Gln-Asp-Leu-Asp-Asp-Ala-Ile-

Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Len-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-

Gln-Gln-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asp-Gly-Ala-Len-Len-Arg-Val-Tyr-Val-

Pro-Arg-Ser-Ser-Len-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Len-Thr-Len-Ala-Ala-Pro-Gln-

Ala-Ala-Gly-Gln-Val-Gln-Arg-Len-Ile-Gly-His-Pro-Len-Pro-Len-Arg-Len-Asp-Ala-

Ile-Thr-Gly-Pro-Gln-Gln-Gly-Gly-Arg-Len-Gln-Thr-Ile-Len-Gly-Trp-Pro-Len-Ala-

Gln-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-

Asp-Len-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Gln-Gln-Ala-Ile-Ser-Ala-Len-Pro-Asp-

Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Arg-Gln-Asp-Len-Lys-end.

Claims (3)

1. stimulate hormone and reorganization Pseudomonas exotoxin to merge the chimeric toxin that forms by small peptide parahormone gonadotropin-releasing hormone or melanochrome, be characterised in that wherein said small peptide parahormone as directed agents partly is partly to be connected with reorganization Pseudomonas exotoxin as cytotoxic agent with two molecules series connection form.

2. according to the chimeric toxin of claim 1, wherein said reorganization Pseudomonas exotoxin is PE40.

3. contain the chimeric toxin of with good grounds claim 1 and the pharmaceutical composition of one or more pharmaceutically acceptable carriers or vehicle.

Images