CN105734059A - GP73 inhibitor and application thereof - Google Patents
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Abstract
The invention provides a GP73 inhibitor which includes: a factor for inhibiting correct cutting of GP73, a factor which is capable of inhibiting or stopping combination of the GP73 and an acceptor, siRNA which is capable of specially inhibiting the GP73, shRNA which is capable of specially inhibiting the GP73, microRNA which is capable of specially inhibiting the GP73, an anti-body which is capable of specially inhibiting the GP73, and an inhibiting factor or a dominant-negative DNA plasmid thereof, which is introduced in whole body or local part, and expresses or activates a GP73-encoding inhibiting factor and a protein complex composition factor thereof. The invention also provides the application of the GP73 inhibitor in preparation of medicines for treating liver cancer, and a progress of hepatic fibrosis and liver cirrhosis, introduced by HPV infection, until hepatocellular carcinoma, and provides a medicine composition. The GP73 inhibitor can be used for liver cancer prevention for HBv positive patients, liver cirrhosis patients, and other liver cancer high-risk people, and as well as treating liver cancer patients and strengthening health after treatment.
Description
Technical field
The invention belongs to biomedicine field.The present invention relates to a kind of GP73 inhibitor and application thereof.
Background technology
Primary hepatocyte hepatocarcinoma (is called for short hepatocarcinoma, Hepatocellularcarcinoma, HCC), is the malignant tumor of fatality rate the 3rd in the world at present, there are about 500,000 patients every year because of HCC death[1].Hepatocellular carcinoma is most to be changed through liver cirrhosis by chronic hepatitis, and the liver cancer patient of about 75% has hepatitis b virus infected medical history；In B-mode and the third type chronic hepatitis patient 20%, in liver cirrhosis patient 80% can develop into hepatocarcinoma.Hepatitis B, liver cirrhosis and hepatocarcinoma are high at China's sickness rate, and there is hepatitis B virus carriers about 9,003,000,000 in China, accounts for the 7.18% of population, are the 2/3 of world's hepatitis B virus infection sum；China's liver cancer patient accounts for the 55% of world patient populations, just has 1 example to occur in China in 2 namely often newly-increased in the world example hepatocarcinoma.Therefore, understand fully the molecule mechanism of the viral hepatitis transition mechanism to hepatocarcinoma and onset of liver cancer, and it is particularly important in China that it is carried out early diagnosis and targeted therapy.
The treatment of hepatocarcinoma is with excision for first-selection, and early stage excision is the key improving survival rate, and tumor is more little, and five year survival rate is more high.Therefore, early diagnosis is the premise that primary hepatocarcinoma obtains early treatment.But the early symptom of hepatocarcinoma is often inconspicuous, once occur in that typical case's symptom of hepatocarcinoma and sign, diagnosis is difficulty, but often non-early stage.For being not suitable for carrying out the patient of resection operation, treatment is based on chemotherapy, and since the fifties in last century, the system chemotherapy of hepatocarcinoma makes little progress.Although some Therapy study centers are in progress to some extent in the early diagnosis treatment of hepatocarcinoma in recent years, the prognosis making part hepatocarcinoma patient makes moderate progress, but for whole onset of liver cancer crowds, treatment means still efficiency is low, erious adverse reaction, hepatocarcinoma is still one of worst tumor of prognosis so far.
From 19 the end of the century RobertKoch, KitasatoShibasaburo, EmilvonBehring and PaulEhrlich et al. it is found that utilize cured since patients serum treats same disease, antibody is always up one of modern medicine and biological development priority as medicine[11,12]。
Along with development and the application of hybridoma technology, monoclonal antibody is possibly realized as medicine.Therapeutic monoclonal antibodies owing to it is safe, special, high-affinity and destruction protein-protein interaction efficient, be increasingly becoming a kind of important drug type.Approval first was for the monoclonal antibody of human clinical therapy in 1986 for U.S. FDA, and the Murononab-CD3 (having another name called: OrthocloneOKT3) in mice source, this antibody is used to the immunosuppressant after organ transplantation[13]。
In recent years, serum GP73 is considered as the very promising liver cancer serum mark of one[2,3].GP73 is present in a kind of transmembrane glycoprotein of Golgi body.It includes a short N-end cytoplasmic domain, a membrane spaning domain (TMD) and a big C-terminal domains towards Golgi body tube chamber.A coiled-coil domain (coiled-coildomain) and an acidic ending, this regions mediate Protein-protein interaction is included in this Gorky's lumen area[4].Coiled-coil domain includes a proprotein convertases (proproteinconvertase, PC) recognition site, can mediate the shearing of GP73, thus causing that GP73 secretes[5].First the cDNA of GP73 was cloned by Kladney etc. in 2000, and Kladney etc. finds, in normal human's hepatic tissue, GP73 is mainly expressed by bile duct epithelial cell, and liver cell expression is not seldom even expressed[6].Hepatocyte is subject to virus (HBV and HCV) infection can cause GP73 high expressed[4,6].Iftikhar etc. then find, in acute hepatitis and Progressive symmetric erythrokeratodermia liver cirrhosis patient, GP73 is high expressed in hepatocyte[7].2004, Maitra proposed the earliest, and GP73 is equal high expressed in the hepatic disease that many factors causes, it is possible to as the blood serum designated object of hepatic disease[8].2005, the rising of GP73 was connected by the Block of the U.S. etc. for the first time with hepatocarcinoma[9].But, so far, but without utilizing GP73 inhibitor to treat the relevant report of hepatocarcinoma, thus research finds that can GP73 inhibitor be used for treating hepatocarcinoma, it is possible to provide a new therapy approach for liver cancer treatment.
Summary of the invention
Therefore, it is an object of the invention to for the present situation that there is presently no the correlational study utilizing GP73 inhibitor for treating hepatocarcinoma, there is provided a kind of for inhibitor suppressing GP73 and application thereof, a new therapy approach safe, special, efficient is provided, to improve the prognostic level of liver cancer patient for liver cancer treatment.
nullUnless specifically stated otherwise，" GP73 " herein be " GP73 by name entirely，Golgi's membrane protein 1 (golgiprotein73,golgimembraneprotein1)”，Have another name called " GOLM1，Gorky's phosphoprotein 2 (golgiphosphoprotein2，Golph2)”，Its GeneID is " 51280 "，The aminoacid sequence of albumen is the aminoacid sequence shown in following SEQIDNO:1，SEQIDNO:1:MMGLGNGRRSMKSPPLVLAALVACIIVLGFNYWIASSRSVDLQTRI MELEGRVRRAAAERGAVELKKNEFQGELEKQREQLDKIQSSHNFQLESVNKLYQDE KAVLVNNITTGERLIRVLQDQLKTLQRNYGRLQQDVLQFQKNQTNLERKFSYDLSQ CINQMKEVKEQCEERIEEVTKKGNEAVASRDLSENNDQRQQLQALSEPQPRLQAAG LPHTEVPQGKGNVLGNSKSQTPAPSSEVVLDSKRQVEKEETNEIQVVNEEPQRDRL PQEPGREQVVEDRPVGGRGFGGAGELGQTPQVQAALSVSQENPEMEGPERDQLVIP DGQEEEQEAAGEGRNQQKLRGEDDYNMDENEAESETDKQAALAGNDRNIDVFNVED QKRDTINLLDQREKRNHTL.
Unless specifically stated otherwise, " GP73 inhibitor " herein refers to " any agent that the conduction of GP73 signal can be caused to suppress ", its principle includes but not limited to suppress GP73 to shear, GP73 is suppressed to be transferred to cell membrane, suppress expression and the function of GP73 and receptor, suppress GP73 to update, shift, shear and endocytosis, change glycosylation or the ubiquitination of GP73 or GP73 functional domain, change expression or the function of GP73 cofactor or effector.
The invention provides a kind of GP73 inhibitor on the one hand, its factor correctly sheared selected from suppression GP73, the factor that can suppress or hinder GP73 to be combined with receptor, the siRNA of energy specificity suppression GP73, energy specificity suppress the antibody of the shRNA of GP73, the microRNA of energy specificity suppression GP73, energy specificity suppression GP73, and whole body or local proceed to, express or the inhibitive factor of activated code GP73 inhibitive factor, its albumen composition composing factor, or the DNA plasmid of its dominant negatives.
Preferably, the factor that the described GP73 of suppression correctly shears includes GP73 and shears the Furin inhibitor 1 of required convertase Furin, it is preferably Dec-RVKR-CMK, Furin inhibitor 2, be preferably many poly arginines peptide, 2,5-deoxystreptamine complex, α-1 antitrypsin silicate, MACl (2), wherein, M is copper or zinc, and A is chelate group, preferably, described MACl (2) includes Cu (TTP) Cl (2) and Zn (TTP) Cl (2), and wherein, TTP is 4'-[p-tolyl]-2,2':6', 2 "-terpyridyls；
Preferably, the siRNA of GP73 can be suppressed, the shRNA of GP73 can be suppressed or the target sequence of microRNA of GP73 can be suppressed to be have at least the sequence of 90% homology with the nucleotide sequence shown in SEQIDNO:2 by specificity by specificity by specificity.
According in one embodiment of the invention, described the antibody of GP73 being suppressed to be anti-GP73 antibody by specificity, described anti-GP73 antibody the antigen of specific recognition can be selected from protein:
() aminoacid sequence is the aminoacid sequence shown in SEQIDNO:1；Or
() in the aminoacid sequence that () limits through replacing, disappearance or one or several aminoacid of superposition and derivative protein, and the protein shown in itself and () has identical function.
Preferably, described anti-GP73 antibody is the anti-GP73 chimeric antibody comprising the aminoacid sequence such as variable region light chain shown in SEQIDNO:3 and the aminoacid sequence such as Variable region heavy shown in SEQIDNO:4.
It is highly preferred that the variable region light chain of described anti-GP73 chimeric antibody is the gene code being classified as SEQIDNO:5 by nucleotides sequence, the Variable region heavy of described anti-GP73 antibody is the gene code being classified as SEQIDNO:6 by nucleotides sequence.
On the other hand, the present invention also provide for a kind of GP73 inhibitor of the present invention preparation for treat hepatocarcinoma, HBV infection cause hepatic fibrosis and liver cirrhosis until hepatocellular carcinoma process medicine in application.
Another further aspect, the present invention also provides for the preparation of a kind of GP73 inhibitor of the present invention for suppressing the application in the medicine of fucosylation and invasion and attack, preferably, described GP73 inhibitor is the chimeric antibody comprising the aminoacid sequence such as variable region light chain shown in SEQIDNO:3 and the aminoacid sequence such as Variable region heavy shown in SEQIDNO:4.
Further aspect, the present invention provides the application in the medicine of the generation prepared for suppressing the subcutaneous formation tumor of hepatoma carcinoma cell of a kind of anti-GP73 antibody of the present invention.
Further aspect, the present invention provide a kind of for treating hepatocarcinoma, hepatic fibrosis that HBV infection causes and liver cirrhosis until the pharmaceutical composition of process of hepatocellular carcinoma, described pharmaceutical composition comprises the GP73 inhibitor of the present invention of effective dose and pharmaceutically acceptable carrier or adjuvant.
Preferably, described GP73 inhibitor is anti-GP73 chimeric antibody.
It is further preferred that the GP73 inhibitor of described effective dose is local concentration is 20-250 μ g/mL.
Beneficial effects of the present invention: hepatocellular carcinoma is one of worst cancer of current prognosis, and occurred frequently in China.GP73 is as the target spot of the molecule that liver cancer genesis and development is played an important role and liver cancer treatment, and its inhibitor is important treatment and prevention medicine, plays basic effect to reducing the onset of liver cancer caused by viral infection with improving liver cancer treatment level.GP73 inhibitor can be used for HBv positive patient, liver cirrhosis patient and the prevention of hcc of other Liver Cancers crowd；Consolidate after the treatment of liver cancer patient and treatment.
Accompanying drawing explanation
Hereinafter, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:
Fig. 1 shows that GP73 can promote tumor development, wherein, A shows the expression of GP73 in the GP73 overexpressing cell line HepG2GP73 that builds and low express cell system BelshGP73, B shows GP73 low express cell system BelshGP73 one-tenth tumor level in nude mice Tumorigenesis, C shows GP73 overexpressing cell line HepG2GP73 survival level in nude mice Tumorigenesis, and D shows GP73 low express cell system BelshGP73 survival level in nude mice Tumorigenesis；
Fig. 2 shows that secretion GP73 promotes cell proliferation, wherein, A shows the GP73 secretion level in the cell culture fluid of GP73 overexpressing cell line HepG2GP73, B shows the cell proliferation level after cultivating PC3 cell, C shows the cell proliferation level after cultivating MEF cell, and D shows the cell proliferation level after cultivating GP73 low express cell system BelshGP73；Figure E show HepG2GP73 cell conditioned medium and GP73 monoclonal antibody 8A10 hatch 16 hours after ProteinGagarose precipitation remove antibody and GP73, cultivate HepG2 cell, cell proliferation level with the cell culture fluid after processing；Figure F shows prokaryotic expression GP73 secretory protein total length, N end, C end, cell proliferation level；Figure G is the cell proliferation level in HepG2 cell culture fluid after albumen shown in addition figure F；
Fig. 3 shows that GP73 inhibitor shGP73 can treat hepatocellular carcinoma；Wherein A and D is for showing that expressing shGP73 in hepatoma cell line HepG2.2.15 (A) and MHCC97H (D) strikes low GP73, detects the expression of GP73 with zero load for comparison (v) westernblot；B and E is HepG2.2.15shGP73 (B) and the cell proliferation curve of MHCC97HshGP73 (E)；C and F is HepG2.2.15shGP73 (C) and the plate clone of MHCC97HshGP73 (F)；G is plate clone experimental result statistics；There is (left side) and time-to-live (right side) in H and I, HepG2.2.15shGP73 (H) and MHCC97HshGP73 (I) tumor in tumor formation in nude mice；
Fig. 4 shows screening and the qualification result of the chimeric antibody of specific recognition GP73, and wherein, A-B shows that detecting it by ELISA and immunoblotting identifies restructuring GP73 molecule ability screening positive clone；C shows and identifies specific recognition antibody to GP73 high expression tumour cell by Flow cytometric analysis.
Fig. 5 shows that GP73 chimeric antibody suppresses the propagation of GP73 high expression tumour cell, migrate, invasion and attack and one-tenth tumor ability, wherein, A shows to add the cell proliferation level after anti-GP73 chimeric antibody is cultivated in the culture medium of GP73 low express cell system Bel7402, B shows to add after GP73 chimeric antibody in the culture medium of human hepatoma cell strain MHCC97h, the impact of corresponding antibodies on cell migration ability is detected through cell scratch experiment, after C shows the culture medium by anti-GP73 chimeric antibody addition MHCC97h, detection MHCC97h traverse Matrigel substrate (BD) coated BDFalconTM24 abilities cultivating cells, after D shows low express cell system BelshGP73 is seeded in nude mice by subcutaneous, after injecting anti-GP73 chimeric antibody, the tumor size of measurement, E shows after processing according to the method identical with D, by sacrifice, peels off tumor acquired results.
Detailed description of the invention
Unless specifically stated otherwise, reagent used in following example is analytical pure level reagent, and can from regular distributor available from.
Embodiment 1GP73 can promote that tumor occurs
Tumorigenic hypothesis is promoted for checking GP73, we are in hepatocellular carcinoma cell line HepG2 (purchased from American DSMZ, ATCC) with reversion virus overexpression GP73 (HepG2GP73 group) in, with zero load reversion virus (HepG2v group) for comparison, build GP73 overexpressing cell line, respectively HepG2GP73 cell line and HepG2v cell line.Specifically comprise the following steps that people gp73cdna (mhs1011-59469, OpenBiosystems, Inc.) coded sequence and FLAG are cloned into plxin-hyg vector expression GP73-FLAG fusion vector.Transfect this plasmid with liposome 2000 and plxin-hyg (comparison HepG2v group) enters packaging cell line PT67 and produces retrovirus.After 48 hours, it is filtered through the filter of 0.45 millimeter containing virulent conditioned medium, is subsequently used for transfectional cell.100 mg/ml hygromycinB are used for screening transfection positive cell.Result as shown in Figure 1A, and becomes tumor level by detecting the cell proliferation of this cell line, plate clone with nude mice, it has been found that GP73 can promote cell proliferation and tumor development (result is shown in Figure 1B-D).
Wherein, Figure 1A is with reversion virus overexpression GP73 (HepG2GP73 group) in hepatocellular carcinoma cell line HepG2, detects GP73 expression with zero load reversion virus for comparison (HepG2v group) westernblot.The cell proliferation curve of B, HepG2GP73 and HepG2v.There is (left side) and time-to-live (right side) in the plate clone of C, HepG2GP73 and HepG2v cell and result statistics (D) HepG2GP73 and HepG2v tumor in tumor formation in nude mice thereof.
And in hepatoma cell line Bel7402 (the purchased from American DSMZ of high expressed GP73, ATCC) in, shRNA strikes low GP73 (BelshGP73), with empty carrier (Belshv) for comparison, build the low express cell system of GP73, respectively BelshGP73 cell line and Belshv cell line, specifically comprises the following steps that the target of shRNA is according to different target gene design.Forward and reverse primer are annealed and be cloned into pLL3.7 carrier.Target sequence is as follows: SEQIDNO:2:GGGAATGAAGCTGTAGCTTCC.Pll3.7-shRNA and package carrier VSVG, REV, andpMDL corotation enter 293T cell and produce slow virus plasmid.Transfect latter 48 hours, filtered by the filter of 0.45 millimeter containing virulent conditioned medium, be subsequently used for transfectional cell；Detect the GP73 level in cell culture fluid by Westernblotting, and detect its one-tenth tumor level by tumor formation in nude mice.Specifically comprise the following steps that immunodeficiency nude mice (BALB/C, 68 week old) (Institute of Experimental Animals, Chinese Academy of Medical Sciences).Every eight random packet of mice.After tumor cell 5x106 subcutaneous injection, a situation arises and tumor size for every weekly check tumor, draws curve, and tumor, more than 1000 cubic millimeters, above has ulcer or tumor, and the loss in weight is designated as dead mouse more than 10%.Result is as shown in figures 5 a-b.
Becoming tumor level by the nude mice of the above cell line of tumor formation in nude mice, result is such as shown in Fig. 1 C-D, it has been found that GP73 can promote that cell proliferation and tumor occur.
The hypersecretion of embodiment 2GP73 can cause cell hyperproliferation
Hypersecretion for proving GP73 can cause the reason of cell hyperproliferation, and we are 2 × 106Individual GP73 high-expression cell line HepG2GP73 and HepG2v is in DMEM culture medium, in 37 DEG C, and 5%CO2After constant incubator is cultivated 48 hours, collection condition culture fluid.Cultivate HepG2, MEF, MHCC97H, MHCC97HshGP73, PC3 cell and normal mouse fibroblast (MEF) with conditioned medium respectively, detect cell proliferation.Specific as follows: 3000-5000 cell is seeded at 96 orifice plates, 37 DEG C, 5%CO2Removing normal culture fluid after hatching one day in constant incubator, add 200 μ L conditioned mediums, comparison is changed without.Within every 24 hours, carry out MTT and detect cell proliferation.Step: in 96 orifice plates, cultivated cell adds 200 μ L fresh cultures and 20 μ LMTT reagent [3 (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazoliumbromide；5 mg/ml are dissolved in PBS, 37 DEG C, add 150 μ DMSO/ holes after 1-4 hour.Shading shakes up 10 minutes.Under 492 nanometers, spectral absorbance is measured by microplate reader.Cell proliferation level is sample and the luminous absorptance value compareed.).By 2 × 106Individual GP73 low express cell system Bel7402 in DMEM culture medium, 37 DEG C, 5%CO2Constant incubator is cultivated 48 hours, collects cell culture fluid, cultivates cell, detect cell proliferation as previously mentioned after the affinity column removal GP73 that GP73 monoclonal antibody couples, and result is as shown in fig. 2 a-d.
Experimental result shows, culture fluid is after cultivating GP73 high expressing cell, and the low express cell culture fluid of relative GP73, on cell proliferation has facilitation.
In order to more directly check the impact of the GP73 on cell proliferation of secretion, using antibody special for GP73 to remove the GP73 in above-mentioned supernatant, it has been found that after removing GP73, this supernatant incubated cell causes that cell proliferation level declines (2E-G).Wherein, Fig. 2 E, after hatching 16 hours such as front HepG2GP73 cell conditioned medium and GP73 monoclonal antibody 8A10, ProteinGagarose precipitation removes antibody and GP73, cultivates HepG2 cell with the cell culture fluid after processing, and detects cell proliferation.2F, prokaryotic expression GP73 secretory protein total length, N end, C end, and check the effect of its on cell proliferation.Our prokaryotic expression removes the secretion GP73 albumen of signal peptide, it has been found that hepatoma carcinoma cell ties up to propagation level in the cell culture fluid after adding GP73 to be increased.
Embodiment 3 can treat hepatocellular carcinoma by GP73 inhibitor-shGP73
Suppress the cell proliferation level after GP73 at cell-based assay, inquire into the probability by GP73 inhibitor for treating hepatocellular carcinoma.We first pass through RNAinterference method and strike low GP73 in hepatoma cell line HepG2.2.15 and MHCC97H, it has been found that shGP73 can suppress the propagation of hepatoma carcinoma cell, plate clone formed with subcutaneous become tumor (Fig. 3 I).
A and D in Fig. 3, expresses shGP73 in hepatoma cell line HepG2.2.15 (A) and MHCC97H (D) and strikes low GP73, detect GP73 expression with zero load for comparison (v) westernblot.The cell proliferation curve of B and E, HepG2.2.15shGP73 (B) and MHCC97HshGP73 (E).The plate clone of C and F, HepG2.2.15shGP73 (C) and MHCC97HshGP73 (F).G, plate clone experimental result is added up；There is (left side) and time-to-live (right side) in H and I, HepG2.2.15shGP73 (H) and MHCC97HshGP73 (I) tumor in tumor formation in nude mice.
The preparation of the chimeric antibody of embodiment 4 energy specific recognition GP73
GP73 monoclonal antibody entrusts Jing Tiancheng bio tech ltd to prepare.The monoclonal antibody people GP73C terminal peptide fragment immunity Balb/c mice by expressing with prokaryote preparation.From the splenocyte of immune mouse and myeloma cell, the hybridoma of gained is taped against 96 orifice plates, detects it by ELISA and immunoblotting (Fig. 4 A-B) and identify restructuring GP73 molecule ability screening positive clone.Positive colony is by sub-clone and amplification, and injection mice produces ascites, obtains antibody after purification.The specific recognition of GP73 high expression tumour cell is identified (Fig. 4 C) by Flow cytometric analysis by antibody, specific as follows: 106Cell is hatched through 8A10GP73 monoclonal antibody (40 μ g/milliliter), and the Mus IgG1 bis-being subsequently adding PE labelling resists.GuavaeasyCyte6HT-2L flow cytometer (EMDMillipore) carries out flow cytometry, and suitably mates Isotype antibody with comparing.The purity of the cell separated and survival rate are respectively > 90%.
Preparing the aminoacid sequence of variable region light chain of chimeric antibody such as shown in SEQIDNO:3, the aminoacid sequence of its Variable region heavy is such as shown in SEQIDNO:4.
With encoding amino acid sequence be SEQIDNO:3 the gene order SEQIDNO:5 of variable region light chain and encoding amino acid sequence be SEQIDNO:4 the gene order SEQIDNO:6 carrier construction of Variable region heavy.
Variable region light chain aminoacid sequence SEQIDNO:3
MDLQVQIISFLLISVTVIVSNGEIVLTQSPTTMAASPGEKITITCSANSSISSNYLHWYQQKPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGPMEAEDVATYYCQQGYSIPLTFGAGTKVELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSCTCEATHKTSTSPIVKSFNRNEC
Variable region heavy aminoacid sequence SEQIDNO:4
MDWVWNLLFLMAAAQSAQAQIQLVQSGPELKKPGETVRISCKASGYTFTNYGMNWVKQAPGKGLKWVGWINTYTGEPTYVDDFKGRFAFSLETSASIAYLQINNLKNEDTATYFCARSFYDNDAYWGHGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGFLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC
Variable region light chain gene order
SEQIDNO:5
AAGAGCATGACGGCAGTGGACGATTATGGATTTACAGGTGCAGATTATCAGCTTCCTGCTAATCAGTGTCACAGTCATAGTGTCTAATGGAGAAATTGTGCTCACCCAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAGAAGATCACTATCACCTGCAGTGCCAATTCAAGTATAAGTTCCAATTACTTACATTGGTATCAGCAGAAGCCAGGATTCTCCCCTAAACTCTTGATTTATAGGACATCCAATCTGGCTTCCGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATTGGCCCCATGGAGGCTGAAGATGTTGCCACTTACTACTGCCAGCAGGGTTATAGTATACCGCTCACGTTCGGTGCTGGGACCAAGGTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAACGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTGTACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTGATGAAATCTCCAGAGGATCGCCGGGA
Variable region heavy gene order SEQIDNO:6
AGAGCATGACGGCAAGTGGACGATTATGGATTGGGTGTGGAACTTGCTATTCCTGATGGCAGCTGCCCAAAGTGCCCAAGCACAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAGGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGCTTAAAGTGGGTGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGTTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGTATTGCTTATCTGCAGATCAACAATCTCAAAAATGAGGACACGGCTACATATTTCTGTGCACGATCGTTCTATGATAACGACGCTTACTGGGGCCACGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATTCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTTAGAATCTCCAGAGGATCGCCGGGAACC
The checking of the anti-GP73 chimeric antibody of embodiment 5
By as previously mentioned at the subcutaneous one-tenth tumor model of Balb/C mice, about 100mm will be about by tumor size3Mice random packet, the then possibility of the oncotherapy of the anti-GP73 chimeric antibody that detection embodiment 4 prepares.
Specifically, by 3 × 105In the DMEM culture medium of Bel7402 cell, adding chimeric antibody with concentration as shown in Figure 5A respectively and cultivate 48 hours, mtt assay detects its cell proliferation as previously mentioned, and result is as shown in Figure 5A, it was shown that this antibody can suppress the propagation of Bel7402 cell.
Will in the DMEM culture medium of human hepatoma cell strain MHCC97h add antibody, and be not added with antibody culture medium for negative control, by cell scratch experiment detect corresponding antibodies on cell migration ability impact, concrete grammar is as follows: add about 5 × 10 in six orifice plates5Individual cell, secondary daily rifle head cut, PBS washes cell 3 times, removes the cell under drawing, and adds plasma-free DMEM medium, puts into 37 degree of 5%CO2Incubator.Sampled by 0,6,12,24 hour, take pictures.Result is as shown in Figure 5 B, it was shown that this antibody can suppress to implant the Bel7402 tumor formed at nude mice by subcutaneous and develop.
By anti-GP73 chimeric antibody, and after the DMEM culture medium of comparison IgG addition MHCC97h, sample in 0h, 12h, violet staining detection MHCC97h traverse Matrigel substrate (BD) coated BDFalconTM24 abilities cultivating cell, thus detecting the corresponding antibodies impact on cell invasion ability, result is as shown in Figure 5 C, it was shown that anti-GP73 monoclonal antibody 8A10 and 15A7 all can suppress migration and the invasion and attack of human hepatoma cell strain MHCC97h.
By 3 × 106After the individual low cell line Bel7402 cell expressing GP73 is seeded in BALB/c nude mice by subcutaneous three weeks, with concentration injection anti-GP73 monoclonal antibody 5G2,6A6 and the 8A10 antibody of 400 μ g/ time/, with same volume PBS for comparison.Twice weekly, continuously after injection three weeks, and tumor sizes are measured in the 5th, 8,11,15,18 and 22 days during injection and after injection, and result as shown in Figure 5 D, and after being processed according to abovementioned steps by BALB/c nude mice, peels off tumor, and result is as shown in fig. 5e.
To sum up, our experiment finds that the cell of high expressed GP73 can promote cell proliferation and become tumor, points out its rising to be likely to relevant to the conversion process of hepatocarcinoma with hepatocarcinoma generation and hepatitis.Our work shows further, cell and animal horizontally through the GP73 chimeric antibody neutralisation treatment prepared by the present invention, can suppressing cell proliferation, migration, invasion and attack and tumor growth, therefore, GP73 inhibitor is probably prevention and the targeted drug for the treatment of hepatocellular carcinoma.
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Claims (8)
1. a GP73 inhibitor, it is characterized in that, described GP73 inhibitor include suppress GP73 correctly shear the factor, the factor that can suppress or hinder GP73 to be combined with receptor, can specificity suppress GP73 siRNA, can specificity suppression GP73 shRNA, energy specificity suppression GP73 microRNA, energy specificity suppression GP73 antibody, and whole body or local proceed to, express or the inhibitive factor of activated code GP73 inhibitive factor, its albumen composition composing factor, or the DNA plasmid of its dominant negatives.
2. GP73 inhibitor according to claim 1, it is characterized in that, the factor that the described GP73 of suppression correctly shears includes GP73 and shears the Furin inhibitor 1 of required convertase Furin, it is preferably Dec-RVKR-CMK, Furin inhibitor 2, it is preferably many poly arginines peptide, 2, 5-deoxystreptamine complex, α-1 antitrypsin silicate, MACl (2), wherein, M is copper or zinc, A is chelate group, preferably, described MACl (2) includes Cu (TTP) Cl (2) and Zn (TTP) Cl (2), wherein, TTP is 4'-[p-tolyl]-2, 2':6', 2 "-terpyridyls；
Preferably, the siRNA of GP73 can be suppressed, the shRNA of GP73 can be suppressed or the target sequence of microRNA of GP73 can be suppressed to be have at least the sequence of 90% homology with the nucleotide sequence shown in SEQIDNO:2 by specificity by specificity by specificity.
3. GP73 inhibitor according to claim 1, it is characterised in that described the antibody of GP73 can be suppressed to be anti-GP73 antibody by specificity, described anti-GP73 antibody the antigen of specific recognition can be selected from protein:
() aminoacid sequence is the aminoacid sequence shown in SEQIDNO:1；Or
() passes through replacement, disappearance or one or several amino acid derived protein of superposition in the aminoacid sequence that () limits and has identical function with the protein shown in ().
4. GP73 inhibitor according to claim 3, it is characterised in that described anti-GP73 antibody is the anti-GP73 chimeric antibody comprising the aminoacid sequence such as variable region light chain shown in SEQIDNO:3 and the aminoacid sequence such as Variable region heavy shown in SEQIDNO:4.
5. GP73 inhibitor according to claim 4, it is characterized in that, the variable region light chain of described anti-GP73 chimeric antibody is the gene code being classified as SEQIDNO:5 by nucleotides sequence, and the Variable region heavy of described anti-GP73 antibody is the gene code being classified as SEQIDNO:6 by nucleotides sequence.
6. the GP73 inhibitor according to any one of Claims 1 to 5 preparation for treat hepatocarcinoma, HBV infection cause hepatic fibrosis and liver cirrhosis until hepatocellular carcinoma process medicine in application.
7. the GP73 inhibitor according to any one of Claims 1 to 5 is preparing the application being used for suppressing in the medicine of fucosylation and invasion and attack, preferably, described GP73 inhibitor is the chimeric antibody comprising the aminoacid sequence such as variable region light chain shown in SEQIDNO:3 and the aminoacid sequence such as Variable region heavy shown in SEQIDNO:4.
8. for treating hepatocarcinoma, hepatic fibrosis that HBV infection causes and liver cirrhosis until the pharmaceutical composition of process for hepatocellular carcinoma, described pharmaceutical composition comprises the GP73 inhibitor described in the claim 1 of effective dose and pharmaceutically acceptable carrier or adjuvant；Preferably, described GP73 inhibitor is anti-GP73 antibody；It is highly preferred that the GP73 inhibitor of described effective dose is local concentration is 20-250ug/mL.
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