CN105463001A - Composition containing human papilloma virus (HPV) plasmodium and immunopotentiator and being used for preventing or treating cervical cancer - Google Patents

Composition containing human papilloma virus (HPV) plasmodium and immunopotentiator and being used for preventing or treating cervical cancer Download PDF

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CN105463001A
CN105463001A CN201510982015.7A CN201510982015A CN105463001A CN 105463001 A CN105463001 A CN 105463001A CN 201510982015 A CN201510982015 A CN 201510982015A CN 105463001 A CN105463001 A CN 105463001A
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hpv
nucleotide sequence
albumen
somatotype
signal peptide
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成永喆
徐相焕
徐裕锡
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Genexine Co Ltd
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/54Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C12N2710/20011Papillomaviridae
    • C12N2710/20034Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Abstract

The invention relates to a composition containing a human papilloma virus (HPV) plasmodium and an immunopotentiator and being used for preventing or treating cervical cancer. The composition is characterized in that fusion protein is used for treating tumors caused by HPV through inducing HPV 16 and 18 type antigen-specific immune response, wherein the fusion protein comprises fusion polypeptide which is recombined to enable the three dimensional structures of HPV 16 and 18 type antigens E6 and E7 to deform; signal peptide which is used for secreting the fusion polypeptide; immunological enhancement peptide.

Description

For prevent or treat cervical cancer, the composition that comprises human papillomavirus plasmodium and immunostimulant
Technical field
The present invention relates to for prevent or treat cervical cancer, comprise human papillomavirus (humanpapillomavirus; HPV) composition of plasmodium and immunostimulant.More specifically, fusion rotein treats by induction HPV16 and 18 type antigen-specific immune responses the tumour caused by HPV.Described fusion rotein comprises fusion polypeptide (Polypeptide), signal peptide and mucin peptide, and described fusion polypeptide is reorganized, to make the three-dimensional structure distortion as antigen E6, E7 of human papilloma virus 16 and 18 types.
Background technology
As everyone knows, cervical cancer (cervicalcancer) is the disease (zurHausen, Hetal.BiochemBiophysActa1996,1288 that cause due to the infection of the high-risk human mammilla papillomavirus by human papilloma virus 16 and 18 types etc.; F55-F78, MarkHetal.JNatlCancerInst1993,85; 958-964).In HPV albumen, the morbidity of E6, E7 albumen to cervical cancer plays a major role, and confirmed to express 99% in the tumor tissues of cervical cancer patient, thus main target material (vonKnebelDoeberitzetal.Int.J.Cancer1992,51 of vaccine become for the preparation for the treatment of and prevention cervical cancer; 831-834).E6 is combined with the known tumor suppressor protein p53 that is used as, and by promoting the decomposition of p53, stops the carrying out of cell cycle by apoptosis (apoptosis) approach.E7 combines with the Retinoblastoma Protein pRb (retinoblastomaprotein) as tumor-inhibiting factor, by making it inactivation and promoting that it decomposes, induced cell cycle enters S phase (Cobriniketal., TrendsBiochemSci1992,17:312-5).
But, be treatment cervical cancer, in use by expressing in the clinical trial of the composition of the nucleotide sequence of E6, E7 albumen of HPV16 simultaneously, its curative effect remarkable (GarciaFetal.ObstetGynecol2004,103; 317-326).This result shows, only using HPV antigen is the antigen-specific immune response that cannot to be enough to treat or suppress cervical cancer.
Therefore, be treatment uterus carcinoma, the immunogenicity (immunogenicity) of E6, E7 need be strengthened, and the carciongenic potency of described albumen need be eliminated.
Summary of the invention
technical problem
The object of the invention is to, be provided for preventing or treat the new fusion protein of disease and the polynucleotide (polynucleotide) of encoding said fusion protein that are caused by HPV.Described fusion rotein can suppress the carciongenic potency of HPVE6, E7 albumen and represent the immunogenicity of enhancing.
Another object of the present invention is, provide express described fusion rotein recombinant vectors (recombinantvector), comprise the host cell of described carrier and the method with described host cell expression fusion rotein.
Another object of the present invention is, provide with described fusion rotein, for preventing or treat the composition of the disease caused by HPV.
Another object of the present invention is, provide with described composition, for preventing or treat the method for the disease caused by HPV.
technical scheme
For achieving the above object, on the one hand, the invention provides fusion rotein, this fusion rotein comprises: fusion polypeptide, namely three-dimensional structure distortion, come from human papilloma virus 16 and 18 types and comprise E6, E7 of SEQIDNO:1 aminoacid sequence; For secreting the signal peptide of described fusion polypeptide; And mucin peptide.
On the other hand, the invention provides polynucleotide, these polynucleotide are for fusion rotein of the present invention of encoding.
On the other hand, the invention provides recombinant vectors, this recombinant vectors comprises polynucleotide of the present invention.
On the other hand, the invention provides host cell, this host cell is transformed by recombinant vectors of the present invention.
On the other hand, the invention provides the method for expressing fusion rotein of the present invention, the method completes by cultivating the host cell through transforming of the present invention.
On the other hand, the invention provides the composition for preventing or treat the disease caused by human papillomavirus, said composition comprise as activeconstituents, be selected from fusion rotein of the present invention, by one or more the material of expressing in host cell and homogenate (homogenate) thereof that the recombinant vectors of described fusion rotein is transformed.
On the other hand, the invention provides the method for preventing or treat the disease caused by human papillomavirus, the method comprises the step of the composition of the present invention using effective dose to the person of needs.
beneficial effect
Fusion rotein prepared by the present invention treats by inducing high HPV16 and 18 type antigen-specific immune responses the tumour caused by HPV, described fusion rotein comprise for a change come from HPV16 and 18 type E6, the three-dimensional structure of E7 albumen and reorganized fusion rotein, for the signal peptide of this fusion rotein of cell exocrine and mucin peptide.
Accompanying drawing explanation
Fig. 1 illustrates the C57BL/6 mouse subcutaneous injection tumor cell line TC-1 in anti-tumor model and the figure of the E6 specific C D8+T cell response of the HPV16 shown after GX-188E treatment of the present invention.
Fig. 2 illustrates the C57BL/6 mouse subcutaneous injection tumor cell line TC-1 in anti-tumor model and the figure of the E7 specific C D8+T cell response of the HPV16 shown after GX-188E treatment of the present invention.
Fig. 3 illustrates the C57BL/6 mouse subcutaneous injection tumor cell line TC-1 in anti-tumor model and the figure of the E6 specific C D8+T cell response of the HPV18 shown after GX-188E treatment of the present invention.
Fig. 4 illustrates the C57BL/6 mouse subcutaneous injection tumor cell line TC-1 in anti-tumor model and the figure of the E7 specific C D8+T cell response of the HPV18 shown after GX-188E treatment of the present invention.
Fig. 5 illustrates the C57BL/6 mouse subcutaneous injection tumor cell line TC-1 in anti-tumor model and the figure of the anticancer effect shown after GX-188E treatment of the present invention.
Embodiment
Content of the present invention will be explained below.
The present invention relates to fusion rotein, this fusion rotein comprises: come from human papilloma virus 16 and 18 types and comprise E6, E7 fusion polypeptide of the three-dimensional structure distortion of SEQIDNO:1 aminoacid sequence; For secreting the signal peptide of described fusion polypeptide; And mucin peptide.
Fusion rotein of the present invention comprises fusion polypeptide, and this fusion polypeptide is reorganized to make the three-dimensional structure of E6, E7 of coming from human papilloma virus 16 and 18 types be out of shape.More specifically, described fusion polypeptide by come from the amino acid of 1st ~ 85 of E6 albumen of HPV 16, the amino acid of 1st ~ 65 of E7 albumen, the amino acid of 71st ~ 158 of E6 albumen, 51st ~ 98 of E7 albumen amino acid and come from 1st ~ 85 of E6 albumen of HPV 18 amino acid, 1st ~ 65 amino acids of E7 albumen, the amino acid of 71st ~ 158 of E6 albumen, 51st ~ 105 of E7 albumen amino acid form.
The most particularly, fusion polypeptide can comprise the aminoacid sequence of SEQIDNO:1.
In addition, described signal peptide refers to comprise about 20 ~ 30 amino acid whose peptides, and described Toplink, by the albumen at cell inner expression, especially will comprise the protein excretion of E6, E7 fusion polypeptide to extracellular.And the nucleotide sequence for coding for said peptides is called as " secretory signal sequence ".Because E6, E7 fusion polypeptide of the present invention is the albumen (nucleusprotein) of expressing in the core of the cell be infected by the virus, its immunizing power is weak.Therefore, E6, E7 secretion of the signal peptide induction three-dimensional structure distortion expressed by secretory signal sequence is to extracellular, thus enhancement antigen specific humoral immune response (humoralimmuneresponse) and cellullar immunologic response (cellularimmuneresponse).
Described signal peptide can use the signal peptide comprised in the higher eucaryotic cells of Mammals etc., as tPA (tissue plasminogen activator), HSVgDs, maybe can use the secretory signal sequence of tethelin etc.Preferably, tPA can be used.More preferably, the aminoacid sequence of SEQIDNO:2 can be comprised.
In addition, mucin peptide refers to activate the cell (e.g., dendritic cell etc.) relevant to immunne response to strengthen the peptide of immunne response.
Described mucin peptide can use CD40L, Flt3 part (fms sample Tyrosylprotein kinase 3 part), flagellin or OX40 etc.Preferably, Flt3 part can be used.Described Flt3 part is can the propagation of inducing dendritic shape cell (dendriticcell, DC) and the ripe factor, can strengthen immunne response, and have the effect that effectively can reduce tumour when merging with tumour antigen through antigen.More preferably, described Flt3 part comprises the aminoacid sequence of SEQIDNO:3.
On the other hand, the present invention relates to the polynucleotide for fusion rotein of the present invention of encoding.
Described polynucleotide are for fusion rotein of the present invention of encoding, and described E6, E7 fusion polypeptide can be encoded by the base sequence of SEQIDNO:4 and be formed, but is not limited thereto.In addition, described signal peptide can be encoded by the base sequence of SEQIDNO:5 and be formed, but is not limited thereto.In addition, described mucin peptide can be encoded by the base sequence of SEQIDNO:6 and be formed, but is not limited thereto.
In addition, polynucleotide of the present invention are prepared by chemical synthesis process or gene engineering.Chemical synthesis process is well known to those skilled in the art, and can use any method.In addition, also by entrusting Commercial nucleic acid synthesis and manufacturers to buy.Such as, when being prepared by gene engineering, by obtaining the nucleic acid fragment of E6 and E7 fusion polypeptide, signal peptide and mucin peptide for encoding known respectively, connect these fragments to meet frame to prepare.The method of described obtained nucleic acid fragment is known in the art, and those skilled in the art easily connect by suitable restriction enzyme.In the specific embodiment of the present invention, by the open method prepared by chemical synthesis.
On the other hand, the present invention relates to the recombinant vectors comprising polynucleotide of the present invention.
In the present invention, " carrier " refers to the genetic construction comprising foreign DNA, and described foreign DNA is inserted in the genome of coded polypeptide.Carrier of the present invention is by secretory signal sequence, the nucleotide sequence of E6 and the E7 fusion polypeptide of the three-dimensional structure distortion of encoding human papilloma virus and the nucleotide sequence etc. of encoding immune enhancing peptide are inserted into the carrier in genome, such as, comprise plasmid vector (plasmidvector), cosmid vector (cosmidvector), phage (bacteriophagevector), yeast vector (yeastvector) or as adenovirus carrier (adenoviralvector), the virus vector (viralvector) of retroviral vector (retroviralvector) and adeno-associated virus vector (adeno-associatedviralvector).
Described secretory signal sequence is the nucleotide sequence of energy encoded peptide, and the tumour antigen at cell inner expression is secreted to extracellular and makes it by immunocyte identification by described Toplink, and it comprises the secretory signal sequence as tPA, HSVgDs or tethelin.Preferably, secretory signal sequence used in mammiferous higher eucaryotic cells can be used.More preferably, tPA (tissue plasminogen activator) can be used.Most preferably, described secretory signal sequence can comprise the base sequence of SEQIDNO:5.In addition, secretory signal sequence of the present invention can be used in host cell genetic codon (codon) replacement with high expression level frequency.
In addition, the nucleotide sequence of described mucin peptide of encoding refers to encode and strengthens the nucleotide sequence of the peptide of immunne response by activating the cell relevant to immunne response.Described mucin peptide can use CD40L, Flt3 part, flagellin (Flagellin) or OX40 etc.More preferably, mucin peptide can use Flt3 part.In addition, the nucleotide sequence of mucin peptide of the present invention of encoding can be used in host cell the genetic codon with high expression level frequency and replaces.
In addition, the described polynucleotide that recombinant vectors of the present invention comprises can be used in host cell the genetic codon with high expression level frequency and replace." being used in host cell the genetic codon with high expression level frequency to replace " or " genetic codon optimization " expressed in the present invention refers to, when the DNA in host cell transcribes or translates into albumen, according to the genetic codon with higher preferences existed in host, in the amino acid whose genetic codon of instruction, replace polynucleotide genetic codon with the genetic codon of higher preferences, thus strengthen by the amino acid of nucleic acid encoding or the expression efficiency of albumen.
Wherein, " host cell " comprises protokaryon or eukaryotic cell, and eukaryotic cell not only refers to comprise the eukaryotic cell such as low of fungi, yeast etc., also refers to the higher eucaryotic cells comprising Mammals etc.
Recombinant vectors of the present invention comprises the nucleic acid by encoding said fusion protein, and this nucleic acid is the form being suitable for the nucleic acid of expressing coding fusion rotein of the present invention in host cell.That is, recombinant vectors of the present invention comprises one or more regulating and controlling sequence for expressing chosen based on host cell, and this sequence operationally links with the nucleotide sequence that will express.
" operationally link " refers to, Target Nucleotide Sequence (such as, transcribe in vitro/translation system in or in host cell) link with described regulating and controlling sequence in a suitable manner, express to complete it.
Term " regulating and controlling sequence " comprises promotor, enhanser and other controlling elements (such as, polyadenylation signal).Regulating and controlling sequence is included in the regulating and controlling sequence indicating constitutive expression target nucleic acid in numerous host cell, and in specific host cell, indicate the regulating and controlling sequence (such as, Tissue-specific regulatory sequence) of express nucleic acid.The design that those skilled in the art can understand expression vector can change according to the factor of the selection of the host cell that will transform and target protein expression level etc.Expression vector of the present invention can be introduced in host cell to express described fusion rotein.
In addition, carrier of the present invention is prepared by standard recombinant dna technology, this standard recombinant dna technology comprises, as flush end is connected with cohesive end, by providing suitable end with the process of restriction enzyme, for preventing the removal of the improper phosphate in combination with alkaline phosphatase treatment and being linked the enzyme link etc. of enzyme by T4DNA.E6 and the E7 fusion polypeptide for coded signal peptide, human papillomavirus obtained by chemical synthesis process or gene engineering and each DNA of mucin peptide can be recombinated with the carrier comprising applicable regulating and controlling sequence, to provide carrier of the present invention.The carrier comprising described regulating and controlling sequence is undertaken buying or preparing by business method, and the present invention prepares and uses pGX27, and pGX27 is as the carrier preparing DNA vaccination.
In one embodiment of the invention, described recombinant vectors can be used for preparing cell strain to manufacture fusion rotein of the present invention, as the carrier for transgenosis of gene therapy, or as the active constituents of medicine be applied to experimenter.
On the other hand, the present invention relates to the host cell being converted into carrier.The kind of described host cell is described above.Transforming available by nucleic acid being imported with body, cell, tissue or intraorganic known method, in the intelligible scope of those skilled in the art, selecting method described in the technology implementation that is applicable to according to host cell.Described method comprises electroporation (electroporation), protoplast fusion, calcium phosphate (CaPO 4) precipitation, calcium chloride (CaCl 2) precipitation, with the stirring of silicon carbide fiber, Agrobacterium-medialed transformation, polyoxyethylene glycol, T 500, liposome transfection (lifofectamin) etc., but be not limited thereto.
On the other hand, the present invention relates to the method for expressing fusion rotein of the present invention, the host cell namely by cultivating conversion of the present invention has come.
By cultivating the host cell transformed in suitable substratum, or by cultivating the host cell transformed be directed in any animal body, can easily express and a large amount of preparation fusion rotein of the present invention.
On the other hand, the present invention relates to the composition for preventing or treat the disease caused by individuals papilloma virus, said composition comprises one or more the material be selected from fusion rotein of the present invention, the host cell transformed by the recombinant vectors for expressing described fusion rotein and homogenate thereof.
In the present invention, " experimenter " comprises the Mammals of people, monkey, mouse, pig, ox and rabbit etc., but is not limited thereto.
In addition, the disease caused by described virus can comprise cervical cancer, hepatic portal and Genital warts (anogenitalwarts), wart (verruca) etc.
In addition, composition of the present invention can comprise pharmaceutically acceptable carrier.Described carrier comprises lactose, glucose, sucrose, Sorbitol Powder, mannitol, starch, Sudan Gum-arabic, alginate, gelatin, calcium phosphate, Calucium Silicate powder, Mierocrystalline cellulose, methylcellulose gum, Microcrystalline Cellulose, polyvinylpyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talcum powder, Magnesium Stearate and mineral wet goods.Further, said composition also can comprise lubricant, wetting agent, seasonings, emulsifying agent and sanitas etc.
Composition of the present invention by such as intravenously, intramuscular, oral, in skin, mucous membrane, in nose, tracheal strips or subcutaneous etc. any approach to snibject, but be not limited thereto.Institute's cultured cells, by being applied in the cell of vitro culture by composition, is then used by composition of the present invention in subject, thus indirectly to experimenter's medication.The present invention can carry out whole body or topical.
Composition of the present invention can be made into for oral or injection as the formulation outside intestines, described oral dosage form comprises granule, pulvis, liquid, tablet, capsule or dry syrup, but is not limited thereto.Preferably, the formulation of composition of the present invention can be liquid or injection liquid formulation.
The effective dose of the fusion rotein as activeconstituents of the present invention or recombinant expression vector can be about 0.05 ~ 500mg/kg, be preferably 0.5 ~ 50mg/kg, with single dose or in batches dosage be as the criterion.But described dosage should be determined by factors such as the seriousness of the age of the situation of the disease of required treatment, patient and body weight, symptom, and therefore described scope can not be limited to this.
On the other hand, the present invention relates to the method for preventing or treat the disease caused by individuals papilloma virus, the method comprises the step of the constituent of the present invention using significant quantity to experimenter.
Effect of pharmaceutical composition of the present invention, said composition, the content of medication and dosage etc. are described above.
In the method for the invention, experimenter comprises the Mammals of people, monkey, mouse, pig, ox and rabbit etc., but is not limited thereto.
In addition, the disease caused by described virus comprises cervical cancer, hepatic portal and Genital warts, wart etc.
Below, according to embodiments of the invention and comparative example, in more detail the present invention will be described, but scope of the present invention is not limited to the following example.
Embodiment
the structure of < embodiment 1>GX-188EDNA
Abbreviation used in embodiments of the invention is defined as follows: optimization nucleotide sequence, and " tPa " or " t " represents the secretory signal sequence of tissue plasminogen activator; " F " or " Flt3L " represents fms sample Tyrosylprotein kinase 3 part.
The Flt3L of the tPa secretory signal sequence comprising the optimization genetic codon of SEQIDNO:5 nucleotide sequence with connected form chemosynthesis and the optimization genetic codon comprising SEQIDNO:6 nucleotide sequence.End add KpnI (5'), NheI (3') site, to be inserted in carrier.With the carrier pGX10 (Korean patent application publication No. 2003-0047667) of KpnI and NheI restriction enzyme treatment for the preparation of DNA vaccination, the tPa-Flt3L synthesized by then connecting with ligase enzyme, thus preparation pGX10/tF.
With the nucleotide sequence of connected form chemosynthesis for the optimization genetic codon of 1st ~ 85 amino acids of the HPV16E6 that encodes, for the nucleotide sequence of the optimization genetic codon of 1st ~ 65 amino acids of the HPV16E7 that encodes, for the nucleotide sequence of the optimization genetic codon of 71st ~ 158 amino acids of the HPV16E6 that encodes, for the nucleotide sequence of the optimization genetic codon of 51st ~ 98 amino acids of the HPV16E7 that encodes, for the nucleotide sequence of the optimization genetic codon of 1st ~ 85 amino acids of the HPV18E6 that encodes, for the nucleotide sequence of the optimization genetic codon of 1st ~ 65 amino acids of the HPV18E7 that encodes, for the nucleotide sequence of the optimization genetic codon of 71st ~ 158 amino acids of the HPV18E6 that encodes, nucleotide sequence for the optimization genetic codon of 51st ~ 105 amino acids of the HPV18E7 that encodes is (following, 16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C:SEQIDNO:4).End add NheI (5'), XbaI (3') site, to be inserted in carrier.With NheI and XbaI ferment treatment pGX10/tF16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C, then the 16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C synthesized by connecting with ligase enzyme, thus preparation pGX10/tFpGX10/tF16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C.By with KpnI and Xbal ferment treatment pGX10/tF, isolate tF16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C, after NheI and XbaI ferment treatment pGX10,16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C is connected with ligase enzyme, thus preparation pGX27/tF16E6N16E7N16E6C16E7C18E6N18E7N18E6C18E7C (all claiming " GX-188E " below).
< embodiment 2>GX-188E is to the confirmation of the result for the treatment of of cervical cancer
For confirming that GX-188E is to the result for the treatment of of cervical cancer, to C57BL/6 mouse subcutaneous injection 5 × 10 5individual TC-1 tumour cell, the GX-188E that after injection the 3rd day and the 8th day intramuscular injection 50 μ g and 100 μ g measure, then carries out electroporation.Observe the volume change of tumour cell till the 27th day from injection tumour cell, remove the spleen of mouse at the 36th day, then by 1 × 10 6e6CD8T cell epitope (epitope, the epitope) (E6 of individual cell and IL-2 and HPV16 48-57; EVYDFAFRDL, Peptron, Korea S) or the E7CD8T cell epitope (E7 of HPV18 49-57; RAHYNIVTF, Peptron, Korea S) or the E6 peptide pond (peptidepool) of HPV18 or the HPV-16 E7 pond of HPV18 be placed in the against murine IFN-g antibody (BDPharmigen of the 5 μ g/ml having used 50 μ l together, Sandiego, California) to wrap in the flat board of quilt and at 37 DEG C and 5%CO 2incubator (Froma, Minnesota, the U.S.) in cultivate 24 hours.After washing described flat board with PBST, the IFN-g containing pendency vitamin H adding the 2 μ g/ml of 50 μ l wherein respectively detects antibody (BDPharmigen, Sandiego, California) and cultivates about 3 hours at normal temperatures.Afterwards, with PBST washing, wherein add the streptavidin-AKP (alkaline phosphatase) being diluted to 1:2000 of 50 μ g and cultivate about 1 hour at normal temperatures.After PBST washing, add NBT (Promega, the Madison of the 66 μ l in the alkaline phosphate buffer of 10ml of 50 μ l respectively, WI) with the BCIP (Promega of 33 μ l, Madison, WI) mixture, react to each other to make it.Flat board is placed in 37 DEG C of incubators about 30 minutes, then uses distilled water (D.W.) wash and count generated spot with counter.
Be used as control group pGX27 process mouse compared with, can confirm obviously to reduce (please refer to Fig. 5) with the gross tumor volume of GX-188E to the mouse that TC-1 tumour cell was treated.
Industrial applicability
Fusion rotein of the present invention can be used for the therapeutical agent of the tumour caused by HPV.

Claims (27)

1. polynucleotide, it comprises: the nucleotide sequence of encode fusion polypeptide, and described fusion polypeptide is used for conversion source from human papilloma virus 16 and 18 types and has three-dimensional (3D) structure of E6 and E7 of aminoacid sequence as shown in SEQIDNO:1; Encoding immune strengthens the nucleotide sequence of peptide; With the nucleotide sequence of coded signal peptide.
2. polynucleotide according to claim 1, is characterized in that, described mucin peptide is selected from CD40L, fms sample Tyrosylprotein kinase 3 (Flt3) part and flagellin.
3. polynucleotide according to claim 1, is characterized in that, the nucleotide sequence of described encoding immune enhancing peptide comprises the nucleotide sequence as shown in SEQIDNO:6.
4. polynucleotide according to claim 1, is characterized in that, described signal peptide is selected from tissue plasminogen activator (tPa) signal peptide, HSV gD s (HSVgDs) signal peptide and tethelin signal peptide.
5. polynucleotide according to claim 1, is characterized in that, the nucleotide sequence of described coded signal peptide comprises the nucleic acid as shown in SEQIDNO:5.
6. polynucleotide according to claim 1, is characterized in that, it is codon-optimized.
7. polynucleotide according to claim 6, is characterized in that, it comprises the nucleic acid as shown in SEQIDNO:4.
8. polynucleotide, comprise: coding corresponds to the nucleotide sequence of the 1st-85 amino acid whose aminoacid sequences of the E6 albumen of human papillomavirus (HPV) 16 somatotype, coding corresponds to the nucleotide sequence of the 1st-65 aminoacid sequences of the E7 albumen of HPV-16 somatotype, coding corresponds to the nucleotide sequence of the 71st-158 aminoacid sequences of the E6 albumen of HPV-16 somatotype, coding corresponds to the nucleotide sequence of the 51st-98 aminoacid sequences of the E7 albumen of HPV-16 somatotype, coding corresponds to the nucleotide sequence of the 1st-85 aminoacid sequences of the E6 albumen of HPV-18 somatotype, coding corresponds to the nucleotide sequence of the 1st-65 aminoacid sequences of the E7 albumen of HPV-18 somatotype, coding corresponds to the nucleotide sequence of the 71st-158 aminoacid sequences of the E6 albumen of HPV-18 somatotype, coding corresponds to the nucleotide sequence of the 51st-105 aminoacid sequences of the E7 albumen of HPV-18 somatotype, encoding immune strengthens the nucleotide sequence of peptide, and the nucleotide sequence of coded signal peptide.
9. polynucleotide according to claim 8, is characterized in that, it is codon-optimized.
10. polynucleotide according to claim 8, is characterized in that, the nucleotide sequence that described encoding immune strengthens peptide is codon-optimized.
11. polynucleotide according to claim 8, is characterized in that, the nucleotide sequence of described coded signal peptide is codon-optimized.
12. polynucleotide according to claim 8, it is characterized in that, it comprises SEQIDNO:4, the nucleotide sequence of SEQIDNO:5 and SEQIDNO:6.
13. carriers comprising polynucleotide as claimed in claim 1.
14. carriers comprising polynucleotide as claimed in claim 8.
15. the carrier according to claim 13 or 14, is characterized in that, described carrier is plasmid.
16. host cells comprising carrier as claimed in claim 13.
17. host cells comprising carrier as claimed in claim 14.
18. 1 kinds of fusion roteins, comprise: have the fusion polypeptide of three-dimensional (3D) structure of E6 and E7 of aminoacid sequence as shown in SEQIDNO:1 for conversion source from human papilloma virus 16 and 18 types; Mucin peptide; And signal peptide.
19. fusion rotein according to claim 18, is characterized in that, described mucin peptide is selected from CD40L, fms sample Tyrosylprotein kinase 3 (Flt3) part and flagellin.
20. fusion roteins according to claim 18, is characterized in that, described mucin peptide comprises the aminoacid sequence as shown in SEQIDNO:3.
21. fusion roteins according to claim 18, it is characterized in that, described signal peptide is selected from tissue plasminogen activator (tPa) signal peptide, HSV gD s (HSVgDs) signal peptide and tethelin signal peptide.
22. fusion roteins according to claim 18, is characterized in that, described signal peptide comprises the aminoacid sequence as shown in SEQIDNO:2.
23. 1 kinds of fusion roteins, comprise: corresponding to the 1st-85 amino acid whose aminoacid sequences of the E6 albumen of human papillomavirus (HPV) 16 somatotype, corresponding to the 1st-65 aminoacid sequences of the E7 albumen of HPV-16 somatotype, corresponding to the 71st-158 aminoacid sequences of the E6 albumen of HPV-16 somatotype, corresponding to the 51st-98 aminoacid sequences of the E7 albumen of HPV-16 somatotype, corresponding to the 1st-85 aminoacid sequences of the E6 albumen of HPV-18 somatotype, corresponding to the 1st-65 aminoacid sequences of the E7 albumen of HPV-18 somatotype, corresponding to the 71st-158 aminoacid sequences of the E6 albumen of HPV-18 somatotype, corresponding to the 51st-105 aminoacid sequences of the E7 albumen of HPV-18 somatotype, encoding immune strengthens the aminoacid sequence of peptide, with the aminoacid sequence of coded signal peptide.
The composition of 24. diseases caused for prevention in individuality in need or treatment HPV, comprises polynucleotide as claimed in claim 1, polynucleotide as claimed in claim 8, carrier as claimed in claim 13, carrier as claimed in claim 14, host cell as claimed in claim 16, host cell as claimed in claim 17, fusion rotein as claimed in claim 18 or fusion rotein as claimed in claim 23.
25. compositions according to claim 24, is characterized in that, the disease that described HPV causes is cervical cancer.
26. compositions according to claim 24, is characterized in that, comprise the carrier that pharmacy allows further.
The method of 27. expressed fusion proteins, comprising: by the polynucleotide transfection as described in claim 1 or 8 to host cell.
CN201510982015.7A 2010-08-13 2010-08-13 Composition containing human papilloma virus (HPV) plasmodium and immunopotentiator and being used for preventing or treating cervical cancer Pending CN105463001A (en)

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