CN101074267A - Transduced peptide-humanized granular leukocyte macrophage colony stimulating factor fusion protein and its medicinal composition - Google Patents

Abstract

A transduction-peptide-humanized colony-forming unit-granulocyte-macrophage stimulating factor fusion protein, nucleic acid molecule of nucleic acid sequence for encoding fusion protein, expression carrier containing the nucleic acid molecule and its usage of fusion protein in preparation of medicine are disclosed. It can be used for leukopenia after malignant tumors radiant chemotherapy, bone narrow transplantation, aregenerative anemia, immune-deficient diseases treatment, traumato-therapy, and anti-viral and anti-fungal treatments.

Description

Transduced peptide-humanized granular leukocyte macrophage colony stimulating factor fusion rotein and pharmaceutical composition thereof

Invention field

The present invention relates to have the fusion rotein of pharmaceutical use, be specially the transduced peptide-humanized granular leukocyte macrophage colony stimulating factor fusion rotein, the nucleic acid molecule of this fusion rotein of encoding.The present invention also relates to contain the pharmaceutical composition of this fusion rotein.

Background of invention

General introduction

Granulocyte-macrophage colony stimutaing factor (Granulocyte macrophage colonystimulating factor, GM-CSF) be one of main hemopoieticgrowth factor, can stimulate the propagation and the differentiation of early stage pluripotential hemopoietic stem cell and grain-monosystem progenitor cell; Strengthen ripe neutrophil leucocyte, eosinophilic granulocyte and monokaryon-macrophage function; Collaborative red system, macronucleus system and the high proliferation potential progenitor cells (HPP-CFC) of stimulating grown; Prolong the scavenger cell survival time and improve its anti-tumor capacity; Simultaneously, GM-CSF can also grow by stimulating endothelial cell, stops apoptosis of many kinds, plays a significant role in hematopoiesis regulation and control and immunomodulatory.GM-CSF can also promote the expression of dendritic cell APC such as (DC) differentiation, maturation and activation and rise CD86 to excite the immunne response to tumour; Promote Th, Tc, NK to soak at tumor locus, thus killing tumor cells.

Hemopoieticgrowth factor as a pluripotency, its main clinical application is to promote marrow hemopoiesis, at present clinically rhGM-CSF be successfully used to treat that malignant tumour is put, the leukopenia due to after the chemotherapy, and be used for the treatment etc. that there are the low immunodeficiency diseases of white corpuscle in bone marrow transplantation, aplastic anemia and some.Discovering in recent years, GM-CSF not only can act on that hematopoiesis is done, progenitor cell promotes marrow hemopoiesis, but also acts on the strongest antigen presenting cell-dendritic cell of function in the present known body, to promote immunne response, regulates immune response [[2,3].In addition, GM-CSF also acts on keratinocyte, inoblast, mucous membrane cell etc.Many in recent years bibliographical informations, rhGM-CSF not only can be used in prevents and treats leukopenia, and in wound healing, treatment such as antiviral, antimycotic better curative effect is arranged also, has important clinical application value.

The character of GM-CSF

GM-CSF is a kind of acid glycoprotein.The hGM-CSF assignment of genes gene mapping is in No. 5 karyomit(e)s long-armed (5q21-32), and mrna length is 2.5kb, and 4 exons and 3 introns are arranged.HGM-CSF is a kind of glycoprotein of being made up of 127 amino acid, according to its degree of glycosylation difference, is divided into types different more than 9, molecular weight 14kD-32kD.Its secondary structure is made up of two antiparallel βZhe Dies and 4 antiparallel α spirals, and it is the cog region of GM-CSF receptor beta subunit that N holds first spiral.Four cysteine residues form two intrachain disulfide bonds (C54, C96) and (C88 C121), plays an important role to stability and the biological activity of GM-CSF.

Known at present, the biological function of GM-CSF is to mediate by the specific receptors that is expressed on the cytolemma.GM-CSF by with receptors bind, activate Na on the target cell ⁺/ H ⁺Conversion carrier activity improves internal pH numerical value, thus cell guiding increment differentiation.

The target cell of GM-CSF mainly contains: the hematopoietic cell of marrow different developmental phases, neutrophil leucocyte, mononuclear macrophage, eosinophilic granulocyte, endotheliocyte, acute myeloid leukaemia cell etc.

The expression system of GM-CSF

The method of utilizing recombinant clone in intestinal bacteria, yeast and mammal cell line system successful expression GM-CSF.Phraseology in intestinal bacteria is mainly two kinds: (1) inclusion body form, GM-CSF is modal at expression in escherichia coli to be to form insoluble inclusion body, obtains GM-CSF through extracting steps such as inclusion body, sex change cracking, renaturation, purifying.(2) secretion type expression promptly utilizes outer membrane protein signal peptide (OmpA) gene, and OmpA gene back is arrived in the GM-CSF gene clone, remove the catenation sequence of the two with the method for positional mutation, obtain sophisticated GM-CSF, but expression amount is not high, every liter of fermented liquid is only about 20mg.

The expression of GM-CSF in Yeast system, be to α-factor signal peptide gene downstream with the GM-CSF gene clone, with the method removal GM-CSF of positional mutation and the catenation sequence between α-factor signal peptide, at ADHZ, under the promoter regulations such as PGK, express justacrine in yeast outside born of the same parents, expression amount can be up to every liter of fermented liquid 50-60mg.

GM-CSF expresses in mammalian cell system such as Chinese hamster ovary cell (CHO) and monkey COS-1 cell, but expression amount is very little.

The GM-CSF that above-mentioned three kinds of systems are expressed, its key distinction is the glycosylation in N-site and O-site.The GM-CSF of escherichia coli expression (E.coli-GM), all not glycosylations of N-site and O-site, molecular weight is 14.6kD; The GM-CSF of yeast expression (Yeast-GM), only N-site glycosylation, and the not glycosylation of O-site, molecular weight 15.6-30kD; The GM-CSF of mammalian cell expression (CHO-GM), N-site and O-site be all by glycosylation, molecular weight 18-24kD.Experiment in vitro shows glycosylation and non-glycosylated GM-CSF no significant difference aspect biologic activity.

The clinical application aspect, Dorr has compared the toxic side effect of E.coil-GM and Yeast-GM, and liquid hold-up appears in the E.coil-GM group as a result, expiratory dyspnea, fever, the average frequency of myalgia/ostalgia/arthrodynia and macula is respectively 18.4%, 55.2%, 40.7%, 28.5% and 12.5%, the Yeast-GM group is corresponding to be respectively 8.3%, 13.4%, 21.7%, 16% and 14.3%.Show that the Yeast-GM toxic side effect is lighter.Hussein etc. are with E.coil-GM, Yeast-GM and CHO-GM are respectively applied for mammary cancer or the melanoma patient behind the autologous bone marrow transplantation, the result is aspect the recovery of leukocyte increasing and neutrophil leucocyte, E.coli-GM and Yeast-GM are apparently higher than CHO-GM, and the CD34+ cell quantity, the CHO-GM group is the highest, and the Yeast-GM group is higher than E.coil-GM again.Its mechanism of action still needs further to inquire into.

The biological action of GM-CSF

GM-CSF mainly contains following biological action: can be in hemopoietic stem cell and the horizontal effective stimulus granulocyte of progenitor cell and monokaryon-macrophage system propagation, differentiation and maturation.But the multidirectional hemopoietic progenitor cell of the normal medullary system of effective stimulus, grain/scavenger cell and eosinophilic granulocyte clone form in external semi-solid the cultivation.Also have the activity of burst promoting factor (BPA), form with the collaborative burst forming unit erythroid that promotes of GM-CSF.GM-CSF can promote that also macronucleus is cell growth and hematoblastic generation.Also stimulate some leukemia cell (HL 60 and KG 1) proliferate, induce HL 60 cells to break up to ripe direction.The marrow endothelial cell is one of stroma cell of hematopoieticmicroenviron-ment, and hematopoiesis is had the important regulating and controlling effect, and exogenous GM-CSF can stimulate its propagation.GM-CSF not only can stimulate the propagation of prematurity precursor cell, also can improve the function of mature blood cell, prolongs its lifetime, has seemed central role in regulating the mature blood cell function.Granulocytic effect mainly shows as GM-CSF to neutrality: increase protein synthesis, prolong lifetime; Promote the ADCC effect; Change cell surface receptor and express, suppress its moving in the inflammation district; Raise the cell surface adhesion protein and express, induce degranulation in the endochylema; Strengthen oxidative metabolism, increase super-oxide and produce; Quicken Ca2+ stream, induce migration, it is synthetic to increase LTB4.Also can strengthen the effect of the engulfing of mononuclear macrophage, cell toxicant and killing tumor cell, promote cytokine expression, stick and oxidative metabolism.To eosinophilic granulocyte mainly is synthetic, the histamine release of enhancing cytotoxicity and leukotriene.In addition, the tumor cell line of multiple non-hematopoietic origin also has sensitivity response to GM-CSF.

GM-CSF is a kind of polypeptide growth factor with broad effect spectrum, and it is by working in conjunction with specific high-affinity receptor.In regulating hematopoiesis and leukocyte function, play an important role, can stimulate myeloid progenitor propagation and differentiation, stimulate neutrality, eosinophilic granulocyte, M Φ and DC propagation and ripe; Also can promote megakaryocyte growth.Growth has the auxiliary adjustment effect to red corpuscle, promoting erythrocyte, reticulocyte effect are arranged, improve the phagocytic function of mononuclear macrophage to tumour cell, increase the granulocyte number, improve expression of adhesion molecule and ADCC and kill the knurl effect, reduce infection chance, improve immunizing power, especially have the function of enhancing body tumour immunity.

Clinical application

GM-CSF is important a member in many Hemopoietic factors, from the nineties be applied to clinical since, particularly treatments such as the oligoleukocythemia due to tumor radiotherapy, the chemotherapy, bone marrow transplantation, myelodysplastic syndrome, aplastic anemia have obtained certain curative effect.Find again in the clinical application in recent years to put, mucositis, fungi infestation, viral hepatitis, diabetes merge fat necrobiotic more property ulcus cruris and pulmonary alveolar proteinosis etc. also do not have curative effect preferably due to the chemotherapy.

1, the hematopoietic disorder due to the tumor chemoradiotherapy

The curative effect of chemotherapy of tumors largely depends on dose intensity, the density of chemotherapeutics, and both increase and often cause the infringement of bone marrow depression, immunologic function, so that the chemotherapy failure.Bone marrow depression granulocytopenia, secondary bacterium and fungi infestation even cause patient death again.The clinical practice of more than ten years has proved uses GM-CSF, and G-CSF can effectively prevent and treat granulocytopenia due to the chemotherapy, shortens neutrophil leucocyte and reduces the time, reduces the generation of infecting.

But the propagation of GM-CSF hemopoietic mainly is by bringing into play its biological effect with the GM-CSF receptors bind on effector cell (normally breeding bone marrow stem cell, neutrophil leucocyte, monokaryon granulocyte, scavenger cell etc.) surface.It is except that the differentiation that stimulates stem cell, progenitor cell, propagation, the activity that also has similar megalokaryocyte clone stimulating factor, leukocyte increasing is situated between plain-3 (1L-3) to megalokaryocyte clone formation effectiveness, collaborative erythropoietin stimulating red corpuscle propagation [Liu Wei, Feng Weijian, the clinical observation of .GM-CSF such as Wang Yuhua and chemotherapy synchronous applications. Chinese tumor biotherapy magazine, 2000; 7 (3): 181].

2, the reconstruction of hemopoietic function after the quickening bone marrow transplantation

Bone marrow transplantation is the main means of current blood system treating malignant tumor, in recent years solid tumor such as mammary cancer, melanoma, lung cancer has also been obtained certain success.In general, need three all neutrophil leucocytes to recover after the bone marrow transplantation, very easily infect during this, die from the infected and account for 10%-15%.High-dose chemotherapy, bone marrow transplantation is used GM-CSF can quicken the marrow hematopoietic reconstitution, the rising peripheral white blood cells, the minimizing infectation of bacteria [Zhao Zhongxin. the novel clinical use of recombinant humangranulocyte-giant cells G CFS. Chinese tumor biotherapy magazine, zooo; 7 (1): 71].

3, treatment marrow abnormal syndrome (MDS) and aplastic anemia (SAM)

It is regenerative power that reported literature GM-CSF can effectively recover patient's MDS grain, can make white corpuscle rising 5-70 doubly, begin to rise with medicine 48h, one week reached stable, but also the someone thinks on the differentiation of GM-CSF promotion white corpuscle, maturation, GM-CSF can induce the potential Leukemia Cell Proliferation of MDS, also is the problem that the clinician worries.

Aplastic anemia is a kind of heterology disease, since the eighties of last century GM-CSF nineties is applied to the clinical report that a lot of relevant GM-CSF treatments " barrier again " are arranged, can make peripheral white blood cell increase 5-20 doubly, the neutrophil leucocyte number increases 5-373 doubly, and other also all have in various degree rising as monocyte, eosinophilic granulocyte, red corpuscle, thrombocyte etc.However, owing to the barrier again due to the different reasons, its treatment result also is not quite similar, have in addition effect relatively poor.Be still the further problem [TroussardX of research of needs with GM-CSF treatment " barrier again ", Macro M, Vie B, et al.Human recombinantganulocyte-macrophage colony stimulating factor (hrGM-CSF) improves double hemibody irradiation (DHBI) tolerance inpatients with stage U1 multiple myeloma:a pilot study.Br JHaematal, 1995; 89:191].

4, the leukopenic treatment due to the acquired immune deficiency syndrome (AIDS) (AIDS)

Oligoleukocythemia is the main intercurrent disease of AIDS, works as oligoleukocythemia, and Chang Yi causes severe infections, threatens patient's AIDS life security, and using GM-CSF not only can increase patient's AIDS peripheral blood neutrophil quantity, and can increase its function.Because neutrophil leucocyte can produce the cell toxicant (ADCC) of antibody dependent cellular mediation, the HIV of AIDS there is antagonistic action.Reported literature GM-CSF and Eidovudin interferon therapy recover granulocyte rapidly recently, but also the someone reports that GM-CSF can make HIV produce antigen (virus replication) level and raise, dark people's research so definite curative effect is still needed.

5, enhancing immunity, antitumor action

Monocyte is one of cell crucial among the human immune system, except that having phagocytic function, regulates the vital role of host immune system in addition, and this effect is because GM-CSF impels the ADCC effect and the synthetic anti-tumor factor that discharges of mononuclear macrophage.Britain Christie hospital application GM-CSF treatment late malignant tumour 20 examples, 7 routine tumours were stablized 70 days, and 1 routine liposarcoma dwindles 50%, and continue 6 months [open keep loft, Song Xilin. granulocyte-macrophage colony stimutaing factor is in oncologic application progress treatment and prevention of tumour magazine, 2000; 7 (5): 518].

6, malignant tumour put, the prevention and the therapeutic action of mucous membrane inflammation, ulcer due to the chemotherapy

Stomatocace is that malignant tumour is put, inevitable complication in the chemotherapy process, and this complication not only influences proceeding of treatment, and easily cause bacterium, infection by microorganisms such as fungi, increase the weight of the state of an illness.At present clinically what good treatment means the processing of this complication is not also had, conventional treatment measure is actively anti-infective, vitimin supplement and strengthens nutrition etc., but the almost few of clinical effectiveness of these ways.

RhGM-CSF not only can promote marrow hemopoiesis, and stomatocace is also had good preventive and therapeutic effect.Employing rhGM-CSF such as Reynos in 1994 add method that physiological saline gargles high-dose chemotherapy, stomatocace that allogeneic bone marrow transplantation and autologous peripheral blood stem cell transplantation caused are studied, and prove that rhGM-CSF has good effect to the control stomatocace.No matter rhGM-CSF is to adopt subcutaneous injection, still adopts to be dissolved in the mode that physiological saline is gargled, and it all can promote ulcer healing effectively, and perhaps this be one clinically and well select.

7, to treatment of fungal infections

At present, fungi infestation has become one of major causes of death of tumour patient.Bibliographical information is arranged, and for persistent fever and with the low patient of white corpuscle, after 1 week, the incidence of systemic fungal infection is about 33% through antibiotic therapy, and for the bone marrow transplantation patient, the incidence of invading human nature fungi infestation is also up to 15%-30%.These phenomenons show clearly, and fungi infestation has produced the prognosis of malignant tumor patient and seriously influenced.

Antifungal preparation now commonly used clinically mainly contain amphotericin, nystatin, gram mould sound of crying or vomiting, the fluorine health sound of crying or vomiting, ketone health mile etc., these medicines all have certain antifungic action, but their all have simultaneously a serious side reaction-bone marrow depression, so that many patients stopped treatment of having to.GM-CSF is a kind of hemopoieticgrowth factor with multinomial potential, and it not only can promote propagation, differentiation and the maturation of hemopoietic forebody cell, recovers because of the bone marrow depression due to the chemotherapy of tumors, and also plays a part certain to the adjusting of human body immune function.Given this, GM-CSF can bring into play good effect in treatment fungi infestation.[Peters BG such as Peter, AdkinsDR, Harrison BR, et al.Antifungal effects of yeast derivedrhuGM-GSF in patients receiving high dose chemotherapy givenwith or without autologous stem cell transplantation; Aretrospective analysis.Bone Marrow Transulant.1996.18 (1): 93] fungi infestation situation that 145 examples are carried out the autologous stem cell transplantation patient carried out retrospective analysis, have 2 examples to have fungi infestation among the 70 routine patients that find to adopt rhGM-CSF to treat, incidence is 2.9%; And, having 9 examples that fungi infestation takes place among the 75 routine patients at control group, incidence is 12%.Through statistical procedures, the P value is 0.023, has notable difference between the two.RhGM-CSF has good curative effect really to fungi infestation, and it can strengthen the effect of antifungal drug.

8, the treatment of viral hepatitis

Tradition HBV, HCV is main treatment means with Interferon, rabbit, but clinically more and more feels not really desirable, many cases have to stop treatment because of occurring bone marrow depression in the treatment.Recent study finds, but GM-CSF activated T cell, endotheliocyte, the function of enhancement antigen presenting cell (APC) increases cell MHC, and collaborative stimulation molecule (CM) participates in immunity of organism and regulates, and strengthens the curative effect of antiviral.GM-CSF has been used for the treatment of chronic hepatitis at present.GM-CSF improves a-INF to chronic hepatitis B antiviral therapy effect, its mechanism may improve the relevant [Shao Chunzhong of antigen presentation ability with the elevating blood amount of mononuclear cells, Liu Wenyan, Chen Xiaoxia etc. the observation of curative effect of rHuGM-CSF and alpha-interferon combination therapy chronic hepatitis B. China's experiment and clinical virology magazine, 2001; 15 (1): 86].

9, GM-CSF is in neonatal application

The newborn infant is because autoimmune characteristics, incidence of infection and case fatality rate height, traditional treatment pattern mainly comprises antibiotic use and the various blood products of infusion (comprising blood plasma, granulocyte, Gammavenin), latter's good effect, but potential side effect is big.Use CSF and can strengthen the phagocytic cell system function, enhance immunity power is for the prevention and the treatment of infection of newborn opens up a new way.But also be in the preliminary stage at present, still need further fundamental research and clinical experiment inquire into [Zhang Yongjun, Xu Yingmei. granulocyte-Granulocyte Colony-stimulating is in neonatal application. foreign medical science paediatrics fascicle, 2001; 28 (2): 99].

The GM-CSF toxic side effect

Overwhelming majority patient can both finely tolerate the rGM-CSF dosage of clinical use.Double blinding, find that with the comparative studies of collimation rGM-CSF treatment group patient diarrhoea, asthenopia, fash and the incidence that does not feel like oneself are higher at random, the type of other side effect and incidence and placebo group comparison no significant difference.Open test shows, uses the patient of rGM-CSF, and the symptom of similar influenza is all arranged mostly, shows as myalgia, ostalgia, tired and headache, part patient's heating, the small portion patient has capillary vessel to ooze out symptom, the liquid storage occurs and stay, edema, pericardium and pleura liquid ooze out, and cause drug withdrawal.Also be reported in the subcutaneous injection position and fash occurs, individual patients has central vein conduit thrombosis and respiratory distress, thereby, advocate at present to use rGM-CSF should be specifically noted that to the previously patient of tuberculosis history is arranged.

Dosage and administration

With the reconstruction of the rGM-CSF treatment autologous bone marrow transplantation postoperative medullary cell in yeast source, 2-4h used 250ug/m after bone marrow infusion was recommended by the U.S. ², every day, 2h was quiet, used 21d continuously.With the patient of rGM-CSF treatment bone marrow transplantation failure, 250ug/m advocates in the U.S. ²/ d, quiet of 2h, continuously 14d if necessary, uses by last method behind the 7d at interval again, the 3rd the course of treatment rGM-CSF dosage rise to 500ug/m ²/ d, rGM-CSF 500ug/m ²/ d is the maximal dose of clinical use, does not have improvement as patient's state of an illness, does not want escalated dose again.The pharmaco-kinetic properties of rGM-CSF is relevant with route of administration.Intravenously is given rGM-CSF, and blood plasma rGM-CSF descends rapidly, and distribution half-life is 5-15min, and the elimination transformation period is 1.5-2h.Subcutaneous to 2h behind the rGM-CSF, rGM-CSF Plasma Concentration peaking descends later on gradually, is 3h.Intravenous injection rGM-CSF 15ug/kg can keep effective concentration 1ug/L 8-22h, and subcutaneous injection equivalent rGM-CSF can keep effective concentration 16h.

The route of administration of GM-CSF comprises vein and subcutaneous injection.The subcutaneous injection method can reduce the dosage of GM-CSF.The use of GM-CSF must be intravenous injection and subcutaneous injection administering mode at present, the long-term injecting pathway that adopts brings misery and inconvenience for patient's (removing the hemodialysis patient), penetrate administration if use atraumatic administering mode such as skin or mucous membrane instead, easily accepted, and enter the transformation period that to improve security and prolong drug in the body by skin by patient.For overcoming the above problems, utilization of the present invention can efficiently be carried protein molecule and be penetrated small molecules transduction peptide (the Protein Transduction Domain that microbial film comprises skin, mucous membrane, hemato encephalic barrier, PTD), prokaryotic expression recombinant GM-CSF is entered in the body by the atraumatic administering mode, thereby reach the purpose for the treatment of anaemia.

The transduction peptide is a kind ofly can efficiently pass biomembranous nexin transduction domain, and it can be striden the polypeptide covalently bound with it, protein and DNA equimolecular film and import nearly all tissue and cell, can also pass hemato encephalic barrier.Green in 1988 finds that the Tat albumen of HIV can free in and out cell efficiently.After Fawell in 1994 was linked to the regional chemistry of 36 Aa of TAT on the heterologous protein, crosslinking protein is also transducible went in the cell.People such as Schwarze SR with the PTD of TAT zone small peptide and macromole tilactase in prokaryotic cell prokaryocyte behind the amalgamation and expression, fusion rotein is injected in the mouse body, finding all has expression in activated enzyme molecule each tissue in vivo, particularly importantly can pass through hemato encephalic barrier.Except that tat peptide, transcribe the peptide sequence of the 43-58 position of factors A ntp from fruit bat feeler foot homology abnormal shape, 267-300 sequence from the conjugated protein VP22 of the single viral DNA of sore rash all has and on all four transduction function of tat peptide and transduction mechanism, and they are collectively referred to as PTD.

Summary of the invention:

One aspect of the invention relates to transduced peptide-humanized GM-CSF fusion rotein.Among the present invention, PTD sequence, the fruit bat homology abnormal shape that the transduction peptide of the described people source GM-CSF that is used to transduce can be selected from trans-activator TAT transcribed PTD sequence or its functional analogue or the fragment of factors A NTP and herpes simplex virus I-type VP22 transcription factor.Preferably, described transduction peptide has the described aminoacid sequence of SEQID NO.2 (YGRKKRRQRRR) or its functional fragment.In fact, as long as the transduction peptide of the people of the present invention source GM-CSF that can transduce all is applicable to the present invention.

Among the present invention, the people source GM-CSF of described fusion rotein contains aminoacid sequence or its functional fragment of SEQ ID NO:4 (APARSPSPSTQPW EHVNAIQEAR RLLNLSRDTA AEMNETVEVI SEMFDLQEPTCLQTRLELYK QGLRGSLTKL KGPLTMMASH YKQHCPPTPE TSCATQIITFESFKENLKDF LLVIPFDCWE PVQE).Preferably, people source granulocyte colony-stimulating factor is made up of SEQ ID NO:4.Transduced peptide-humanized GM-CSF fusion rotein of the present invention has the sequence (YGRKKRRQRRR GGS APARSPSPSTQPW EHVNAIQEAR RLLNLSRDTA AEMNETVEVI SEMFDLQEPTCLQTRLELYK QGLRGSLTKL KGPLTMMASH YKQHCPPTPE TSCATQIITFESFKENLKDF LLVIPFDCWE PVQE) of SEQ ID NO:6.

-GM-CSF,SEQ ID NO：5 ( TAC GGCCGC AAG AAA CGC CGC CAG CGC CGC CGC GGT GGTACC gcaccggcacgtagcccgag cccgagcacg cagccgtggg agcatgtgaa tgccatccaggaggcccgtc gtctgctgaa cctgagccgt gacaccgcgg cggagatgaatgaaaccgtg gaagtcatca gcgaaatgtt tgacctgcag gagccgacctgcctgcagac ccgcctggag ctgtacaagc agggcctgcg tggcagcctgaccaagctga agggcccgct gaccatgatg gccagccact acaagcagcactgcccgccg accccggaaa ccagctgtgc aacccagacc atcacctttgaaagcttcaa agagaacctg aaggactttc tgctggtcat cccgtttgactgctgggagc cggtccagga gtaa ) 。

Above-mentioned nucleotide sequence can insert in the expression vector.Preferably, expression vector of the present invention is protokaryon or carrier for expression of eukaryon, wherein is preferably pGEX, pET, pBV220 and peDNA.

The present invention among the pGEX-4T-1 etc., cultivates the people source GM-CSF that obtains with transduction peptide amalgamation and expression by described nucleic acid molecule is imported prokaryotic expression carrier pET28 by prokaryotic cell prokaryocyte.

The method easy handling, cost is low, transduction efficiency is high.Protein purification carries out under the sex change condition, and the purge process of the fusion rotein that exists with the inclusion body form is simplified greatly.Make the native conformation of metaprotein become chain-like structure relatively flexibly, break away from the restriction of space conformation, possess higher energy, transduction efficiency is improved greatly.After fusion rotein changes cell over to, folding again, recover native conformation and make GM-CSF bring into play its biologic activity, and the protein transduction domain covalently bound with it does not influence the normal configuration or the function of cell.

Another aspect of the invention relates to the pharmaceutical composition that contains fusion rotein of the present invention, described pharmaceutical composition is used for that malignant tumour is put, the leukopenia due to after the chemotherapy, there are the treatment of the low immunodeficiency diseases of white corpuscle in bone marrow transplantation, aplastic anemia and some, and wound healing, treatment such as antiviral, antimycotic.

Described pharmaceutical composition can contain non-injection type pharmaceutically acceptable carrier or vehicle commonly used.Preferably, formulation of the present invention is for can pass through all cytolemma and biomembranous formulations such as skin, mucous membrane, cornea, conjunctiva, serous coat, sarolemma, blood vessel and lymph periosteum, choroid, neu, hemato encephalic barrier.Particularly multiple mode medications such as sublingual administration, lung suction, nasal spray, anus bolt and skin absorption.

By fusion rotein of the present invention, can make clinical application safer, convenient people source GM-CSF input body through non-injecting pathway.

Embodiment

The extraction of the total RNA of embodiment 1 human peripheral blood mononuclear cell

Healthy people's anticoagulation cirumferential blood is isolated monocyte with lymphocyte separation medium.Containing 20% calf serum, 10 ^-8In the RPMI-1640 of mol/L TPA and 2ug/ul PHA, at 37 ℃ with contain 50%CO ₂The saturated humidity condition under cultivate 20h, collect adherent monocyte.Adopt the RNAgentsR Total RNA Isolation System of Promega company to extract total RNA.

The clone of embodiment 2 hGM-CSF cDNA

With reference to hGM-CSF cDNA sequences Design primer among the Genebank: front induced one restriction enzyme EcoR I site and initiator codon ATG, contain partial sequence in natural GM-CSFcDNA 3 ' the end non-coding region in the downstream primer, restriction enzyme BamH I site is introduced in the back, and its sequence is:

Primer 1:5 '-G GAATTC ATGGCA CCA GCT CGT TCT CCA-3 ' (SEQ IDNO:7)

Primer 2: 5 '-CT GGATCC CGATCGGGATCATGAGAGAG CAGCTC-3 ' (SEQID NO:8)

The RNA that obtains with embodiment 1 is as template, and reference reagent box manufacturer's recommended scheme is carried out the RT-PCR amplification, obtains the hGM-CSF cDNA of total length, 5 ' and 3 ' end carry EcoRI, BamH I site respectively.

Reverse transcription and pcr amplification: with the reverse transcription PCR test kit of Perkin ElmerCetus company production.Getting the cell total rna 1-2ug of extraction, add in the 20ul reverse transcription system, is primer with oligo dT, in 42 ℃ of effect 1h, synthetic cDNA first chain.99 ℃ of 5min stop reverse transcription reaction.20ul reverse transcription product is joined in the PCR reaction system of 100ul, wherein contain the upstream and downstream primer.Add the Taq archaeal dna polymerase behind 96 ℃ of sex change 10min, carry out the PCR circulation, reaction parameter is 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, and 72 ℃ are extended 1min, and 30 circulations are extended 7min for back 72 ℃ again.

Embodiment 3 reorganization hG-CSF expression plasmids make up and identify

The RT-PCR product of hGM-CSF with the expression vector pBV220 fragment that cuts back to close through same enzyme, connects by 4: 1 mol ratios behind EcoRI and BamHI double digestion, transformed competence colibacillus bacillus coli DH 5 alpha, screening positive recombinant pBV hGM-CSF.Serve the order-checking of Hai Shengong company.

The abduction delivering of embodiment 4 pBV hG-CSF in intestinal bacteria

Positive single colony inoculation of embodiment 3 screening of incubated overnight (is contained Amp 60mg/L) in the LB liquid nutrient medium, be cultured to OD in 30 ℃ ₆₀₀After=0.45, be warming up to 42 ℃ rapidly, induce about 5h, to OD ₆₀₀＞1.2.In 4 ℃ of centrifugal 10min of following 6000g, collect thalline.The SDS-PAGE electrophoresis is identified the expression level of rhGM-CSF, and identifies expression-form.

Renaturation and the purifying of embodiment 5 rhGM-CSF

With the thalline ultrasonic disintegration behind the above-mentioned abduction delivering.The centrifugal 20min of 12000g abandons supernatant.Precipitation is through washing, dissolving, and (3.6 * 100cm), elutriant is the TE damping fluid that contains 8mol/L urea, 1mmol/L DTT, flow velocity 0.5ml/min to Sephacryl S-200 post with sample on the solubilization of inclusion bodies liquid.It is that urea concentration remains on 1mol/L below the 100ug/ml that above-mentioned collection liquid is diluted to protein concentration with TE, adds 0.1mmol/L Sleep-promoting factor B (G-S-S-G), 1mmol/L reduced glutathion (GSH), 10 ℃ of renaturation 2 hours.With TE damping fluid balance QSepharose Fast Flow chromatography column (1.5 * 20cm).With sample on the renaturation solution, wait to pass after the peak, respectively with containing 0.3mol/L, 0.5mol/L, the TE wash-out of 1.0mol/L NaCl, flow velocity 1ml/min collects the purpose peak, and PBS is dialysed.

Embodiment 6 biological activity determinations

Adopt mouse leukemia cell strain NFS-60 dependency MTT assay method.NFS-60 is gone down to posterity in containing the perfect medium RPMI-1640 of 10ng/ml GM-CSF.Detect the same day, NFS-60 washes (the centrifugal 5min of 1000r/min) 3 times with RPMI-1640, is adjusted into 5 * 10 with the substratum that contains 15% serum ⁴/ ml.Sample is diluted to 10ng/ml, the equal doubling dilution of hGM-CSF standard substance and testing sample according to the measuring and calculating protein concentration.Negative control does not add GM-CSF.Cell is at 50%CO ₂, 37 ℃ of saturated humidity environment are cultivated 72h down.Add MTT colour developing liquid and lysate dimethyl sulfoxide (DMSO), survey the OD value with the 570nm wavelength filter.

Be calculated as follows rhGM-CSF activity unit:

The units of calculating every milligram of rhG-CSF according to protein concentration is specific activity (U/mg).

Sequence table

＜110〉Inst of Toxic Medicinal Materials, P.L.A. Academy of Military Medical Sciences

＜120〉transduced peptide-humanized granular leukocyte macrophage colony stimulating factor fusion protein and pharmaceutical composition thereof

<160>8

<210>I

<211>33

<212>DNA

＜213〉nucleotide sequence of coding PTD

<400>1

TACGGCCGCA?AGAAACGCCG?CCAGCGCCGC?CGC 33

<210>2

<211>11

＜212〉aminoacid sequence

＜213〉aminoacid sequence of PTD

<400>2

YGRKKRRQRR?R 11

<210>3

<211>384

<212>DNA

＜213〉nucleotide sequence of coding people source rHuGM-CSF

<400>3

gcaccggcac?gtagcccgag?cccgagcacg?cagccgtggg?agcatgtgaa?tgccatccag

gaggcccgtc?gtctgctgaa?cctgagccgt?gacaccgcgg?cggagatgaa?tgaaaccgtg

gaagtcatca?gcgaaatgtt?tgacctgcag?gagccgacct?gcctgcagac?ccgcctggag

ctgtacaagc?agggcctgcg?tggcagcctg?accaagctga?agggcccgct?gaccatgatg

gccagccact?acaagcagca?ctgcccgccg?accccggaaa?ccagctgtgc?aacccagacc

atcacctttg?aaagcttcaa?agagaacctg?aaggactttc?tgctggtcat?cccgtttgac

tgctgggagc?cggtccagga?gtaa 384

<210>4

<211>127

＜212〉aminoacid sequence

＜213〉aminoacid sequence of people source rHuGM-CSF

<400>4

APARSPSPST?QPWEHVNAIQ?EARRLLNLSR?DTAAEMNETV?EVISEMFDLQ?EPTCLQTRLE

LYKQGLRGSL?TKLKGPLTMM?ASHYKQHCPP?TPETSCATQT?ITFESFKENL?KDFLLVIPFD

CWEPVQE 127

<210>5

<211>426

<212>DNA

＜213〉nucleotide sequence of coding PTD-people source granular leukocyte macrophage colony stimulating factor fusion protein

<400>5

tacggccgca?agaaacgccg?ccagcgccgc?cgcggtggta?ccgcaccggc?acgtagcccg

agcccgagca?cgcagccgtg?ggagcatgtg?aatgccatcc?aggaggcccg?tcgtctgctg

aacctgagcc?gtgacaccgc?ggcggagatg?aatgaaaccg?tggaagtcat?cagcgaaatg

tttgacctgc?aggagccgac?ctgcctgcag?acccgcctgg?agctgtacaa?gcagggcctg

cgtggcagcc?tgaccaagct?gaagggcccg?ctgaccatga?tggccagcca?ctacaagcag

cactgcccgc?cgaccccgga?aaccagctgt?gcaacccaga?ccatcacctt?tgaaagcttc

aaagagaacc?tgaaggactt?tctgctggtc?atcccgtttg?actgctggga?gccggtccag

gagtaa 426

<210>6

<211>141

＜212〉aminoacid sequence

＜213〉aminoacid sequence of transduced peptide-humanized granular leukocyte macrophage colony stimulating factor

<400>6

YGRKKRRQRR?RGGSAPARSP?SPSTQPWEHV?NAIQEARRLL?NLSRDTAAEM?NETVEVISEM

FDLQEPTCLQ?TRLELYKQGL?RGSLTKLKGP?LTMMASHYKQ?HCPPTPETSC?ATQTITFESF

KENLKDFLLVIPFDCWEPVQE 141

<210>7

<211>28

<212>DNA

＜213〉primer

<400>7

6GAATTCATG?GCACCAGCTC?GTTCTCCA 28

<210>8

<211>34

<212>DNA

＜213〉primer

<400>8

CTGGATCCCG?ATCGGGATCA?TGAGAGAGCA?GCTC 34

Claims (12)

1. fusion rotein, it contains transduction peptide and human granulocyte macrophage colony stimulating factor.

2. PTD sequence, the fruit bat homology abnormal shape that the fusion rotein of claim 1, wherein said transduction peptide are selected from trans-activator TAT transcribed PTD sequence or its functional analogue or the fragment of factors A NTP and herpes simplex virus I-type VP22 transcription factor.

3. the fusion rotein of claim 2, wherein said transduction peptide has sequence or its functional fragment of SEQ ID NO:2.

4. each fusion rotein among the claim 1-3, wherein said human granulocyte macrophage colony stimulating factor has the sequence of SEQ ID NO:4.

5. the fusion rotein of claim 1, it has the sequence of SEQ ID NO:6.

6. nucleic acid molecule, each fusion rotein among its coding claim 1-5.

7. the nucleic acid molecule of claim 6, it has the sequence of SEQ ID NO:5.

8. the expression vector that comprises the nucleic acid molecule of claim 7.

9. pharmaceutical composition, it contains among the claim 1-5 each fusion rotein.

10. the pharmaceutical composition of claim 9, its formulation is for can pass through all cytolemma and biomembranous formulations such as skin, mucous membrane, cornea, conjunctiva, serous coat, sarolemma, blood vessel and lymph periosteum, choroid, neu, hemato encephalic barrier.

11. the pharmaceutical composition of claim 9, it is suitable for sublingual administration, lung suction, nasal spray, anus bolt and skin absorption medication.

12. the application of each fusion rotein in the preparation medicine among the claim 1-5, there are the low immunodeficiency diseases of white corpuscle in leukopenia due to described medicine is used for the treatment of behind the concurrent chemoradiotherapy of malignant tumor, bone marrow transplantation, aplastic anemia and some, and wound healing, antiviral, antifungal therapy.