CN105879053A - Use of CMTM1-V5 gene and its encoded protein - Google Patents

Abstract

The invention relates to a use of a CMTM1-V5 polypeptide or a CMTM1-V5 polypeptide encoding polynucleotide as a biological medicine in treatment on lymphocytic leukemia and other leukemia. The invention also relates to a use of the CMTM1-V5 polypeptide or the CMTM1-V5 polypeptide encoding polynucleotide in treatment on inflammation and autoimmune diseases through NFKB and NFAT activation inhibition. The invention also relates to a pharmaceutical composition for treating leukemia, inflammation and autoimmune diseases.

Description

CMTM1-V5 gene and the application of encoding proteins thereof

Technical field

The present invention relates to the new opplication of CMTM1-v5, specifically, the present invention relates to CMTM1-v5 polypeptide or encode its polynucleotide and prepare for treating leukemia, inflammation and the application of autoimmune disease.

Background technology

Lymphoma (lymphoma) is the malignant tumor of blood-lymphatic system, recently as social senilization and the deterioration of environment, lymphadenomatous sickness rate rises year by year, statistics according to U.S.'s tumor new cases in 2012, the annual morbidity of non-Hodgkin lymphoma exceedes the summation of whole blood malignant tumor, occupies the 7th in all tumors.The sickness rate of China's malignant lymphoma rises the most year by year, has become as one and threatens the important sick of national health to plant.Lymphadenomatous hypotype is numerous, and heterogeneity is relatively strong, and some patients is more sensitive to chemotherapy, but chemotherapeutics majority is cytotoxic drug, kills poor specificity, and through the research of nearly half a century, development space is limited.The nearly more than ten years are the targeted therapy epoch, perfect, targeting new drug and the application of hematopoietic stem cell transplantation along with chemotherapy system, lymphadenomatous raising evident in efficacy, some drugs such as Mabthera, Bortezomib has been achieved for good therapeutic effect in clinic, and within 5 years, survival rate is up to 60-70%；But still have some patients can produce drug resistance or recur after alleviation, become difficult point and the emphasis of clinical treatment.Therefore find specific target therapeutic agent still to shoulder heavy responsibilities.

CMTM1 (CKLF sample MARVEL wears spanning domain superfamily member 1, former name CKLFSF1) is that inventor utilizes bioinformatics method to clone the new gene obtained for 2004.CMTM1 is high expressed in Human Testis tissue, is substantially not detectable expression in other normal structures.The structure of this gene is extremely complex, and RT-PCR and cDNA clone order-checking find this gene at least 23 shearing variants, and being named as CMTM1-v1-v23, CMTM1-v5 respectively is one of them spliced body.About 600 bases of the full-length cDNA of CMTM1-v5, are sheared by the 1st, 6,7 exon of this gene and form, and CMTM1-v5 causes to transcribe owing to cDNA shears and stops in advance, 61 aminoacid of coding altogether.

Inventor utilizes the various functional gene Screening Platform discovery CMTM1-v5 activation to NF-κ B and NFAT in the Screening Platform of luciferase reporter gene to have the strongest inhibitory action, which results in our interest.Owing to lymphadenomatous pathogenesis is complicated, influence factor is many, and the signal transduction pathway of various kinds of cell all develops relevant with it, and wherein the overactivity of NF-κ B has played important function by inhibited apoptosis path in lymphadenomatous development.NF-κ B is transcription regulaton factor important in nucleus, is the important moderator of cell survival, cell cycle, cell adhesion and migration.NF-κ B can be with the expression of the regulating cell factor, chemotactic factor, adhesion molecule etc.；Such as IAPs, Bcl-2, Bcl-xL, c-myc, cyclin D1 etc., cell proliferation, inhibited apoptosis can be promoted with transcriptional activation apoptosis suppressive gene.Therefore, the abnormal activation of NF-κ B and the generation of Lymphocytic leukemia, development, drug resistance, recurrence are closely related.The activation of NF-κ B has two main signal paths, I-κ B upstream kinases molecule IKK (I-κ B Kinase) complex is key molecule therein: (1) is under some stimulus signal effect, such as virus infection etc., IKK activation in IKK complex, making I-κ B phosphorylation, the I-κ B of phosphorylation by proteasomal degradation, is made NF-κ B be released by further ubiquitination, (P65/P50) complex is indexed into core, activates numerous target gene.Some virus protein can make IKK keep the state of activation, causes effective phosphorylation and degraded I-κ B to make NF κ B continuous activation；(2) the non-classical approach of NF-kB activation is to be regulated and controled by NF-κ B inducible kinase NIK and IKK α, and is initiateed transcribing of downstream target gene by RelB/P52 complex nuclear translocation.Can reach to treat the purpose of tumor by the activity of suppression NF-κ B.Have now been found that and the compound of NF-kB activity can be suppressed to have more than 100 to plant, but these compounds non-specific suppression NF-κ B, therefore it is difficult to clinic.So, sight has been turned to searching endogenic NF-κ B Inhibitory molecules by researchers, it is desired to be able to develops and treats lymphadenomatous novel drugs.Bortezomib is a kind of protease inhibitor being applied to clinic in recent years, mainly suppress cell proliferation by stoping the degraded of I-κ B to block the activation of NF-κ B, cause apoptosis, play antitumor action, it also can make tumor cell more sensitive to chemotherapy, radiotherapy and immunization therapy, overcome the cell tolerance to medicine, the treatment of myeloma and part lymphoma hypotype shows preferable curative effect and clinical tolerability, but to how many lymphadenomatous unsatisfactory curative effects.Since CMTM1-v5 also has the strongest inhibitory action to the activation of NF-κ B and NEAT, getting a good chance of becoming the lymphadenomatous potential new drug for the treatment of, highly we further investigate.

In view of the foregoing, this seminar has carried out the functional study of system to CMTM1-v5.The function of CMTM1-v5 is have studied in various kinds of cell system is such as Jurkat, Raji, HeLa, PC3,293T, HEK293, result shows, in Jurkat and Raji cell, process LAN CMTM1-v5 can substantially suppress the multiplication effect of cell, there are quick, the serious phenomena of mortality in inducing cell, other cell lines are then without substantially changing, and prompting CMTM1-v5 inducing cell death has obvious cell-specific.The comparison having two path researchs in apoptosis is clear, and one is membrane receptor path, by TNF α and FASL equimolecular active cell apoptosis；Another is then the mitochondrial pathways, and multiple mitochondrial molecule is the important participant of this path.The multiple dead signal mitochondrion to be transferred to of cell also causes mitochondrial membrane permeability to increase, once mitochondrial membrane is impaired, the apoptosis inducing factor such as cytochrome C and AIF is from mitochondrial inner membrane gap release to endochylema, a series of proteolysis will be caused, discharge the cytochrome c to endochylema and can form complex with Apaf-1, activate caspase-9 and the caspase-3 molecule in downstream.And ultimately result in nuclear damage (such as DNA segment) and cell death.The most also it is found that the apoptosis pathway of endoplasmic reticulum.Further Mechanism Study finds that CMTM1-v5 can cause cell mitochondrial transmembrane potential to decline and cytochrome c discharges, pro-caspase-9 and pro-caspase-3 proenzyme reduces, and caspase-3 activates and the cutting of substrate PARP.This shows, CMTM1-V5 causes the apoptosis of the classical pathway of mitochondrion and the dependence of caspase molecule.

Cell membrane is made up of double-layer of lipoid, wherein inlays protein and glycoprotein, and in general albumen cannot pass through cell membrane.But some bigger albumen, cell can be entered by endocytosis.Such as HIV (human immunodeficiency virus) (HIV) transcription activating protein Tat, herpes simplex virus (HSV) Viral structural protein VP2 2 etc..These albumen have some general character: they are discharged into extracellular with a kind of non-classical secretory pathway (having the participation of secretion signal)；Be combined with target cell in specific receptor non-dependent mode；Typically having high alkalinity region, these regions show the ability being combined with polyanion, as heparin/Heparan sulfate, poly secrete sialic acid and nucleic acid.The approach of albumen, polypeptide and other macromolecules into cells is always the study hotspot of association area.The mechanism of endocytosis is complicated, and the endocytic pathway relied on including several different approach: clathrin (clathrin or clathrin) originates in plasma membrane and is coated with caving in of clathrin, is then assembled into the vesicle of clathrin parcel；The approach that non-clathrin relies on, the endocytosis mediated including lipid raft (Lipid Rafts) and caveolae (little cellar for storing things).

The endocytosis of Clathrin mediation is the main mechanism of a kind of cell membrane specific receptors internalization.Clathrin-coated pits participates in the ensuing degraded of receptor internalisation and recirculation, and the input of nutrition, the formation of synaptic vesicle.Lipid Rafts (lipid raft) and Caveolae functional similarity, be formed by Plasmalemma invagination rich in cholesterol and the region of the film of the anti-detergent of sphingomyelins, participate in signal transduction and the intracellular transport of Lipid Rafts correlation molecule.Caveolae is one group of membrane structure with Lipid Rafts characteristic, typical ampuliform membrane invagination.Caveolin-1 is the marker protein of caveolae.Methyl-B-cyclodextrin (M β CD) exhausts cholesterol, destroys Lipid Rafts.

In order to study whether CMTM1-v5 polypeptide can enter cells play effect, inventor has synthesized CMTM1-v5 polypeptide, purity ＞ more than 90%.Functional experiment proves that it has biologic activity.CMTM1-v5 polypeptide can enter Jurkat, Raji cell line with specificity, it is possible to causes apoptosis, this effect to have dose dependent.It is interesting that CMTM1-v5 can only enter lymphoma cell strain Jurkat, Raji cells play effect, it is impossible to enter the various kinds of cell such as cell strain such as Hela, 293T, PC3 in other tumors source, this entrance is described and to act on be all specific.Experiment in vivo also show this albumen and has preferable protective effect to mice.Experimental group is divided into basic, normal, high various dose group; CMTM1-v5 polypeptide is injected again after tail vein injection Raji cell; negative control group has injected injecting normal saline after Raji cell; positive controls injects amycin after having injected Raji cell; found that CMTM1-v5 polypeptide has extraordinary protective effect to mice; negative control group Mus is the most dead at 22 days; 60 days mouse of positive controls are the most dead; experimental group: low dose group death when nearly 30 days, middle dosage group when 60 days dead 3；High dose group did not had Died Of Disease when 60 days.Result explanation CMTM1-v5 polypeptide has extraordinary protective effect to the mice having injected lymphoma cell.

Summary of the invention

It is an object of the present invention to provide CMTM1-v5 polypeptide or encode its polynucleotide in treatment Lymphocytic leukemia, the application in inflammation and autoimmune disease.

In an aspect, the invention provides CMTM1-v5 polypeptide or encode its polynucleotide and preparing the purposes of medicament for treatment of leukemia.

In one aspect of the method, the invention provides CMTM1-v5 polypeptide or encode its polynucleotide in preparation purposes in the medicament of inflammation (including acute inflammation and chronic inflammatory disease) damage.

In a further aspect, the invention provides CMTM1-v5 polypeptide or encode its polynucleotide in preparation for the purposes treating a kind of medicine of autoimmune disease.

The invention still further relates to a kind of for treating leukemia and treating leukemic pharmaceutical composition, it comprises the CMTM1-v5 polypeptide selected from antibiotics and antimetabolitas or the chemotherapeutic of its combination in any and enhanced sensitivity effective dose of therapeutically effective amount.

In one embodiment, described CMTM1-v5 polypeptide is selected from lower group: (1) has the polypeptide of aminoacid sequence shown in SEQ ID NO:2；Or (2) comprise the polypeptide of the aminoacid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 97% homology with aminoacid sequence described in SEQ ID NO:2, it has the essentially identical biological function of polypeptide described with (1) or activity；Or functional fragment, variant, analog or the derivant of (1) or (2) described polypeptide, they have the biological function essentially identical with (1) or (2) described polypeptide or activity.

In another embodiment, described polynucleotide include: the nucleotide sequence of aminoacid sequence shown in (a) coding SEQ ID No:1；(b) under strict conditions with the nucleotide sequence of the nucleotide sequence hybridization described in (a)；Or (c) has the conforming nucleotide sequence of at least 70%, 75%, 80%, 85%, 90%, 95% or 97% with nucleotide sequence shown in (a).

In the present invention, the biological function of described polypeptide or activity such as but not limited to, as described herein, inducing leukemia cell/apoptosis of tumor cells, antiinflammatory, treatment autoimmune disease.

Accompanying drawing explanation

Accompanying drawing 1, in-vitro transfection CMTM1-v5 can suppress the growth of Jurkat and Raji cell

Normal Jurkat and the Raji cell cultivated, uses electric shocking method transient transfection CMTM1-v5, pCDB and Bax eukaryon expression plasmid, uses the situation of mtt assay detection cell growth.As seen in figure la and lb, both results are consistent, and i.e. compared with empty vector control group, the growth of Jurkat and the Raji cell of overexpression CMTM1-v5 is significantly suppressed, and cell quantity elapses the most in time and increases.Utilizing cell viability calculating instrument that cell carries out tire and expect that blue coloration result displays that, the Cell viability of the Jurkat cell of overexpression CMTM1-v5 is decreased obviously, such as Fig. 1 c.

Accompanying drawing 2, in-vitro transfection CMTM1-v5 can induce Jurkat and Raji apoptosis

Annexin-V can with film outside PS specific binding, utilize fluorescein-labeled Annexin-V to combine Fluorescein activated cell sorter and cell PS can be turned up and quantitative determine, judge the integrity of cell membrane simultaneously with propidium iodide (PI) dyeing.As in figure 2 it is shown, abscissa represents the fluorescence intensity of FITC-Annexin-V, vertical coordinate represents the fluorescence intensity of PI, and lower left region represents normal cell, and bottom right, upper right, top left region represent early apoptosis, middle and advanced stage apoptosis and the cell of death respectively.Jurkat cell transient transfection pCDB, pCDB-CMTM1-v5 and Bax plasmid, about 8 hours after transfection, CMTM1-v5 group PI-Annexin V is mono-positive and the double positive, and cell proportion was i.e. many than matched group, elapses over time, CMTM1-v5 group cell death is more and more obvious, reaches 45%；Similar result have also been obtained confirmation in Raji cell, the apoptosis that CMTM1-v5 is induced occur very early and substantially.

Accompanying drawing 3, CMTM1-v5 process LAN inducing cell mitochondrial transmembrane potentials (Δ ψ m) decline

We use DIOC6 (3) dyestuff to the Jurkat cell of transfection CMTM1-v5, the change situation of Δ ψ m in conjunction with flow cytometry.Result is as it is shown on figure 3, process LAN CMTM1-v5 and Bax can substantially induce Δ ψ m to decline.This shows that mitochondrion sustains damage during the apoptosis of CMTM1-v5 induction.

Accompanying drawing 4, in Jurkat cell, process LAN CMTM1-v5 causes cytochrome C to be discharged into endochylema from mitochondrion

We utilize Western blot to have detected the release of cytochrome C.Endochylema and mitochondrial protein is extracted respectively after Jurkat cell transfection CMTM1-v5, (Fig. 4) display process LAN CMTM1-v5 group is compared with empty carrier group, in cytoplasmic components, the content of cytochrome C substantially increases, illustrate that cytochrome C intermembrane space inside and outside mitochondrion is discharged into cytoplasm, and and Caspase-9, Apaf-1 equimolecular combines, and forms complex, activates the caspase-3 molecule in downstream and participates in the signal transduction of apoptosis process.

Accompanying drawing 5, in-vitro transfection CMTM1-v5 can activate caspases

After Jurkat and Raji cell transfecting 12 hours, harvesting extracted albumen, carried out DEVDase activity (i.e. caspase-3 activity) detection.Its fluorogenic substrate Ac-DEVD-AMC is cut by activation caspase-3, discharge free AMC and can be inspired fluorescence by spectrofluorophotometer, strong and weak by fluorescence in detection reaction system, can determine whether out the power of caspase-3 enzymatic activity, thus react the Activation of caspase-3.Fig. 5 a result shows, compared with empty carrier group, when apoptosis occurs comparing in early days (12h after transfection), CMTM1-v5 group and Bax group can cause intracellular caspase-3 activity significantly raised (Jurkat cell 7 times, in Raji cell 5 times).

Western blot result (Fig. 5 b) display process LAN CMTM1-v5 can make caspase-3, caspase-9, caspase-12 proenzyme content reduce, and i.e. shows all to be activated.Caspases activation can also cut specific substrate, thus causes dysfunction or the structural damage of cell.The cutting situation of one of our substrate being detected simultaneously by caspase-3 PARP.In the most visible PARP cutting of CMTM1-v5 process LAN group and Bax group, the small fragment PARP content after cutting increases than empty carrier group.

The experiment of accompanying drawing 6, luciferase reporter gene proves that transfection CMTM1-v5 can suppress NF-AT signal path

We compare CMTM1-v5 and the CAML interaction impact on NFAT signal path to utilize dual-luciferase reporter system.After result display PMA+Ionomycin stimulates, blank pcDB group uciferase activity adds about 4 times, illustrates that cell has been fully activated.And compared with the control, process LAN CMTM1-v5 can suppress not add uciferase activity (Fig. 6 a) after stimulation (endogenous) and stimulation.The cell fluorescence element enzymatic activity of process LAN CAML group is the most significantly raised, and this is also consistent with document report.During coexpression CMTM1-v5 and CAML, the depression effect of NFAT is then reversed (Fig. 6 b) by CMTM1-v5.And the activity as the renilla luciferase of internal reference does not change significantly (data do not show), illustrate that weakening of this luciferase signal is special, be not that the dosage effect of the cellulotoxic effect by overexpression CMTM1-v5 or two kinds of plasmids of corotation causes.

The experiment of Fig. 7, luciferase reporter gene proves that transfection CMTM1-v5 can suppress the activation of NF-κ B

293T cell routine Secondary Culture contains in the DMEM of 10%FBS again, with 0.25% pancreatin+EDTA peptic cell, spreads 96 porocyte culture plates by 1.5 × 105 cells/ml, and cultivating 24 hours ionomycin with 100ng/ml PMA and 1nM after transfection is stimulus object.Stimulating 6-8 hour harvesting, every hole adds 30 μ l 1 × 1PLB cell pyrolysis liquids, and each sample sets 6 multiple holes, and wherein 3 holes add pNF-kB-Luc reporter plasmid, and remaining 3 hole adds pLuc-myc reporter plasmid.Negative control is pcDNA3.1 empty carrier, activates and the positive control that suppresses is respectively IkkB and IkB.Luciferase reporter gene result shows, overexpression CMTM1-v5 and v5-N end, and v5-C end can suppress the activation of NF κ B.P+I represents with 100ng/ μ l PMA and 1nM ionomycin as stimulus object.BLANK represents and does not adds stimulus object, and result prompting CMTM1-v5 can substantially suppress the activation of NF-κ B signal Signal Transduction Pathways.

Accompanying drawing 8, yeast two-hybrid and co-immunoprecipitation checking CMTM1-v5 interact with CAML in cell

We have arrived the interaction target molecule of CMTM1-v5 by yeast two-hybrid screening.CMTM1-v5 and CAML has interaction.In order to verify the interaction of the two albumen further in mammalian cell, we utilize co-immunoprecipitation experiment to verify.Result shows, by IgG and GST antibody I P, then use HA antibody test, on the position identical with INPUT GST-CMTM-v5, can occur in that corresponding band (Fig. 8), prompting CAML and CMTM1-v5 can be combined with each other under intracellular environment in GST antibody I P group.

Accompanying drawing 9, CMTM1-v5 Yu CAML are total to positioning analysis in HeLa cell

We use two kinds of molecules of confocal laser scanning microscope in the situation of intracellular targeting.CMTM1-v5 Yu CAML of process LAN is all located in cytoplasm, and in the point-like gathering of core week, both distributions are the most completely overlapped, and prompting CMTM1-v5 Yu CAML is in intracellular location altogether.

Accompanying drawing 10, in Jurkat cell coexpression CAML Yu CMTM1-v5 on apoptotic impact

We, by the eukaryon expression plasmid cotransfection of CMTM1-v5, CAML to Jurkat cell, detect apoptotic situation.When CAML Yu CMTM1-v5 transfects jointly to Jurkat cell, the apoptosis of CMTM1-v5 induction has significantly been restrained (cell of the double dye of AV+PI is reduced to 17.50%) by 31.27%.CAML Yu CMTM1-v5 transfects jointly can affect the apoptosis that CMTM1-v5 is induced really.

Capillary electrophoresis technique detection FITC labelling CMTM1-v5 polypeptide

With FITC labelling CMTM1-v5 polypeptide, capillary electrophoresis technique after gel-purified, is utilized to be detected, it was demonstrated that labelling success.

The CMTM1-v5 polypeptide of labelling can enter lymphoma cell

Observing under laser confocal microscope, fluorescently-labeled CMTM1-v5 polypeptide can enter Raji cell, can enter cell, but be mainly attached to around cell membrane to enter cell when 4 DEG C when 37 DEG C.

Accompanying drawing 11, flow cytometry and laser confocal microscope find that the CMTM1-v5 polypeptide of labelling can enter raji and jurkat cell.

The albumen of labelling is added to cultivate in cell, flow cytomery, and result proves compared with matched group, and experimental group cell average fluorescent strength adds more than 7 times.

Accompanying drawing 12, use CMTM1-v5 polypeptide can induction of lymphocyte Leukemia Cell Lines raji and jurkat apoptosis

CMTM1-v5 polypeptide can induce Raji apoptosis, and has obvious dose-dependence.CMTM1-v5 polypeptide is added in Raji cell, within 24,48 hours, can cause Raji apoptosis, and along with the increase of protein concentration, apoptotic cell ratio dramatically increases, and has obvious dose-dependence.

Accompanying drawing 13, CMTM1-v5 polypeptide can be obviously prolonged the life cycle having injected raji cell SCID mice in vivo

Experiment in vivo result: the negative control group of injecting normal saline: mice was the most dead in 22 days；The positive controls of injection chemotherapeutic amycin: mice was the most dead in 58 days；CMTM1-v5 polypeptide low dose group: mice was the most dead in 28 days；Dosage group in CMTM1-v5 polypeptide: 58 days 3 dead mouses；CMTM1-v5 polypeptide high dose group: 58 days without mice Died Of Disease；Result proves that CMTM1-v5 polypeptide middle and high dosage group can clearly protect the mice having injected lymphoma cell, extends life cycle.

Specific embodiment

In the present invention, CMTM1-v5 includes CMTM1-v5 gene or CMTM1-v5 polypeptide.Described CMTM1-v5 polypeptide is the aminoacid sequence as shown in SEQ ID N0:2, and it encodes 61 aminoacid.Described CMTM1-v5 gene is the polynucleotide sequence of the SEQ ID NO:2 of code book invention, it can be for amino acid whose coded sequence shown in SEQ ID NO:2, or in addition to the coded sequence of above-mentioned aminoacid sequence, non-coding sequence, the non-coding sequence etc. that such as intron, coded sequence 5 ' or 3 ' are held can also be included.Described polynucleotide sequence can be DNA or RNA, and wherein DNA includes the DNA of cDNA, genomic DNA and synthesis.Wherein preferably gene is the polynucleotide sequence shown in SEQ ID NO:1, and 603 nucleotide of this sequence, coded sequence is 1-186 position nucleotide (the CDS: the 1-183 position nucleotide).Or, the nucleotide sequence of a kind of separation, it comprises coding CMTM1-v5 protein sequence, the such as coded sequence shown in SEQ ID NO:1: nucleotide 1-186.Those of ordinary skill in the art are it is known that the nucleotide sequence of CMTM1-v5 of the present invention can be entirely identical to the coded sequence as shown in SEQ ID NO:1, it is also possible to due to the degeneracy of genetic code, be not exclusively equal to the coded sequence of above-mentioned nucleotide.

The CMTM1-v5 polynucleotide sequence of the present invention can with the PCR amplification technique of establishing criteria by cDNA, mRNA or genomic DNA as template, and choose the amplification of suitable oligonucleotide primers and obtain.The nucleotide so obtained can be cloned in suitable carrier, and carries out sequence description by DNA analysis technology.Can also be obtained by standard DNA synthesis technique.The CMTM1-v5 albumen of the present invention can be obtained by conventional method, such as by transformed host cell and after transfected host cell growth to suitable cell density, with suitable method (such as temperature change or chemistry speciality induction) evoked promoter, then proceed to cultivate.After cultivation completes, available centrifuging collection cell, and by any known method, such as freeze-thaw method, Ultrasonic treatment, lysozyme dissolution method or mechanical crushing method smudge cells.Can reclaim from host cell cultures and the CMTM1-v5 albumen of the purification present invention by various known methods, these methods include ammonium sulfate or ethanol precipitation, acid extraction method, ultrafiltration, ion exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography method, gel filtration, affinity chromatography and high pressure fluid column chromatography.Can also be transformed in escherichia coli by prokaryotic expression carrier, by suitable method (as temperature change or chemistry speciality are induced) evoked promoter, then proceed to cultivate.After cultivation completes, available centrifuging collection cell, and by any known method, such as freeze-thaw method, Ultrasonic treatment, lysozyme dissolution method or mechanical crushing method smudge cells.Can reclaim from host cell cultures and the CMTM1-v5 albumen of the purification present invention by various known methods, these methods include ammonium sulfate or ethanol precipitation, acid extraction method, ultrafiltration, ion exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography method, gel filtration, affinity chromatography and high pressure fluid column chromatography.

The CMTM1-v5 of the present invention can also pass through the carrier mammalian cell-infectings such as slow virus, adenovirus, intracellular and internal play a role.

The CMTM1-v5 polypeptide of the present invention can also be obtained by the method synthesis of chemistry.

In an embodiment of the invention, CMTM1-v5 can suppress NFKB and NFAT signal path, therefore can suppress diseases associated with inflammation and autoimmune disease.

According to a further aspect in the invention, the present invention also provides for one and treats leukemic pharmaceutical composition.This pharmaceutical composition comprises the recombiant protein of CMTM1-v5 or the full-length polypeptide of synthesis.One or more pharmaceutically acceptable carriers or excipient can also be comprised.Described carrier or excipient can include normal saline, isotonic glucose solution, buffer saline, glycerol, ethanol and the combination of above-mentioned solution.Described pharmaceutical composition can also comprise penetration enhancer, antioxidant and/or protease inhibitor etc. further.

For therapeutic purposes, described pharmaceutical composition can use suitable route of administration to use.Described pharmaceutical composition can be made various dosage form, such as solution, Liposomal formulation, polymer formulations, microcapsule and other slow releasing preparation etc..

The pharmaceutical composition of the present invention can such as pass through intravenous injection with Formulations for systemic administration；Or topical, such as to suffering from cancer position local injection.The suitableeest dosage will be according to the state of an illness of patient, age, sex or body weight, and depending on administering mode and required effect, those skilled in the art can just can determine that optimal method of application in its ken without creative work.

Below with reference to embodiment, the present invention will be further elaborated.

One aspect of the present invention provides CMTM1-v5 polypeptide or encodes its polynucleotide and preparing the purposes of medicament for treatment of leukemia.In some embodiments, described CMTM1-v5 polypeptide or encode in described biologically active drug is delivered to tumor cell by its polynucleotide and induce its apoptosis.In other embodiments, described CMTM1-v5 polypeptide or encode its polynucleotide by suppression NFAT and NFKB path suppression inflammation and autoimmune disease.

One aspect of the present invention provides and uses CMTM1-v5 polypeptide or encode its leukemic method of polynucleotide therapy, and described method includes the CMTM1-v5 polypeptide to experimenter's administering therapeutic effective dose or encodes its polynucleotide.In some embodiments, described CMTM1-v5 polypeptide or encode in biologically active drug is delivered to cancer cell by its polynucleotide and realize suppression growth of tumour cell.In other embodiment, described polynucleotide can be various ways, includes but not limited to the forms such as cloning vehicle, expression vector, adenovirus, slow virus, plasmid, phasmid, cosmid, naked DNA.In other embodiments, described CMTM1-v5 polypeptide or encode its polynucleotide and realize suppression tumor growth by apoptosis-induced.In other embodiment, treatment leukemia is realized by both the above mode.Preferably, described method is implemented in vivo or in vitro.

An additional aspect of the present invention provides to use CMTM1-v5 polypeptide in subject or encode its polynucleotide and can suppress inflammation and autoimmune disease.Described method includes using the CMTM1-v5 polypeptide of effective dose to experimenter or encoding its polynucleotide.

An additional aspect of the present invention provides a kind of for treating leukemic pharmaceutical composition, its CMTM1-v5 polypeptide comprising therapeutically effective amount and chemotherapeutics.

In some embodiments of the present invention, described CMTM1-v5 polypeptide is selected from lower group: (1) has the polypeptide of aminoacid sequence shown in SEQ ID NO:2；Or (2) comprise the polypeptide of the aminoacid sequence having at least 70% homology with aminoacid sequence described in SEQ ID NO:2, it has the essentially identical biological function of polypeptide described with (1) or activity；Or the functional fragment of (1) and (2) described polypeptide, variant, sum analogous to general Dedekind sum, they have the biological function essentially identical with (1) or (2) described polypeptide or activity；Wherein said polynucleotide include: (a) encodes the polynucleotide of the CMTM1-v5 polypeptide described in above-mentioned (1), (2) or (3)；(b) under strict conditions with the multi-nucleotide hybrid described in (a) have at least 70% conforming polynucleotide with it；(c) polynucleotide passage of (a) or (b) described polynucleotide is comprised.

Preferably, in some embodiments of the present invention, described CMTM1-v5 polypeptide comprises has at least 75% with aminoacid sequence described in SEQ ID NO:2, preferably at least 80%, more preferably at least 85%, still more preferably at least 90%, even more preferably at least 95%, the aminoacid sequence of most preferably at least 97% homology.

Preferably, in some embodiments of the present invention, the polynucleotide encoding described CMTM1-v5 polypeptide have and nucleotide sequence at least 70% shown in SEQ IDNO:1, preferably at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 95%, even at least 97% conforming sequence, the most wherein said polynucleotide have nucleotide sequence shown in SEQ ID NO:1.

The present inventor enters cell through research display people's CMTM1-v5 polypeptide and shows the specificity of cell type, in the cell of checking at present, mainly entering lymphocytic leukemia cell, such as Jurkat T lymphoma cell, Raji B lymphoma cell all shows the ability entering cell.And to the such as HeLa of attached cell the most after testing, HT-29, A549, PC-3 then can not enter.Process LAN CMTM1-v5 energy inducing cell apoptosis, the many Toplink of exogenous human CMTM1-v5 special entrance target cell, this characteristic that CMTM1-v5 polypeptide possesses has important function at treatment Lymphocytic leukemia.

As used herein, term " experimenter " refers to mammal, such as the mankind, it may also be other animal, such as domestic animal (such as Canis familiaris L., cat etc.), domestic animal (such as cattle, sheep, pig, horse etc.) or laboratory animal (such as monkey, rat, mice, rabbit, Cavia porcellus etc.).

As used herein, term " polypeptide " refers to by the interconnective a string at least two amino acid residue of covalent bond (such as peptide bond), can be recombinant polypeptide, natural polypeptides or synthesis polypeptide.

As used herein, the functional fragment of CMTM1-v5 polypeptide, variant, derivant and analog may is that one or more amino acid residue is guarded or nonconservative amino acid residue replaces (preferably conservative amino acid residue), and substituted amino acid residue yes or no is by the aminoacid coded by genetic code；One or more amino acid residues are with substituted radical；Mature polypeptide merges with another compound；One or more other aminoacid blend with mature polypeptide, such as help sequence or the proprotein sequence of Purified mature albumen；At one end or two ends or internal insert and/or add and/or lack one or more aminoacid.In terms of these disclosures, in such fragment, derivant and analog believe the ken being in those skilled in the art.

As it is used herein, term " concordance ", " Percent Identity ", " homology " or " homogeneity " refers to the sequence iden between two aminoacid sequences or between nucleotide sequence.Can determine that Percent Identity, Percent Identity refer to that compared sequence has the quantity of position identical residue (i.e. aminoacid or nucleotide) by two sequences of comparison.Can use this area canonical algorithm (such as Smith and Waterman, 1981, Adv.Appl.Math.2:482；Needleman and Wunsch, 1970, J.MoI.Biol.48:443；Pearson and Lipman, 1988, Proc.Natl.Acad.Sci., USA, 85:2444) or by computerization version (Wisconsin Genetics Software Package Release 7.0, the Genetics Computer Group of these algorithms, 575Science Drive, Madison, WI) carry out sequence alignment and compare, described computerization version is publicly available for BLAST with FASTA.It addition, can be used for gene comparision by NIH (Bethesda MD) available ENTREZ.When using BLAST and Gapped BLAST programs, the default parameter of each program (such as BLASTN, available on the internet site at American National Biotechnology Information center) can be used.In one embodiment, the GCG that Gap Weight can be used to be 1 determines that the percentage ratio of two sequences is with all one's life so that each aminoacid breach give weight as it be that the monamino acid between two sequences does not mates.Or, ALIGN program (2.0 editions) can be used, it is a part for GCG (Accelrys, San Diego, CA) sequence alignment program bag.

" hybridize " and refer to that two single stranded polynucleotide Non-covalent binding are to form the process stablizing double-stranded polynucleotide.Term " hybridizes " can also refer to three chain hybridization.The polynucleotide of produced (typically) double-strand are " crossbred " or " duplex "." hybridization conditions " generally comprises below about 1M, more typically less than about 500mM and the salinity of below about 200mM.Hybridization temperature can as little as 5 DEG C, but usually above about 22 DEG C, the most greater than about 30 DEG C, preferably above about 37 DEG C.Hybridization is generally carried out under stringent condition (condition that i.e. probe can hybridize with its target sequence).Stringent hybridization condition depends on sequence, and there are differences in different situations.Longer fragment may need higher hybridization temperature to carry out specific hybrid.Owing to other factors (including base composition and length, the existence of organic solvent and the base mispairing degree of complementary strand) may affect the stringency of hybridization, therefore the combination of parameter is more even more important than the absolute value of any one individual parameter.It is said that in general, stringent condition is chosen to lower than the Tm of this particular sequence under the ionic strength determined and pH about 5 DEG C.Exemplary stringent condition includes the Na ion concentration (or other salt) of pH7.0 to 8.3 time at least 0.01M to not higher than 1M and the temperature of at least 25 DEG C.For stringent condition, refering to such as Sambrook, Fritsche and Maniatis. " Molecular Cloning A laboratory Manual " second edition Cold Spring Harbor Press (1989) and Anderson " the Nucleic Acid Hybridization " first edition, BIOS Scientific Publishers Limited (1999), they are all integrally incorporated herein by reference for all above-mentioned purposes.

Pharmaceutical composition

Present invention also offers the pharmaceutical composition comprising CMTM1-v5 polypeptide (or nucleic acid of coding CMTM1-v5 polypeptide).In addition to the active component, according to the present invention and pharmaceutical composition used according to the invention can comprise pharmaceutical acceptable excipient, carrier, buffer agent, stabilizer or other well known to a person skilled in the art material.These materials should be atoxic, and should not disturb the curative effect of described active component.The definite character of described carrier or other material can depend on route of administration, and described approach can be such as per os, intravenous or local.

Described preparation can be liquid, such as, contain physiological solt solution or the freeze-dried powder of the non-phosphate buffer of pH6.8-7.6.

Dosage

CMTM1-v5 polypeptide as herein described is preferably administered to individuality with " therapeutically effective amount " or " effective dose ".Described compositions is preferably administered to individuality, described therapeutically effective amount or enhanced sensitivity effective dose with " therapeutically effective amount " or effective dose be enough to show its benefit for described individuality.The actual amount used, and the speed used and time course can depend on own situation and the order of severity of institute's therapist.The prescription (the such as decision etc. to dosage) for the treatment of is finally general practitioner and the responsibility of other doctor and relies on it and make a decision, and generally considers the disease treated, the situation of individual patients, site of delivery, application process and other factors known to for doctor.

In some embodiments, the dosage range of CMTM1-v5 polypeptide can be 80mg/kg/4 days to 160mg/kg/4 days.

Use

Polypeptide in the present invention is administered alone, and it generally includes according to the drug excipient being suitable for selected by the planned manner used, diluent or carrier.Polypeptide can by any applicable by the way of be applicable to treatment patient.Accurate metering will depend upon which multiple factor, including the accurate character of this polypeptide.

In some embodiments of the present invention, CMTM1-v5 polypeptide and chemotherapeutics separate administration.In other embodiments, CMTM1-v5 polypeptide is used with chemotherapeutic drug combination.

Some suitable occupation modes include, but is not limited to be administered orally, rectum, nose, locally (include oral cavity and Sublingual), subcutaneous, vagina or parenteral (in including subcutaneous, muscle, vein, Intradermal, sheath and exterior dura) use.

For intravenous injection and the injection in ailing position, the aqueous solution form that active component will accept for a kind of parenteral, it is without thermal source and has suitable pH value, isotonia and stability.

In this area, person skilled uses, such as: isotonic vehicle such as sodium chloride injection, ringer's injection, lactated ringer's injection, it is possible to the solution that well preparation is suitable for.As requested, preservative, stabilizer, buffer agent, antioxidant and/or some other additive can be added.Orally administered pharmaceutical composition can be to be the forms such as tablet, capsule, powder or oral liquid.Tablet can include solid carrier, such as gelatin or adjuvant.Composition of liquid medicine generally includes liquid-carrier, such as water, oil, animal or plant oil, mineral oil or artificial oil.Normal saline solution, glucose or other sugar juice or glycols can also be included, such as ethylene glycol, propylene glycol or Polyethylene Glycol.

Above mentioned technology and the example of scheme and some other can find in 1980. at Remington ' s Pharmaceutical Sciences, 16th edition, Oslo, A. (ed) according to technology used in the present invention and scheme.

Embodiment 1, overexpression CMTM1-v5 can suppress the growth of Jurkat and Raji cell

The propagation mtt assay of Jurkat and the Raji cell of overexpression CMTM1-v5 and cell viability calculating instrument measure.

MTT is faint yellow methyl tetrazole salt, and the mitochondrial dehydrogenase of living cells can produce blue crystalloid first with MTT, and first is dissolved through dimethyl sulfoxide (DMSO) can carry out colorimetric.The amount that first generates is directly proportional to the metabolism power of living cells, and only living cells has this to react.Result microplate reader is measured, can be according to the power of the depth reflection cell metabolic activity of colour developing.According to the density of 2000 cells/well, the cell after transfection is inoculated in 96 orifice plates, often group cell sets 5 parallel multiple holes, cultivate 6 days continuously, every 24 hours addition MTT, 37 DEG C, after 5%CO2 hatches 4-6 hour, add cell pyrolysis liquid, second day measures optical density value in ELISA Reader 570nm, draws the growth curve of cell, the proliferation activity of reflection cell after different groups, each time point result being added up.

Cell viability counts: the permeability of trypan blue cell is raised by dead cell, can be dyed to blueness, and living cells then refuses dye.Utilize Vi-CELLXRTM cell viability calculating instrument that the cell of different time points is detected, cell size, living cells proportion and total viable count can be shown, thus draw cell growth curve.Jurkat and the Raji cell of transfection CMTM1-v5 and control plasmid is inoculated in 12-well Tissue Culture Plate according to the density of 2x105cells/well, cultivates 6 days continuously.Within every 24 hours, take machine testing on cell, and draw cell growth curve analysis cell proliferative condition.

Normal Jurkat and the Raji cell cultivated, uses electric shocking method transient transfection CMTM1-v5, pcDB and Bax eukaryon expression plasmid, uses the situation of mtt assay detection cell growth.Both results are consistent, and i.e. compared with empty vector control group, the growth of Jurkat and the Raji cell of overexpression CMTM1-v5 is significantly suppressed, and cell quantity elapses the most in time and increases.Utilizing cell viability calculating instrument that cell carries out tire and expect that blue coloration result displays that, the Cell viability of the Jurkat cell of overexpression CMTM1-v5 is decreased obviously (Fig. 1).

Embodiment 2, overexpression CMTM1-v5 can induce Jurkat and Raji apoptosis

It is one of typical Biochemical Characteristics of apoptosis that Phosphatidylserine (PS) turns to the outer surface of cell membrane from the endochylema side of cell membrane.Annexin-V can with film outside PS specific binding, utilize fluorescein-labeled Annexin-V to combine Fluorescein activated cell sorter and cell PS can be turned up and quantitative determine, judge the integrity of cell membrane simultaneously with propidium iodide (PI) dyeing.As shown in (Fig. 2), abscissa represents the fluorescence intensity of FITC-Annexin-V, vertical coordinate represents the fluorescence intensity of PI, and lower left region represents normal cell, and bottom right, upper right, top left region represent early apoptosis, middle and advanced stage apoptosis and the cell of death respectively.Jurkat cell transient transfection pcDB, pcDB-CMTM1-v5 and Bax plasmid, about 8 hours after transfection, CMTM1-v5 group PI-Annexin V is mono-positive and the double positive, and cell proportion was i.e. many than matched group, elapses over time, CMTM1-v5 group cell death is more and more obvious, reaches 45%；Similar result have also been obtained confirmation in Raji cell, the apoptosis that CMTM1-v5 is induced occur very early and substantially.

Embodiment 3, CMTM1-v5 overexpression inducing mitochondrial transmembrane potential (Δ ψ m) decline

Mitochondrion is one of key regulatory person of apoptosis pathway, and mitochondrial transmembrane potentials (Δ ψ m) is impaired is one of the biochemical character of apoptosis earliest period of mitochondrion mediation.In order to determine the participation of apoptosis that CMTM1-v5 mediates whether mitochondrial, we use DIOC6 (3) dyestuff to the Jurkat cell of transfection CMTM1-v5, the change situation of Δ ψ m in conjunction with flow cytometry.After transfection, different time points harvesting is in centrifuge tube, and 1500rpm is centrifuged 5 minutes, abandons supernatant；PBS washed cell 2 times, is resuspended in 400l PBS, prepares single cell suspension.Add the DiOC6 (3) of 20nM, after 37 DEG C of lucifuges hatch 15 minutes, carry out flow cytometry immediately.Gather in the crops 1 × 104 cell, calculate the cell percentages that transmembrane potential reduces.Process LAN CMTM1-v5 and Bax can substantially induce Δ ψ m to decline.This shows to sustain damage at the apoptosis process Mitochondria of CMTM1-v5 induction.

Embodiment 4, CMTM1-v5 overexpression induced cytochrome C are discharged into endochylema from mitochondrion

Mitochondrion Δ ψ m decline makes mitochondrial membrane permeability change, and makes mitochondrion PTP open and discharges some Apoptosis-Related Factors in kytoplasm, such as cytochrome C.It is the key link starting endogenous apoptosis path that cytochrome C is discharged into kytoplasm from mitochondrion.Under normal circumstances, cytochrome C is positioned intermembrane space inside and outside mitochondrion；When the apoptotic program that mitochondrion participates in is activated, outer membrane permeability increases, cytochrome C intermembrane space inside and outside mitochondrion is discharged into cytoplasm, and and Caspase-9, Apaf-1 equimolecular combines, and forms complex, activates the caspase-3 molecule in downstream and participates in the signal transduction (22 of apoptosis process, 23,24).We utilize Western blot to have detected the release conditions of cytochrome C.Jurkat cell transfection CMTM1-v5 after extract endochylema and mitochondrial protein respectively, Fig. 4 show process LAN CMTM1-v5 group compared with empty carrier group, in cytoplasmic components, the content of cytochrome c substantially increases.Result also shows (Fig. 5), and the proenzyme of downstream caspase-9 molecule also has significantly minimizing in CMTM1-v5 overexpression group.Total protein of cell extracts: collects cell, washes cell 2 times with PBS, and is transferred to EP pipe, is centrifuged and abandons most supernatant；Cell is resuspended in cell pyrolysis liquid (20mM Tris-HCl, pH 7.4,150mM NaCl, 1mM EDTA, 1mM EGTA, 1%Triton X-100, Cocktail), ice bath 30 minutes；15,000g are centrifuged 10 minutes, collect supernatant, BCA standard measure.

Mitochondrial protein and cytoplasmic protein extract: utilize the method for differential centrifugation cell can be divided into different components (21).Specifically comprise the following steps that results 1 × 106 cell, after PBS washs 2 times, with 1ml buffer I (20mM Hepes-KOH, pH7.2,210mM sucrose, 70mM mannitol, 10mM KCl, 1.5mM MgCl2,1mM EDTA, 1mM EGTA, 1mM DTT, proteinase inhibitor C ocktanl) balance washed once；Resuspended with 200 μ l buffer I, ice bath 30 minutes；Being homogenized cell 50 times by Dounce micro-homogenizer gentleness, collect homogenate, 750g, 4 DEG C are centrifuged 5 minutes；Take supernatant continue 4 DEG C, 10,000g are centrifuged 10 minutes, it is thus achieved that be precipitated as mitochondrial fractions.Sucking-off supernatant continues 4 DEG C, and 10000g is centrifuged 30 minutes, then takes supernatant as without mitochondrion cytoplasm fraction.

Western blot detects: after collecting protein quantification equilibrium, add SDS sample loading buffer (β mercaptoethanol, glycerol, 20%SDS, 0.1 bromophenol blue), boil 5 minutes, after being centrifuged, take supernatant, after the SDS-PAGE glue separation equal protein sample of 12.5%, with 100v, 1.5 hours wet type electrotransfers, on nitrocellulose filter, after closing overnight with 5% skim milk 4 DEG C, are combined 1 hour or 4 DEG C overnight with an anti-room temperature respectively, afterwards with TBST (Tris-HCl, NaCl, 0.1%Tween-20) wash film three times, each 10 minutes；Adding the corresponding anti-lucifuge of IRDyeTM 800/700conjugated bis-to react one hour, TBST detects with Odyssey Imaging System instrument after washing film.

Embodiment 5, CMTM1-v5 induction caspase activation

Caspase molecule, particularly Caspase-3 act as carnificial role in apoptosis process, are finally to cause one of apoptotic molecule.When initial caspase molecule such as caspase-8,9,10 cuts and activate downstream effect caspase molecule such as caspase-3, after 6,7, the caspase molecule of activation can make intracellular a series of protein substrate degrade, cell is caused to present apoptotic a series of morphology and molecular biological characteristics, caspase-3 degraded ICAD such as activation, make CAD discharge and incising cell core DNA forms DNA segment (25,26,27).Whether the apoptosis induced for clear and definite CMTM1-v5 is along with the activation of caspase-3, and after Jurkat and Raji cell transfecting about 12 hours, harvesting extracted albumen, carries out DEVDase activity (i.e. caspase-3 activity) detection.Its fluorogenic substrate Ac-DEVD-AMC is cut by activation caspase-3, discharge free AMC and can be inspired fluorescence by spectrofluorophotometer, strong and weak by fluorescence in detection reaction system, can determine whether out the power of caspase-3 enzymatic activity, thus react the Activation of caspase-3.(Fig. 5 a) result shows, compared with empty carrier group, when apoptosis occurs comparing in early days (12h after transfection), CMTM1-v5 group just can cause intracellular caspase-3 activity significantly raised (Jurkat cell 7 times, in Raji cell 5 times).Western blot result also shows that CMTM1-v5 overexpression one of the substrate making caspase-3 PARP, caspase9,12 all can there occurs activation, i.e. there occurs that fragment is cut.

Caspase-3 is a member of cell Ban Guang aspartic protease family, in apoptosis process can the D1E2V3D4-X sequence of specific recognition substrate protein white matter, and cut D4-X covalent bond.Utilizing Caspase-3 substrate-fluorescence coupling tetrapeptide Ac-DEVD-AMC that above-mentioned principle design synthesizes, when covalent bond, AMC is not released, and therefore can not be inspired fluorescence.When Ac-DEVD-AMC is disappeared by the special cutting of Caspase-3, peptide bond D4-X, and the free AMC discharged can send fluorescence after being excited.By the size of fluorescence intensity in detection reaction system, can the intensity of indirect reaction Caspase-3 activity.This experiment i.e. designs based on above-mentioned principle, and concrete operations are as follows:

Cell cracks 30 minutes in full cell pyrolysis liquid (10mM Tris-HCl, pH7.5,130mM NaCl, 1%Triton X-100,1mM PMSF) on ice, and protein concentration BCA protein detection kit measures (Pierce, USA).Taking 5g total protein of cell, with Caspase-3 Activity determination buffer (25mM HEPES, pH 7.5,100mM NaCl, 1mM EDTA, 0.1%CHAPS, 10mM DTT), fluorogenic substrate Ac-DEVD-AMC (20M) is hatched jointly at 37C.Use POLARStar (BMG Labtechnology, Germany) spectrofluorophotometer detection AMC burst size, exciting light 380nm, launch light 460nm.Within every 10 minutes, measure once, continuously monitoring 120 minutes.Representing Caspase-3 enzymatic activity with the value added of fluorescence intensity, each sample at least does three repeating holes.

Embodiment 6, process LAN CMTM1-v5 can suppress NF κ B signal pathway activated

By the eukaryon expression plasmids such as pcDB-CMTM1-v5, pcDB empty carrier and p-NF κ B-Luc reporter plasmid, pRL-TK internal reference plasmid transfects the most jointly to 293T cell or Jurkat cell.Transfecting latter 24 hours and add final concentration PMA (100ng/ml)+Inomycin (1mM) stimulation, in the multiple hole of 6 of each plasmid to be measured, 3 add stimulation, and 3 are not added with stimulating.Gather in the crops after stimulating 6-8 hour and by Promega double reporting system explanation harvesting, illustratively use Veritas Microplate Illuminometer machine detection uciferase activity, and use Excel software to carry out data process.

Embodiment 7, yeast two-hybrid and co-immunoprecipitation checking CMTM1-v5 interact with CAML in cell

Inventor's interaction target molecule by yeast two-hybrid screening CMTM1-v5.Result is pointed out in yeast cells, CMTM1-v5 and CAML of process LAN has interaction.In order to verify the interaction of the two albumen further in mammalian cell, carry out again co-immunoprecipitation experiment and verified.Result shows, by IgG and GST antibody I P, then use HA antibody test, on the position identical with INPUT GST-CMTM-v5, can occur in that corresponding band (Fig. 7), prompting CAML and CMTM1-v5 can be combined with each other under intracellular environment in GST antibody I P group.

Embodiment 8, CMTM1-v5 Yu CAML are total to positioning analysis in HeLa cell

Can be with CAML in intracellular interaction for being further characterized by CMTM1-v5, we construct pEGFP-N1-CMTM1-v5 (green) and CAML-Flag plasmid, and transfect to HeLa cell simultaneously, utilize the fluorescence localization of indirect immunofluorescene assay CAML-Flag, and use two kinds of molecules of confocal laser scanning microscope in the situation of intracellular targeting.The HeLa cell of transfection pEGFP-N1-CMTM1-v5 plasmid creep plate on coverslip is cultivated, hatch 30 minutes at 37 DEG C with endoplasmic reticulum specific dye ER Tracker Blue (100nM) and mitochondrion specific dye Mito Tracker Red (100nM) respectively, under Laser Scanning Confocal Microscope, observe positioning scenarios altogether.Fig. 8 shows: CMTM1-v5 Yu CAML of process LAN is all located in cytoplasm, and in the point-like gathering of core week, both distributions are the most completely overlapped, and prompting CMTM1-v5 Yu CAML is in intracellular location altogether.The Subcellular Localization situation of comprehensive CMTM1-v5, it is presumed that CMTM1-v5 Yu CAML is positioned endoplasmic reticulum jointly, and interacts wherein.This is the albumen being positioned at endoplasmic reticulum with document report CAML, and can be consistent with other protein-interactings many.CAML, is the protein being positioned at endoplasmic reticulum that can combine with Cyclophilin-B (CyP, ciclosporin A associated proteins) found by yeast two-hybrid in lymphocyte.CAML take part in the activation of the numerous transcription factor of lymphocyte.Particularly CAML can rely on calcineurin, and then activation NF-AT by activating calcium.

Embodiment 9, in Jurkat cell coexpression CAML Yu CMTM1-v5 on apoptotic impact

In order to verify CMTM1-v5, CAML interaction functionally, the eukaryon expression plasmid of CMTM1-v5, CAML is transfected respectively and cotransfection is in Jurkat cell, detect apoptotic situation.Similar to aforesaid result, in Jurkat cell, process LAN CMTM1-v5 can cause obviously apoptosis (cell of the double dye of AV+PI reaches 31%), individually transfection CAML is compared with matched group, the most significantly affects apoptosis (13.30%, 4.27%).And when CAML Yu CMTM1-v5 transfects jointly to Jurkat cell, the apoptosis of CMTM1-v5 induction has significantly been restrained (cell of the double dye of AV+PI is reduced to 17.50%) by 31.27%.Numerically, the reverse of the apoptosis-induced effect of CMTM1-v5 is obviously rather than the neutralization of simple two numerical value, and we have carried out strict control to the plasmid amount added when cotransfection, so we can be with initial guess, CAML Yu CMTM1-v5 transfects jointly can affect the apoptosis that CMTM1-v5 is induced really.

Embodiment 10, process LAN CMTM1-v5 have inhibitory action to NF-AT signal path, and suppress the CAML activation to NF-AT path

Research before this finds that CMTM1-v5 has inhibitory action to NF-AT signal path.We compare the activity of luciferase when individually transfecting both CAML, CMTM1-v5 and corotation to utilize dual-luciferase reporter system, after result display PMA+Ionomycin stimulates, blank pcDB group uciferase activity adds about 4 times, illustrates that cell has been fully activated.And compared with the control, process LAN CMTM1-v5 can suppress not add uciferase activity after stimulation (endogenous) and stimulation.The cell fluorescence element enzymatic activity of process LAN CAML group is the most significantly raised, and this is also consistent with document report.During coexpression CMTM1-v5 and CAML, the depression effect of NFAT is then reversed by CMTM1-v5.By detection CMTM1-v5 and CAML coexpression on apoptosis and the impact of NEAT reporter gene activity, we demonstrate that CMTM1-v5 and CAML interaction has biological effect, and this interaction is probably a kind of negativity regulation, i.e. CMTM1-v5 may inhibit some function of CAML by interacting with CAML, such as intracellular calcium concentration etc., thus show the activity of suppression NFAT and apoptosis-induced effect.

Embodiment 11, CMTM1-v5 polypeptide internalization kinetics

Jurkat and Raji cell is incubated at containing 10% heat-inactivated hyclone (FBS) (Hyclone, USA), 100U/ml penicillin, 100g/ml streptomycin, in the RPMI 1640 (Life Technologies, USA) of 2mM L-glutaminate.FITC-CMTM1-v5 polypeptide is added in cell, its internalization of flow cytometry: cultivate Jurkat and Raji cell respectively with 24 orifice plates, then hatch at 37 DEG C with 5g/ml FITC-CMTM1-v5 polypeptide, after hatching end, wash after cell 2 times with FACSCalibur flow cytometer (BD Biosciences) detection with PBS.Result shows, FITC-CMTM1-v5 polypeptide can enter Jurkat and Raji cell.

Laser confocal microscope analyzes the FITC-CMTM1-v5 polypeptide of internalization in intracellular location: Raji cell is cultivated micropore ware (the MatTek corporation at special glass bottom, U.S.A.), on, washed cell also adds fresh 10%FBS culture medium and FITC-CMTM1-v5 polypeptide extremely final concentration of 20ug/ml.Washing 2 times with PBS, 2% paraformaldehyde room temperature fixes 15min.Then cell TCS-SP laser scanning co-focusing microscope is observed, core imaging after Hoechst 33342 (red, false color) dyeing process.Process the microscopic analysis after cell with 20ug/ml FITC-CMTM1-v5 polypeptide and show that fluorescence localization, in cytoplasm, dissipates spot distribution.

Embodiment 12, CMTM1-v5 polypeptide can be with inducing cell apoptosis

CMTM1-V5 polypeptide hatches the corresponding time with Raji cell altogether in 37 DEG C, harvesting is also prepared as single cell suspension, the PBS of pre-cooling washs 2 times, it is resuspended in 200 μ l Binding Buffer (10mM HEPES, pH 7.4,140mM NaCl, 1mM MgCl2,5mM KCl, 2.5mM CaCl2), add FITC-Annexin V to final concentration 0.5 μ g/ml, 4 DEG C of lucifuges hatch 30 minutes, add PI (propidium iodide) to final concentration 1 μ g/ml, flow cytometer detection Level of Apoptosis.Dye with Annexin-V/PI (Bao Sai company), flow cytomery apoptosis.Gather in the crops more than 10,000 cells, calculate apoptotic cell percentage ratio.

Embodiment 13, animal model build

SICD/Beige mice, female, 6-8 week, 5 groups, often group 7, totally 35.Experimental group be divided into low (tail vein injection CMTM1-V5 polypeptide 400 μ g/ only), in (tail vein injection CMTM1-V5 polypeptide 800 μ g/ is only), high (tail vein injection CMTM1-V5 polypeptide 1600 μ g/ is only) various dose group, the 0th day equal tail vein injection Raji cell 1*10^7/.Injecting CMTM1-v5 polypeptide, injecting normal saline after negative control group injection Raji cell after experimental group tail vein injection Raji cell again, positive controls only injects amycin 48ug/ after having injected Raji cell, and injection cumulative volume 100ul/ is only.Within 1st, 2 days, polypeptide group respectively injects polypeptide once.Natural law booster injection polypeptide the most at differing intervals.Hydrochloride for injection doxorubicin was treated continuously by the 21 days/course for the treatment of.Observe the life cycle of mice.Dead Mus takes its tissue immediately and internal organs process.

Found that CMTM1-v5 polypeptide has extraordinary protective effect to mice.

Prepared by embodiment 14, Organs of Mice cell suspension

Winning mouse spleen and liver, be placed in the PBS of pre-cooling, with 5ml plunger gentle abrasion on filter screen, collect cell suspension, then go over filter screen, 1600rpm is centrifuged 5min, abandons supernatant；Adding 5ml erythrocyte cracked liquid ACK (0.15M NH4Cl, 10mM KHCO3,0.1mMEDTA, pH 7.2-7.4) re-suspended cell, room temperature places 5min, adds 5ml PBS, and 1600rpm is centrifuged 5min, abandons supernatant；Precipitation 5mlPBS is resuspended, carries out cell counting.Mouse femur medullary cell separation method is as follows: successively peeling off skin and leg muscle, distal femur cut off, near-end is broken disconnected at hip joint, take out femur, thrust by near-end epiphysis along key direction with 1ml syringe, bone marrow is discharged, is incubated in 1640 containing 10%FBS.

Embodiment 15, statistical analysis

The difference of statistical significance whether is there are between each group of Student ' s T check analysis；Survival curve GraphPad Prism software analysis significant difference.P ＜ 0.05 thinks have significant difference.

Claims (8)

1.CMTM1-v5 polypeptide or encode its polynucleotide in preparation for treating the leukemic medicine of experimenter Purposes, wherein said polypeptide is selected from lower group:

(1) there is the polypeptide of aminoacid sequence shown in SEQ ID NO:2；Or (2) comprise and SEQ ID NO:2 Described aminoacid sequence has at least 70%, 75%, 80%, 85%, 90%, 95% or 97% homology The polypeptide of aminoacid sequence, it has the essentially identical biological function of polypeptide described with (2) or activity；Or

(2) functional fragment of described polypeptide, variant, analog or derivant, they have described many with (2) Biological function that peptide is essentially identical or activity.

2. the purposes of claim 1, wherein said polynucleotide include:

(1) nucleotide sequence of aminoacid sequence shown in coding SEQ ID No:1；(2) under strict conditions with (1) The nucleotide sequence of described nucleotide sequence hybridization；Or nucleotide sequence shown in (1) has at least 70%, 75 The conforming nucleotide sequence of %, 80%, 85%, 90%, 95% or 97%.

3.CMTM1-v5 polypeptide or encode its polynucleotide in preparation for suppressing experimenter's inflammation and/or treatment to be subject to The purposes of the medicine of examination person's autoimmune disease, wherein said polypeptide is selected from lower group:

(1) there is the polypeptide of aminoacid sequence shown in SEQ ID NO:2；Or (2) comprise and SEQ ID NO:2 Described aminoacid sequence has at least 70%, 75%, 80%, 85%, 90%, 95% or 97% homology The polypeptide of aminoacid sequence, it has the essentially identical biological function of polypeptide described with (2) or activity；Or

(2) functional fragment of described polypeptide, variant, analog or derivant, they have described many with (2) Biological function that peptide is essentially identical or activity.

4. the purposes of claim 3, wherein said polynucleotide include:

(1) nucleotide sequence of aminoacid sequence shown in coding SEQ ID No:1；(2) under strict conditions with (1) The nucleotide sequence of described nucleotide sequence hybridization；Or nucleotide sequence shown in (1) has at least 70%, 75 The conforming nucleotide sequence of %, 80%, 85%, 90%, 95% or 97%.

5. treating a leukemic pharmaceutical composition, this pharmaceutical composition comprises the aminoacid sequence of CMTM1-v5 Or encode the polynucleotide sequence of this sequence, and one or more pharmaceutically acceptable carriers or excipient；Its Described in polypeptide selected from lower group:

(1) there is the polypeptide of aminoacid sequence shown in SEQ ID NO:2；Or (2) comprise and SEQ ID NO:2 Described aminoacid sequence has at least 70%, 75%, 80%, 85%, 90%, 95% or 97% homology The polypeptide of aminoacid sequence, it has the essentially identical biological function of polypeptide described with (2) or activity；Or

(2) functional fragment of described polypeptide, variant, analog or derivant, they have described many with (2) Biological function that peptide is essentially identical or activity.

6. the pharmaceutical composition of claim 5, wherein said polynucleotide include:

The nucleotide sequence of aminoacid sequence shown in (a) coding SEQ ID No:1；(2) under strict conditions with (1) The nucleotide sequence of described nucleotide sequence hybridization；Or nucleotide sequence shown in (1) has at least 70%, 75 The conforming nucleotide sequence of %, 80%, 85%, 90%, 95% or 97%.

7. treating a pharmaceutical composition for inflammation and autoimmune disease, this pharmaceutical composition comprises CMTM1-v5's Aminoacid sequence or encode the polynucleotide sequence of this sequence, and one or more pharmaceutically acceptable carriers or Excipient；Wherein said polypeptide is selected from lower group:

(1) there is the polypeptide of aminoacid sequence shown in SEQ ID NO:2；Or (2) comprise and SEQ ID NO:2 Described aminoacid sequence has at least 70%, 75%, 80%, 85%, 90%, 95% or 97% homology The polypeptide of aminoacid sequence, it has the essentially identical biological function of polypeptide described with (2) or activity；Or

(2) functional fragment of described polypeptide, variant, analog or derivant, they have described many with (2) Biological function that peptide is essentially identical or activity.

8. the pharmaceutical composition of claim 7, wherein said polynucleotide include:

(1) nucleotide sequence of aminoacid sequence shown in coding SEQ ID No:1；(2) under strict conditions with (1) The nucleotide sequence of described nucleotide sequence hybridization；Or nucleotide sequence shown in (1) has at least 70%, 75 The conforming nucleotide sequence of %, 80%, 85%, 90%, 95% or 97%.