CN100387716C - Preparation of KTV series products for inhibiting tumor cell growth and use - Google Patents

Preparation of KTV series products for inhibiting tumor cell growth and use Download PDF

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CN100387716C
CN100387716C CNB2004100622966A CN200410062296A CN100387716C CN 100387716 C CN100387716 C CN 100387716C CN B2004100622966 A CNB2004100622966 A CN B2004100622966A CN 200410062296 A CN200410062296 A CN 200410062296A CN 100387716 C CN100387716 C CN 100387716C
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CN1587411A (en
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于在林
富岩
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Fortune Rock China Co ltd
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Tianjin Sino Biotechnology Ltd Fortunerock Inc
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Abstract

The present invention provides a construction method KTV series recombinant viruses for inhibiting the growth of tumor cells by taking viruses as carriers. A recombinant insect baculovirus and a recombinant human adenovirus which carry specific inhibiting gene Rb1 or Rb2 of a specific human tumor can be separately or jointly used for inhibiting the growth of tumor cells or the hyperplasia of tumor bodies, or can be used for clinically treating solid tumors and cancer.

Description

The preparation and the purposes that suppress the KTV series product of growth of tumour cell
Invention field
The present invention relates to utilize insect baculovirus or adenovirus hominis to suppress the recombinant virus KTV (Killing Tumor Viruses) of growth of tumour cell for the series of vector construction.Carry people's tomour specific suppressor gene Rb1 or the KTV of Rb2, can suppress the growth of tumour cell, can be used for the clinical treatment of tumour.For example, KTV-4 in experiment (LdMNPV-Rb2), KTV-7 (AcMNPV-Rb2) can suppress the growth of liver cancer cell, and KTV-8 (Ad-Rb1) and KTV-9 (Ad-Rb2) can be used for suppressing the growth of multiple solid tumor cell.Animal experiment shows that early stage use KTV-9 also can suppress the tumour growth in animal body and the formation of knurl body.
Association area is described
1. virus vector
To in the artificial transfered cell of foreign gene several different methods can be arranged, for example, electric shocking method (Electroporation), calcium precipitation method, liposome method and utilize the gene that the infection ability of virus will be to be expressed to bring (MichaelKriegler in the cell into, in " Gene Transfer and Expression:a laboratory manual ", W.H.Freeman and Company, NY, NY).Multiple virus vector, as adenovirus hominis (Ad), adenovirus hominis associated virus (AAV), retrovirus (RV), Epstein-Barr virus, born of the same parents' exanthema virus (HSV), insect baculovirus (BV) etc. Virus vectorBe applied to CarryGoal gene AndTransduction enters in the cell paste.And (Yamada et al., PNAS, 82:3567,1985 (Ad) in the clinical trial of human gene therapy have been applied to; Kasid et al., PNAS, 87:473,1990 (RV); Sugden et al., Mol.Cell.Biol.5:410,1985 (EB); Spaete andFrenkel, Cell 30:295,1982 (HSV); Hofmann et al., PNAS, 92:10099-103,1995 (BV)).
Adenovirus hominis is to use virus vector comparatively widely.The form of adenovirus is the double-stranded DNA of a linearity of distinctive icosahedron viruses housing, its 5 ' end and a kind of terminal protein (TP) covalent attachment.The member of adenovirus family (Adenoviridae) can infect the cell after the kind mitotic division quite widely, even comprises from the cell in the well differentiated tissue, for example Skeletal Muscle Cell, pneumonocyte, brain cell and heart cell.Because adenovirus can be delivered to the genome of self in the nucleus, and duplicates expeditiously, so adenovirus becomes the main candidate of expressing and transmitting therapeutic gene.But some adenovirus is tumorigenesis in animal body, at external energy transformant.Be used for gene therapy and be to use adenovirus 5 types human body safety as the adenovirus of gene therapy medicament.
Adenovirus carrier can transmit the ability (especially external) with expressing gene efficiently, is confirmed fully in the past 15 years and puts down in writing.Yet intravital immune response has limited the practical application and the development of adenovirus carrier.Therefore, require the transgenosis continuous expression with the case that remedies damaged gene activity in, control that very crucial a bit is exactly or inhibition are at virus vector and genetically modified immune response.Opposite, cancer therapy but can because of active immunoreactive induce to produce increase curative effect.Utilize different virus to carry out the also existing multiple patent application of clinical therapy of tumor as carrier, now list as a reference: US6740320,6638502,6663856,6297357,5532340,6511847 and 5457049, and the disclosed patent application publication number of China: 1244215,1258737,1260998 and 1471977.But use the adenovirus that contains tumor suppressor gene that insect viruses and tumor suppressor gene reach simply as the oncotherapy means and structure can be used for clinical cancer therapy efficiently, do not appear in the newspapers as yet and practical application.
Insect baculovirus (Baculovirus) has been widely used in the control (Yu Zailin of agriculture and forestry injurious insect, forestry scientific research, 2 (3): 1-6,1989) and also have a wide range of applications at exogenous gene expression (O ' Reilly etal., Baculoviurs Expression Vectors:a laboratory manual, W.H.Freeman andCompany NY, NY, 1992).The security of insect baculovirus has obtained multiple zooperal proof.Multiple baculovirus has been finished the research as virus expression carrier, and efficiently expressing and novel recombinant insect virus sterilant of practical application what external source recombinant protein.For example autographa californica nuclear polyhedrosis virus (AcMNPV) expression system (Smith et al., Mol.Cell.Biol., 3:2156-2165,1983) and gypsymoth nucleopolyhedrosis virus (LdMNPV) expression system (Yu et al., J.Gen.Virol., 73:1509-1514,1992).Recently new research and discovery are also arranged aspect the vertebrate cells utilizing baculovirus to enter, and become supporting evidence of the present invention (Ghosh et al., Mol.Ther., 6:5-11,2002) as the carrier transduction foreign gene.
2. promotor and enhanser
Promotor and enhanser are that foreign gene obtains the prerequisite expressed in cell, and promotor can be an external source, the cell institute inherent that also can be transduceed.The enhancing promotor (Enhancer-Promoter) of people CMV (cytomegalovirus) be stronger, use also extensive.Other promotor and enhanser have from virus, for example: SV40 virus, Polyoma virus, retrovirus, Papilloma virus, hepatitis B virus, HIV's and CMV's promotor and enhanser; Also have enhanser in the cell in addition, be divided into the startup enhanser of two para-immunity system genes and the startup enhanser of the intrinsic gene of cell.The former has beta-interferon, T-cell receptors gene, immunoglobulin gene etc.; The latter has the promotor and the enhanser of β-actin, serum albumin, GAPDH gene.Promotor can be that induction type also can be non-induction type.
3. human tumor suppressor gene
Human tumor suppressor gene (TSGs) is considered to a class and relates to the control normal cell at growth and the splitted gene of different steps when (cell cycle cycle).Their common feature is just to cause the generation (or growth) of tumour and maybe in being imported into tumour cell the time, can suppressing the growth of tumour cell or make it enter apoptosis when their heritable variation take place or disappearance takes place.Human retina glucagonoma Rb1 is human first tumor suppressor gene (Lee, W.H., the et al. that finds and report, Human retinoblastoma susceptibility gene:cloning, identification, and sequence, Science 235:1394-1399,1987).The another name of Rb1 gene is Rb, is seated in karyomit(e) 13q14.2 site, and gene expression characteristics is described as retinoblastoma 1 (comprising osteosarcoma).Rb2 and Rb2/p130 and p130 are the another names of gene RBL2, and it is seated in karyomit(e) 16q12.2 site, and gene expression characteristics is described as Retinoblastoma-like 2 (p130) (Mayol, et al., Oncogene, 8:2561-2566,1993; Howard et al., Journal of the NationalInstitute, 90 (19): 1451-1460,1998; Giordano and Kaiser, in vivo 10:223-228,1996; Masciulio et al., International Journal of Oncology, 17:897-902,2000).Tumor suppressor gene is multiple in addition, p53 for example, Rb1, Rb2, p107, WT1, NF1 etc.Rb (Rb1, Rb2) and p53 gene are and the tumour the closest tumor suppressor gene that is related that the tumour more than 50% exists unusual (the comprising point mutation, allelotrope disappearance, rearrangement, insertion, gene fusion etc.) of p53 gene.The Rb2 gene is that variation has taken place in 90% tumour.When the external source method imported in Rb and p53 gene or the protein tumour cell, the growth of kinds of tumor cells all was suppressed.The generation of Rb and p53 gene and tumour, development and clinical efficacy all have substantial connection (Esposito et al., International Journal of Oncology, 9:439-443,1996).People constantly explore application Rb and p53 gene pairs tumour is treated, the external at present main recombinant adenovirus-p53 (Ad-p53) of application and recombinant adenovirus-Rb carry out clinical tumor and study (Habib NA, Hodgson HJ, LemoineNA, et al.Phase I/II study of hepatic artery infusion with wtp53-CMV-Ad inmetastatic malignant liver tumours.Hum Gene Ther, 1999,10:2019-2034; Roth, et al., p53 tumor suppressor gene therapy for cancer.Oncology, 1999,13:148-154).
The description of summary of the invention
The present invention relates to can be used for the field of tumour and gene therapy for cancer, more specifically, the present invention relates to make up with virus, be carrier particularly with insect baculovirus and adenovirus hominis, carry people's tomour specific suppressor gene, be used to suppress the preparation and the purposes of the KTV series product of growth of tumour cell.
The security of insect baculovirus and adenovirus hominis makes it obtain practical application in medical science and clinical medicine domain.Is in present existing technology the most effective and strong as carrier with importing in the cell of specific gene with virus.This provides the basis and the technology of science for variation, disappearance, fusion owing to gene cause genetic treatment of tumor, and has obtained the support of clinical study widely.Discover that Rb1 and Rb2 gene have point mutation in kinds of tumor cells, sequence deletion or karyomit(e) dislocation or dystopy merge, and cause Rb1 and the Rb2 gene biological function in cell to be lost, the cell cycle cycle enters (Howard et al. out of control, Journal of the National CancerInstitute, 90 (19): 1451-1460,1998).When test showed Rb1 or Rb2 gene importing tumour cell, the growth of tumour cell just was suppressed and causes tumour cell to enter apoptosis (Apoptosis) (Claudio, PPet al., Cancer Research, 60:8-12,2000).The present invention uses insect baculovirus and adenovirus hominis to carry Rb1 or Rb2 gene, and growth of tumour cell suppresses and the clinical treatment purpose provides new technology and means in order to be used for.
The invention provides and can be used for genetic treatment of tumor medicine KTV, it can overcome the defective of existing gene therapy product, for example, uses p53 tumor suppressor gene treatment noumenal tumour, and 50% validity is only arranged.And use KTV, will have the growth-inhibiting effect to 90% solid tumor cell.The KTV tumor suppression product of technique construction provided by the invention has range of application widely.
Tumor suppressor gene can use several different methods directly to import or carry importing by carrier.Carrier can be a virus, comprises, but does not limit to what, adenovirus (Ad), adeno-associated virus (AAV), retrovirus (RV), Epstein-Barr virus, born of the same parents' exanthema virus (HSV) or insect viruses.First-selected insect viral vectors is, but do not limit to what, insect baculovirus (Bacloviruses): nucleopolyhedrosis virus (single-or Multiple PolyhandrenViruses) and granulosis virus(GV) (Granuloviruses), nucleopolyhedrosis virus preferably, more preferably, AcMNPV (autographa californica nuclear polyhedrosis virus) and LdMNPV (gypsymoth nucleopolyhedrosis virus).First-selected Human virus's carrier is adenovirus and adeno-associated virus, most preferably adenovirus.
First-selected is human tumor suppressor gene, anti-metastasis gene or have inducing tumor cell and enter apoptotic gene, comprise but do not limit to what, Rb1, Rb2, p53, p21, p16, MST2, Hippo, Salvador, Warts, PTEN, TSCl (Tuberous Sclerosis 1), TSC2, Kail, WT1, NF1 etc.Preferably, Rb2 or Rb1 tumor suppressor gene.
Promotor and enhanser can be used for the expression of tumor suppressor gene in the tumour cell that is imported into.Promotor and enhanser comprise, but be not limited to promotor and the enhanser of the enhancing promotor (Enhancer-Promoter) of CMV (cytomegalovirus), SV40 virus, Polyoma virus, retrovirus, Papilloma virus, hepatitis B virus or HIV; Enhanser in the cell comprises the startup enhanser of two para-immunity system genes and the startup enhanser of the intrinsic gene of cell: the promotor and the enhanser of beta-interferon, T-cell receptors gene, immunoglobulin gene and β-actin, serum albumin, GAPDH gene.Promotor can be that induction type also can be non-induction type.Preferably CMV starts and enhanser.
KTV can be used for suppressing the growth and the transfer of kinds of tumors, and is the means of purpose and method as treatment.These tumours or cancer include, but are not limited to, the tumour or the cancer at brain, lung, liver, stomach, kidney, spleen, intestines, eye, mammary gland, ovary, uterus, prostate gland, uterine neck, neck position (comprising dispute), osteosarcoma, soft tissue sarcoma and melanoma.Can be used for treating primary, recurrent or metastatic tumo(u)r or cancer.The method of treatment can be local injection, intravenously administrable, perfusion, spray, be coated with or absorb administration through the stomach and intestine of mouth and nose.
The construction process of KTV is mainly,
1. the molecular cloning of people's tumor suppressor gene.
2. people's tumor suppressor gene places CMV to start between enhanser and SV40PolyA signal terminating, forms a genetic expression assembly.The expression of the messenger RNA(mRNA) of promoters driven tumor suppressor gene also stops at the terminator place.
3. will express assembly is inserted into the special metastasis transplanting physique grain of virus or tumor suppressor gene is inserted in the viral special metastasis transplanting physique grain that contains promotor and terminator.
4. metastasis transplanting physique grain is recombinated with the homologous sequence of virus in insect cell (AcNPV/LdNPV) or 293 cells (adenovirus).
5. screening and purifying contain tumor suppressor gene and the recombinant virus of expressing tumor suppressor gene in cell.
6. virus formulationization and be used in the cell and the intravital test of animal.
7. carry out the security and the validity of clinical trial checking recombinant virus inhibition tumor growth.
8. the recombinant virus of the present invention's description can be used as gene therapy medicament and uses the what tumor treatment.
The description of accompanying drawing
Fig. 1. the dna nucleotide sequence of human tumor suppressor gene Rb1 and Rb2, protein amino acid sequence
Fig. 2. insect baculovirus lymantria dispar nuclear polyhedrosis virus, autographa california nuclear polyhedrosis virus and adenovirus hominis metastasis transplanting physique grain collection of illustrative plates.
Fig. 3. behind the recombinant virus transfection H23 tumour cell of carrier's tumor suppressor gene, use the protein immunoblotting test and detect the proteinic overexpression of Rb2.(A) the Rb2 protein of purifying; (B) KTV-5; (C) KTV-7; (D) KTV-9; (E) KTV-9 injection back tumour somatocyte.
Fig. 4. behind the recombinant virus transfection H23 tumour cell of carrier's tumor suppressor gene, use the mRNA expression that PCR method detects human tumor suppressor gene Rb1.(A) untransfected H23 tumour cell; (B) 1Kb DNA standard molecular weight (kilobase is to Kb); (C) KTV-4; (D) KTV-6; (E) KTV-8; (F) the tumour somatocyte of injection KTV-9.
Fig. 5. use that injection will be carried or not the recombinant virus of carrier's tumor suppressor gene import in artificial graft's tumour body gross tumor volume variation tendency in different time.A), after the tumour body that shows the artificial graft injects the recombinant virus of carrier's tumor suppressor gene not and injects KTV respectively, the tumour body disappears scope about 60-95%.
Specific embodiment
Embodiment 1 molecule clone technology
Conventional molecule clone technology comprises the extraction of DNA, RNA, sepharose and polyacrylamide gel electrophoresis, the connection of dna fragmentation, digestion with restriction enzyme reacts equal reference literature (Maniatis, et al., " molecular cloning laboratory manual " cold spring harbor laboratory publishes, the cold spring port, New York, 1982).The enzyme that archaeal dna polymerase chain reaction (PCR) (reference literature Saikiet al., science, 230:1350,1985) is used and react required PCR instrument and be Perkin Elmer product.And with reference to producer's schedule of operation.The oligonucleotide primer of dna sequencing and the required usefulness of DNA cloning is finished by functional body.Changeing buied by GIBCO/BRL company by attitude E.coli bacterium.The purifying of plasmid DNA, the recovery of dna fragmentation etc. all adopt the preparation of commodity Qiagen purification column.Adenovirus recombination vector used in the present invention is from company (Q-Biogene, Canada).Insect baculovirus gypsymoth LdMNPV recombinant plasmid used in the present invention is from (the Yu et al. of the big BTI in U.S. Connell institute, J.Gen.Virol., 73:1509-1514,1992), autographa california AcMNPV recombinant plasmid system and contain CMV and start the vector plasmid of enhanser and LdMNPV-LacZ, AcMNPV-LacZ or adenovirus hominis Ad-LacZ strain and all make up (U.S. Tom Si Jefferson university, Kimmer Cancer center protein expression research department) in the research department of Yu Zailin.
The molecular cloning of embodiment 2 human tumor specific suppressor gene Rb1 and Rb2
People's Rb1 gene is to clone from people's newborn placenta eDNA library according to the PCR primer of gene pool (GenbankUS) gi:190958DNA sequences Design.The dna sequence dna of primer is as follows:
Seq ID No 5:5 '- GGATCCATGCCGCCCAAAACCCCCCG-3 ' (the BamHI restriction enzyme shows it with underscore) and
Seq ID No 6:5 '- CCCGGCTCATTTCTCTTCCTTGTTTGAGG-3 ' (Sma I restriction enzyme shows it with underscore).People's Rb2 gene clone is to be that the template clone obtains according to the PCR primer of gene pool (Genbank US) gi:397147DNA sequences Design with the plasmid DNA that contains the Rb2 gene.The dna sequence dna of primer is as follows: Seq ID No 7:5 '- GGATCCATGGACGAGGCGGCGCGGGC-3 ' (the BamHI restriction enzyme shows it with underscore) and Seq ID No 8:5 '
- CCCGGGTCAGTGGGAACCACGGTCATTAGC-3 ' (Sma I restriction enzyme shows it with underscore).
The reaction of pcr amplification gene is by the 1XPCR damping fluid in different test tubes, 200 micromolar dNTP, the dna profiling of 5 micrograms (Rb1 gene clone end user placenta cDNA, plasmid DNA is used in the Rb2 gene clone), the PCR primer mixture of 100 nanomoles, the Taq DNA polymerase of 5 units adds water to final volume 50 microlitres.The PCR response procedures is 94 ℃ of 5 minutes denatured DNAs, carry out then 25 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ of circulations of 3.5 minutes.Last 72 ℃ of insulations 10 minutes also are cooled to room temperature or 4 ℃.Use agarose gel electrophoresis and identify the molecular weight of PCR product.The long 2.8kb of PCR product of Rb1 gene, the long 3.2kb of PCR product of Rb2 gene, this is consistent with bibliographical information.Dna nucleotide sequence, the protein amino acid sequence of Rb1 and Rb2 gene are seen Fig. 1.
Embodiment 3 recombinant baculovirus KTV-4,5,6,7 structure
PLd-CMV (LdMNPV) and pAc-CMV (AcMNPV) insect viral vectors plasmid all contain the compound cloning site of restriction enzyme and CMV promotor and enhanser applied molecular biology method are inserted in the metastasis transplanting physique grain of insect baculovirus LdMNPV and AcMNPV and keep compound cloning site (seeing Fig. 2, A, B).The homologous sequence reorganization partly takes place in the polyhedrin promotor in the genome what metastasis transplanting physique grain of virus, and CMV promotor, enhanser and external source are treated that expressing gene (Rb1, Rb2 or LacZ) is inserted in the genome of virus.People Rb1 and Rb2 gene by the PCR clone obtains are inserted between the BamHI and SmaI restriction endonuclease sites of pAcYZ carrier.Use different restriction endonucleases and identify that it has correct direction of insertion, finishes the structure of recombinant transfer vector plasmid.
With the reorganization of virus genomic homologous sequence be with the virus vector plasmid and contain the linearizing LdMNPV of LacZ gene or AcMNPV (Gibco/BRL, participation USA) is cotransfection gypsy moth cell (IPLB-LD-652Y) or SF9 cell respectively down at liposome Lipofectin.Use the recombinant virus that the separation and purification of plaque method contains Rb1 or Rb2.Difference called after KTV-4 (LdMNPV-Rb1) ,-5 (LdMNPV-Rb2) ,-6 (AcMNPV-Rb1) and-7 (AcMNPV-Rb2).
The structure of embodiment 4 recombinant human adenovirus KTV-8, KTV-9
It is that the metastasis transplanting physique grain of adenovirus hominis (is seen Fig. 2, C) that vector plasmid contains CMV promotor and enhanser.The homologous sequence reorganization partly takes place in genome and the dna sequence dna in the metastasis transplanting physique grain of virus, and with CMV start enhanser, external source treats that expressing gene (Rb1, Rb2 or LacZ) and terminator SV40PolyA are inserted in the viral genome.By people Rb1 and the Rb2 gene that the PCR clone obtains, be inserted into the Sma I restriction endonuclease sites of pAdYZ carrier through " tackization ".Use the transfer vector plasmid that different restriction endonucleases identifies correct direction of insertion.Vector plasmid DNA and adenovirus Ad-LacZ be cotransfection 293 cells in the presence of liposome Lipofectin, use the recombinant adenovirus that the separation and purification of plaque method contains Rb1 or Rb2, called after KTV-8 (Ad-Rb1) and KTV-9 (Ad-Rb2).
The mensuration of embodiment 5 recombinant virus transduction efficiencies
With AcMNPV-Rb1 is representative, and (Multiplicity of infection MOI) infects liver cancer cell H23 or T98 cell by different MOI.Behind the 48h, discard substratum, behind 10% formalin fixed 10min, (1mg/mL X-gal is dissolved in the 0.1mol/L dimethyl formamide to add the X-gal dye liquor, 1.3mmol/L MgCl2,3mmol/L K3Fe (CN) 6,3mmol/L K4Fe (CN) 6), put 37 ℃ of incubator effect 2h to spending the night. and the percentage of counting blue-stained cell under the mirror.The transduction rate of virus all reaches 95%.Use the RT-PCR method and verify that also the gene of transduction obtains expressing in cell.
Embodiment 6 protein immunoblot analytical tests
With the proteic polyclonal antibody of the anti-people Rb2 of rabbit is example, and being used for expressed protein of the definite two kinds of viruses of protein immunoblotting experiment and people's Rb2 proteantigen is homologous protein.Typical protein immunoblotting experiment is that the isolating protein electrophorese of SDS-PAG gel electrophoresis is transferred on nylon or the nitrocellulose membrane, and with specific antibody (first antibody) incubation, the antibody (second antibody) that adds anti-first antibody then combines with first antibody.Because second antibody is through fluorescently-labeled, whole specificity bonded mixture can stay the marking after the X-ray film exposure.The standard protein molecular weight is used to determine the molecular weight of agnoprotein.Fig. 3 shows recombinant baculovirus or adenovirus hominis expressed recombinant protein in institute's cells transfected, and its molecular weight fulfills the expectation, and identical with the proteic antigen of the Rb2 that derives from the people (Fig. 3).
Embodiment 7 tumour cell survival rates are measured
In 6 porocyte culture plates, inoculate 4 * 10 4 power brain cancer cell T98, prostate cancer cell PC3 or liver cancer cell H23, and cultivated 24 hours.Inoculate KTV-6, KTV-7, KTV-8 or the KTV-9 of 1 * 10 6 power virus infection units respectively, and blank (the PBS damping fluid, AcNPV-LacZ or the Ad-LacZ virus that only add equal volume).Each is handled and repeats 3 culture plates.96 hours harvested cells are also counted.Be used to measure the overexpression of the foreign gene of importing after the lysis.
Test-results shows that insect baculovirus and adenovirus hominis can efficiently import foreign gene, and foreign gene is started under the enhanser effect at CMV, can detect exist (the seeing Fig. 3,4) of protein and mRNA in transfectional cell.
Embodiment 8 RT-polymerase chain reaction (RT-PCR)
Detect the Rb1 of people in the tumor tissues or the expression of Rb2mRNA, got the 3rd day and treatment in the 10th day afterwards, tumor tissues.RNA extracts and operates according to Trizol test kit specification sheets.After extracting and detecting the quality of total RNA through agarose gel electrophoresis, be stored in-70 ℃ standby.According to the cDNA sequences Design primer of Rb1 and Rb2, the Rb1 upstream primer is Seq ID No9:5 '-TTGAGGTTGTAATGGCCACA-3 '; The Rb1 downstream primer is Seq ID No 10:5 '-CAGACAGAAGGCGTTCACAA-3 '; The Rb2 upstream primer is Seq ID No 11:5 '-TGCAAGGTATTGCCAATGAA-3 '; The Rb2 downstream primer is Seq ID No 12:5 '-GAATGAGGTCTCCCCTCTCC-3 '.Product size by primer amplification is respectively 543bp (Rb1) and 780bp (Rb2).With β-actin gene primer as internal reference with the check quality of cDNA and the reliability of whole PCR operating process.The cDNA building-up reactions is the total RNA extracts of 1 μ l 0ligo (dT), 20,1.5 μ g, adds DEPC water to cumulative volume 10 μ l.65 ℃, the rearmounted what of 5min adds the synthetic damping fluid 4 μ l of 5 * cDNA on ice subsequently, 0.1mol/L DTT 1 μ l, 40U/ μ l RNA enzyme 1 μ l, 10mmol/L dNTP mixture 2 μ l, 15U/ μ lThermoscript RT 1 μ l adds DEPC water to cumulative volume 20 μ l.React after being mixed, 50 ℃ of 60min, 85 ℃ of 5min add 1 μ l RNA enzyme inhibitors, 37 ℃ of insulation 20min.Preserve or be directly used in the PCR reaction for 20 ℃.Pcr amplification is undertaken by embodiment 2.Amplified production detects through 1% agarose gel electrophoresis.Result's conform to the dna molecular amount of prediction (Fig. 4 is the one-time detection result to Rb1 genetic expression).
Embodiment 9 nude mice subcutaneous transplantation knurl therapeutic tests
Collection is in H23 (ATCC, the U.S.) tumour cell in logarithmic growth stage, washs once and make the single-cell suspension liquid of every milliliter 5 * 10 6 power number of cells with the PBS damping fluid.Subcutaneous in nude mice back of the body both sides respectively by 0.1 milliliter of tumor cell suspension of every site injection.When treating tumor growth to 20 cubic millimeter (about about 15 days), with its branch group of associating with, 3 every group, respectively through 0.1 milliliter of the intratumor injection AcNPV-LacZ of (containing 10 9 power infectious unit pfu), KTV-6 or 7, Ad-LacZ, KTV-8 or 9 recombinant virus injection liquid.Use the diameter of kind of calliper tumour at regular intervals, continue about 3 weeks.Excessive as the knurl body, then humanity is put to death.
Test-results shows that all growth has obvious suppression almost to reach 60%-90% to growth of tumor to inject the KTV that contains tumor suppressor gene.And the tumour body of injection PBS damping fluid, AcMNPV-LacZ, LdMNPV-LacZ and Ad-LacZ recombinant virus is similar to the control group of not doing any processing, and growth of tumor is not suppressed.Fig. 5 has shown that containing the LacZ gene with adenovirus is contrast, and the KTV that insect viruses or adenovirus is contained tumor suppressor gene is injected into tumour body (mm 3) after, the situation that tumor growth is suppressed.
The subcutaneous inhibition transplanted tumor of embodiment 10 nude mices forms test
Press the method for embodiment 9 and prepare tumour cell.Under the butt of nude mice both sides, press 0.1 milliliter of tumor cell suspension of every site injection.Be divided into 4 groups two days later, the recombinant virus injection liquid in the side injection tumour cell position of same nude mice injection KTV-8 or 9, opposite side injection PBS damping fluid.Continue the formation of observation tumour.Injection KTV one side, tumour is not seen the obvious formation that tumour is arranged yet after 20 days.And the formation of tumour is arranged all at PBS damping fluid injection site and control group.
The production and the purifying of embodiment 11 recombinant viruses
Serum free medium suspension cell culture method is adopted in the production of recombinant virus.SF9 insect cell line and SF900-II (Gibco/BRL, the U.S.) liquid culture are used in the production of insect viruses AcMNPV; The production of LdMNPV uses Ip to cultivate down at 27 ℃.The production of adenovirus uses the cell strain of using the acquisition of single cell clone method from 293 cell strains (ATCC, the U.S.) to produce at 37 ℃ of culture condition, and this cell strain is suitable for the suspension culture growth, and viral unit output height is higher than living.The purifying of insect baculovirus and adenovirus uses column chromatography.Cesium chloride gradient centrifugation is used in the evaluation of purity and virion.
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14.Smith?et?al.,Mol.Cell.Biol.,3:2156-2165,1983。
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16.Ghosh?et?al.,Mol.Ther.,6:5-11,2002。
17.Lee,W.H.,et?al.,Science,235:1394-1399,1987。
18.Habib?NA,et?al.,Hum?Gene?Ther,10:2019-2034,1999。
19.Roth,et?al.,Oncology,13:148-154,1999。
20.Saikiet?al.,Science,230:1350,1985。
21.Maniatis,et?al.,“Molecular?Cloning”Cold?Spring?Harbor?LsborstoryPress,Cold?Spring?Harbor,New?YorK,1982。
22. Yu Zailin, forestry scientific research, 2 (3): 1-6,1989
Patent documentation:
US6740320、 US6638502、 US6663856、
US6297357、 US5532340、 US6511847、
US5457049、 CN1244215、 CN1258737、
CN1260998、 CN1471977
Sequence table
<110〉Yu Zailin, Fu Yan
<120〉preparation and the purposes of the KTV series product of inhibition growth of tumour cell
<130> ZYU-051704
<160> 12
<170> Patent?In?version?3.2
<210> 1
<211> 2787
<212> DNA
<213〉people (Homo sapiens)
<400> 1
atgccgccca?aaaccccccg?aaaaacggcc?gccaccgccg?ccgctgccgc?cgcggaaccc 60
ccggcaccgc?cgccgccgcc?ccctcctgag?gaggacccag?agcaggacag?cggcccggag 120
gacctgcctc?tcgtcaggct?tgagtttgaa?gaaacagaag?aacctgattt?tactgcatta 180
tgtcagaaat?taaagatacc?agatcatgtc?agagagagag?cttggttaac?ttgggagaaa 240
gtttcatctg?tggatggagt?attgggaggt?tatattcaaa?agaaaaagga?actgtgggga 300
atctgtatct?ttattgcacg?agttgaccta?gatgagatgt?cgttcacttt?actgagctac 360
agaaaaacat?acgaaatcag?tgtccataaa?ttctttaact?tactaaaaga?aattgatacc 420
agtaccaaag?ttgataatgc?tatgtcaaga?ctgttgaaga?agtatgatgt?attgtttgca 480
ctcttcagca?aattggaaag?gacatgtgaa?cttatatatt?tgacacaacc?cagcagttcg 540
atatctactg?aaataaattc?tgcattggtg?ctaaaagttt?cttggatcac?atttttatta 600
gctaaagggg?aagtattaca?aatggaagat?gatctggtga?tttcatttca?gttaatgcta 660
tgtgtccttg?actattttat?taaactctca?cctcccatgt?tgctcaaaga?accatataaa 720
acagctgtta?tacccattaa?tggttcacct?cgaacaccca?ggcgaggtca?gaacaggagt 780
gcacggatag?caaaacaact?agaaaatgat?acaagaatta?ttgaagttct?ctgtaaagaa 840
catgaatgta?atatagatga?ggtgaaaaat?gtttatttca?aaaattttat?accttttatg 900
aattctcttg?gacttgtaac?atctaatgga?cttccagagg?ttgaaaatct?ttctaaacga 960
tacgaagaaa?tttatcttaa?aaataaagat?ctagatcgaa?gattattttt?ggatcatgat 1020
aaaactcttc?agactgattc?tatagacagt?tttgaaacac?agagaacacc?acgaaaaagt 1080
aaccttgatg?aagaggtgaa?tataattcct?ccacacactc?cagttaggac?tgttatgaac 1140
actatccaac?aattaatgat?gattttaaat?tctgcaagtg?atcaaccttc?agaaaatctg 1200
atttcctatt?ttaacaactg?cacagtgaat?ccaaaagaaa?gtatactgaa?aagagtgaag 1260
gatataggat?acatctttaa?agagaaattt?gctaaagctg?tgggacaggg?ttgtgtcgaa 1320
attggatcac?agcgatacaa?acttggagtt?cgcttgtatt?accgagtaat?ggaatccatg 1380
cttaaatcag?aagaagaacg?attatccatt?caaaatttta?gcaaacttct?gaatgacaac 1440
atttttcata?tgtctttatt?ggcgtgcgct?cttgaggttg?taatggccac?atatagcaga 1500
agtacatctc?agaatcttga?ttctggaaca?gatttgtctt?tcccatggat?tctgaatgtg 1560
cttaatttaa?aagcctttga?tttttacaaa?gtgatcgaaa?gttttatcaa?agcagaaggc 1620
aacttgacaa?gagaaatgat?aaaacattta?gaacgatgtg?aacatcgaat?catggaatcc 1680
cttgcatggc?tctcagattc?acctttattt?gatcttatta?aacaatcaaa?ggaccgagaa 1740
ggaccaactg?atcaccttga?atctgcttgt?cctcttaatc?ttcctctcca?gaataatcac 1800
actgcagcag?atatgtatct?ttctcctgta?agatctccaa?agaaaaaagg?ttcaactacg 1860
cgtgtaaatt?ctactgcaaa?tgcagagaca?caagcaacct?cagccttcca?gacccagaag 1920
ccattgaaat?ctacctctct?ttcactgttt?tataaaaaag?tgtatcggct?agcctatctc 1980
cggctaaata?cactttgtga?acgccttctg?tctgagcacc?cagaattaga?acatatcatc 2040
tggacccttt?tccagcacac?cctgcagaat?gagtatgaac?tcatgagaga?caggcatttg 2100
gaccaaatta?tgatgtgttc?catgtatggc?atatgcaaag?tgaagaatat?agaccttaaa 2160
ttcaaaatca?ttgtaacagc?atacaaggat?cttcctcatg?ctgttcagga?gacattcaaa 2220
cgtgttttga?tcaaagaaga?ggagtatgat?tctattatag?tattctataa?ctcggtcttc 2280
atgcagagac?tgaaaacaaa?tattttgcag?tatgcttcca?ccaggccccc?taccttgtca 2340
ccaatacctc?acattcctcg?aagcccttac?aagtttccta?gttcaccctt?acggattcct 2400
ggagggaaca?tctatatttc?acccctgaag?agtccatata?aaatttcaga?aggtctgcca 2460
acaccaacaa?aaatgactcc?aagatcaaga?atcttagtat?caattggtga?atcattcggg 2520
acttctgaga?agttccagaa?aataaatcag?atggtatgta?acagcgaccg?tgtgctcaaa 2580
agaagtgctg?aaggaagcaa?ccctcctaaa?ccactgaaaa?aactacgctt?tgatattgaa 2640
ggatcagatg?aagcagatgg?aagtaaacat?ctcccaggag?agtccaaatt?tcagcagaaa 2700
ctggcagaaa?tgacttctac?tcgaacacga?atgcaaaagc?agaaaatgaa?tgatagcatg 2760
gatacctcaa acaaggaaga gaaatga 2787
<210> 2
<211> 928
<212> PRT
<213〉people (Homosapiens)
<400> 2
Met?Pro?Pro?Lys?Thr?Pro?Arg?Lys?Thr?Ala?Ala?Thr?Ala?Ala?Ala?Ala
1 5 10 15
Ala?Ala?Glu?Pro?Pro?Ala?Pro?Pro?Pro?Pro?Pro?Pro?Pro?Glu?Glu?Asp
20 25 30
Pro?Glu?Gln?Asp?Ser?Gly?Pro?Glu?Asp?Leu?Pro?Leu?Val?Arg?Leu?Glu
35 40 45
Phe?Glu?Glu?Thr?Glu?Glu?Pro?Asp?Phe?Thr?Ala?Leu?Cys?Gln?Lys?Leu
50 55 60
Lys?Ile?Pro?Asp?His?Val?Arg?Glu?Arg?Ala?Trp?Leu?Thr?Trp?Glu?Lys
65 70 75 80
Val?Ser?Ser?Val?Asp?Gly?Val?Leu?Gly?Gly?Tyr?Ile?Gln?Lys?Lys?Lys
85 90 95
Glu?Leu?Trp?Gly?Ile?Cys?Ile?Phe?Ile?Ala?Arg?Val?Asp?Leu?Asp?Glu
100 105 110
Met?Ser?Phe?Thr?Leu?Leu?Ser?Tyr?Arg?Lys?Thr?Tyr?Glu?Ile?Ser?Val
115 120 125
His?Lys?Phe?Phe?Asn?Leu?Leu?Lys?Glu?Ile?Asp?Thr?Ser?Thr?Lys?Val
130 135 140
Asp?Asn?Ala?Met?Ser?Arg?Leu?Leu?Lys?Lys?Tyr?Asp?Val?Leu?Phe?Ala
145 150 155 160
Leu?Phe?Ser?Lys?Leu?Glu?Arg?Thr?Cys?Glu?Leu?Ile?Tyr?Leu?Thr?Gln
165 170 175
Pro?Ser?Ser?Ser?Ile?Ser?Thr?Glu?Ile?Asn?Ser?Ala?Leu?Val?Leu?Lys
180 185 190
Val?Ser?Trp?Ile?Thr?Phe?Leu?Leu?Ala?Lys?Gly?Glu?Val?Leu?Gln?Met
195 200 205
Glu?Asp?Asp?Leu?Val?Ile?Ser?Phe?Gln?Leu?Met?Leu?Cys?Val?Leu?Asp
210 215 220
Tyr?Phe?Ile?Lys?Leu?Ser?Pro?Pro?Met?Leu?Leu?Lys?Glu?Pro?Tyr?Lys
225 230 235 240
Thr?Ala?Val?Ile?Pro?Ile?Asn?Gly?Ser?Pro?Arg?Thr?Pro?Arg?Arg?Gly
245 250 255
Gln?Asn?Arg?Ser?Ala?Arg?Ile?Ala?Lys?Gln?Leu?Glu?Asn?Asp?Thr?Arg
260 265 270
Ile?Ile?Glu?Val?Leu?Cys?Lys?Glu?His?Glu?Cys?Asn?Ile?Asp?Glu?Val
275 280 285
Lys?Asn?Val?Tyr?Phe?Lys?Asn?Phe?Ile?Pro?Phe?Met?Asn?Ser?Leu?Gly
290 295 300
Leu?Val?Thr?Ser?Asn?Gly?Leu?Pro?Glu?Val?Glu?Asn?Leu?Ser?Lys?Arg
305 310 315 320
Tyr?Glu?Glu?Ile?Tyr?Leu?Lys?Asn?Lys?Asp?Leu?Asp?Arg?Arg?Leu?Phe
325 330 335
Leu?Asp?His?Asp?Lys?Thr?Leu?Gln?Thr?Asp?Ser?Ile?Asp?Ser?Phe?Glu
340 345 350
Thr?Gln?Arg?Thr?Pro?Arg?Lys?Ser?Asn?Leu?Asp?Glu?Glu?Val?Asn?Ile
355 360 365
Ile?Pro?Pro?His?Thr?Pro?Val?Arg?Thr?Val?Met?Asn?Thr?Ile?Gln?Gln
370 375 380
Leu?Met?Met?Ile?Leu?Asn?Ser?Ala?Ser?Asp?Gln?Pro?Ser?Glu?Asn?Leu
385 390 395 400
Ile?Ser?Tyr?Phe?Asn?Asn?Cys?Thr?Val?Asn?Pro?Lys?Glu?Ser?Ile?Leu
405 410 415
Lys?Arg?Val?Lys?Asp?Ile?Gly?Tyr?Ile?Phe?Lys?Glu?Lys?Phe?Ala?Lys
420 425 430
Ala?Val?Gly?Gln?Gly?Cys?Val?Glu?Ile?Gly?Ser?Gln?Arg?Tyr?Lys?Leu
435 440 445
Gly?Val?Arg?Leu?Tyr?Tyr?Arg?Val?Met?Glu?Ser?Met?Leu?Lys?Ser?Glu
450 455 460
Glu?Glu?Arg?Leu?Ser?Ile?Gln?Asn?Phe?Ser?Lys?Leu?Leu?Asn?Asp?Asn
465 470 475 480
Ile?Phe?His?Met?Ser?Leu?Leu?Ala?Cys?Ala?Leu?Glu?Val?Val?Met?Ala
485 490 495
Thr?Tyr?Ser?Arg?Ser?Thr?Ser?Gln?Asn?Leu?Asp?Ser?Gly?Thr?Asp?Leu
500 505 510
Ser?Phe?Pro?Trp?Ile?Leu?Asn?Val?Leu?Asn?Leu?Lys?Ala?Phe?Asp?Phe
515 520 525
Tyr?Lys?Val?Ile?Glu?Ser?Phe?Ile?Lys?Ala?Glu?Gly?Asn?Leu?Thr?Arg
530 535 540
Glu?Met?Ile?Lys?His?Leu?Glu?Arg?Cys?Glu?His?Arg?Ile?Met?Glu?Ser
545 550 555 560
Leu?Ala?Trp?Leu?Ser?Asp?Ser?Pro?Leu?Phe?Asp?Leu?Ile?Lys?Gln?Ser
565 570 575
Lys?Asp?Arg?Glu?Gly?Pro?Thr?Asp?His?Leu?Glu?Ser?Ala?Cys?Pro?Leu
580 585 590
Asn?Leu?Pro?Leu?Gln?Asn?Asn?His?Thr?Ala?Ala?Asp?Met?Tyr?Leu?Ser
595 600 605
Pro?Val?Arg?Ser?Pro?Lys?Lys?Lys?Gly?Ser?Thr?Thr?Arg?Val?Asn?Ser
610 615 620
Thr?Ala?Asn?Ala?Glu?Thr?Gln?Ala?Thr?Ser?Ala?Phe?Gln?Thr?Gln?Lys
625 630 635 640
Pro?Leu?Lys?Ser?Thr?Ser?Leu?Ser?Leu?Phe?Tyr?Lys?Lys?Val?Tyr?Arg
645 650 655
Leu?Ala?Tyr?Leu?Arg?Leu?Asn?Thr?Leu?Cys?Glu?Arg?Leu?Leu?Ser?Glu
660 665 670
His?Pro?Glu?Leu?Glu?His?Ile?Ile?Trp?Thr?Leu?Phe?Gln?His?Thr?Leu
675 680 685
Gln?Asn?Glu?Tyr?Glu?Leu?Met?Arg?Asp?Arg?His?Leu?Asp?Gln?Ile?Met
690 695 700
Met?Cys?Ser?Met?Tyr?Gly?Ile?Cys?Lys?Val?Lys?Asn?Ile?Asp?Leu?Lys
705 710 715 720
Phe?Lys?Ile?Ile?Val?Thr?Ala?Tyr?Lys?Asp?Leu?Pro?His?Ala?Val?Gln
725 730 735
Glu?Thr?Phe?Lys?Arg?Val?Leu?Ile?Lys?Glu?Glu?Glu?Tyr?Asp?Ser?Ile
740 745 750
Ile?Val?Phe?Tyr?Asn?Ser?Val?Phe?Met?Gln?Arg?Leu?Lys?Thr?Asn?Ile
755 760 765
Leu?Gln?Tyr?Ala?Ser?Thr?Arg?Pro?Pro?Thr?Leu?Ser?Pro?Ile?Pro?His
770 775 780
Ile?Pro?Arg?Ser?Pro?Tyr?Lys?Phe?Pro?Ser?Ser?Pro?Leu?Arg?Ile?Pro
785 790 795 800
Gly?Gly?Asn?Ile?Tyr?Ile?Ser?Pro?Leu?Lys?Ser?Pro?Tyr?Lys?Ile?Ser
805 810 815
Glu?Gly?Leu?Pro?Thr?Pro?Thr?Lys?Met?Thr?Pro?Arg?Ser?Arg?Ile?Leu
820 825 830
Val?Ser?Ile?Gly?Glu?Ser?Phe?Gly?Thr?Ser?Glu?Lys?Phe?Gln?Lys?Ile
835 840 845
Asn?Gln?Met?Val?Cys?Asn?Ser?Asp?Arg?Val?Leu?Lys?Arg?Ser?Ala?Glu
850 855 860
Gly?Ser?Asn?Pro?Pro?Lys?Pro?Leu?Lys?Lys?Leu?Arg?Phe?Asp?Ile?Glu
865 870 875 880`
Gly?Ser?Asp?Glu?Ala?Asp?Gly?Ser?Lys?His?Leu?Pro?Gly?Glu?Ser?Lys
885 890 895
Phe?Gln?Gln?Lys?Leu?Ala?Glu?Met?Thr?Ser?Thr?Arg?Thr?Arg?Met?Gln
900 905 910
Lys?Gln?Lys?Met?Asn?Asp?Ser?Met?Asp?Thr?Ser?Asn?Lys?Glu?Glu?Lys
915 920 925 928
<210> 3
<211> 3249
<212> DNA
<213〉people (Homo sapiens)
<400> 3
atggacgagg cggcgcgggc cgaggcctgg gacagctacc gcagcatgag cgaaagctac 60
acgctggagg gaaatgatct tcattggtta gcatgtgcct tatatgtggc ttgcagaaaa 120
tctgttccaa ctgtaagcaa agggacagtg gaaggaaact atgtatcttt aactagaatc 180
ctgaaatgtt cagagcagag cttaatcgaa ttttttaata agatgaagaa gtgggaagac 240
atggcaaatc tacccccaca tttcagagaa cgtactgaga gattagaaag aaacttcact 300
gtttctgctg taatttttaa gaaatatgaa cccatttttc aggacatctt taaataccct 360
caagaggagc aacctcgtca gcagcgagga aggaaacagc ggcgacagcc ctgtactgtg 420
tctgaaattt tccatttttg ttggatgctt tttatatatg caaaaggtaa tttccccatg 480
attagtgatg atttggtcaa ttcttatcac ctgctgctgt gtgctttgga cttagtttat 540
ggaaatgcac ttcagtgttc taatcgtaaa gaacttgtga accctaattt taaaggctta 600
tctgaagatt ttcatgctaa agattctaaa ccttcctctg accccccttg tatcattgag 660
aaactgtgtt ccttacatga tggcctagtt ttggaagcaa aggggataaa ggaacatttc 720
tggaaaccct atattaggaa actttatgaa aaaaagctcc ttaagggaaa agaagaaaat 780
ctcactgggt ttctagaacc tgggaacttt ggagagagtt ttaaagccat caataaggcc 840
tatgaggagt atgttttatc tgttgggaat ttagatgagc ggatatttct tggagaggat 900
gctgaggagg aaattgggac tctctcaagg tgtctgaacg ctggttcagg aacagagact 960
gctgaaaggg tgcagatgaa aaacatctta cagcagcatt ttgacaagtc caaagcactt 1020
agaatctcca caccactaac tggtgttagg tacattaagg agaatagccc ttgtgtgact 1080
ccagtttcta cagctacgca tagcttgagt cgtcttcaca ccatgctgac aggcctcagg 1140
aatgcaccaa gtgagaaact ggaacagatt ctcaggacat gttccagaga tccaacccag 1200
gctattgcta acagactgaa agaaatgttt gaaatatatt ctcagcattt ccagccagac 1260
gaggatttca gtaattgtgc taaagaaatt gccagcaaac attttcgttt tgcggagatg 1320
ctttactata aagtattaga atctgttatt gagcaggaac aaaaaagact aggagacatg 1380
gatttatctg gtattctgga acaagatgcg ttccacagat ctctcttggc ctgctgcctt 1440
gaggtcgtca ctttttctta taagcctcct gggaattttc catttattac tgaaatattt 1500
gatgtgcctc tttatcattt ttataaggtg atagaagtat tcattagagc agaagatggc 1560
ctttgtagag aggtggtaaa acaccttaat cagattgaag aacagatctt agatcatttg 1620
gcatggaaac cagagtctcc actctgggaa aaaattagag acaatgaaaa cagagttcct 1680
acatgtgaag aggtcatgcc acctcagaac ctggaaaggg cagatgaaat ttgcattgct 1740
ggctcccctt tgactcccag aagggtgact gaagttcgtg ctgatactgg aggacttgga 1800
aggagcataa catctccaac cacattatac gataggtaca gctccccacc agccagcact 1860
accagaaggc ggctatttgt tgagaatgat agcccctctg atggagggac acctgggcgg 1920
atgcccccac agcccctagt caatgctgtc cctgtgcaga atgtatctgg ggagactgtt 1980
tctgtcacac cagttcctgg acagactttg gtcaccatgg caaccgccac tgtcacagcc 2040
aacaatgggc aaacggtaac cattcctgtg caaggtattg ccaatgaaaa tggagggata 2100
acattcttcc ctgtccaagt caatgttggg gggcaggcac aagctgtgac aggctccatc 2160
cagcccctca gtgctcaggc cctggctgga agtctgagct ctcaacaggt gacaggaaca 2220
actttgcaag tccctggtca agtggccatt caacagattt ccccaggtgg ccaacagcag 2280
aagcaaggcc agtctgtaac cagcagtagt aatagaccca ggaagaccag ctctttatcg 2340
cttttcttta gaaaggtata ccatttagca gctgtccgcc ttcgggatct ctgtgccaaa 2400
ctagatattt cagatgaatt gaggaaaaaa atctggacct gctttgaatt ctccataatt 2460
cagtgtcctg aacttatgat ggacagacat ctggaccagt tattaatgtg tgccatttat 2520
gtgatggcaa aggtcacaaa agaagataag tccttccaga acattatgcg ttgttatagg 2580
actcagccgc aggcccggag ccaggtgtat agaagtgttt tgataaaagg gaaaagaaaa 2640
agaagaaatt ctggcagcag tgatagcaga agccatcaga attctccaac agaactaaac 2700
aaagatagaa ccagtagaga ctccagtcca gttatgaggt caagcagcac cttgccagtt 2760
ccacagccca gcagtgctcc tcccacacct actcgcctca caggtgccaa cagtgacatg 2820
gaagaagagg agaggggaga cctcattcag ttctacaaca acatctacat caaacagatt 2880
aagacatttg ccatgaagta ctcacaggca aatatggatg ctcctccact ctctccctat 2940
ccatttgtaa gaacaggctc ccctcgccga atacagttgt ctcaaaatca tcctgtctac 3000
atttccccac ataaaaatga aacaatgctt tctcctcgag aaaagatttt ctattacttc 3060
agcaacagtc cttcaaagag actgagagaa attaatagta tgatacgcac aggagaaact 3120
cctactaaaa?agagaggaat?tcttttggaa?gatggaagtg?aatcacctgc?aaaaagaatt 3180
tgcccagaaa?atcattctgc?cttattacgc?cgtctccaag?atgtagctaa?tgaccgtggt 3240
tcccactga 3249
<210> 4
<211> 1082
<212> PRT
<213〉people (Homosapiens)
<400> 4
Met?Asp?Glu?Ala?Ala?Arg?Ala?Glu?Ala?Trp?Asp?Ser?Tyr?Arg?Ser?Met
1 5 10 15
Ser?Glu?Ser?Tyr?Thr?Leu?Glu?Gly?Asn?Asp?Leu?His?Trp?Leu?Ala?Cys
20 25 30
Ala?Leu?Tyr?Val?Ala?Cys?Arg?Lys?Ser?Val?Pro?Thr?Val?Ser?Lys?Gly
35 40 45
Thr?Val?Glu?Gly?Asn?Tyr?Val?Ser?Leu?Thr?Arg?Ile?Leu?Lys?Cys?Ser
50 55 60
Glu?Gln?Ser?Leu?Ile?Glu?Phe?Phe?Asn?Lys?Met?Lys?Lys?Trp?Glu?Asp
65 70 75 80
Met?Ala?Asn?Leu?Pro?Pro?His?Phe?Arg?Glu?Arg?Thr?Glu?Arg?Leu?Glu
85 90 95
Arg?Asn?Phe?Thr?Val?Ser?Ala?Val?Ile?Phe?Lys?Lys?Tyr?Glu?Pro?Ile
100 105 110
Phe?Gln?Asp?Ile?Phe?Lys?Tyr?Pro?Gln?Glu?Glu?Gln?Pro?Arg?Gln?Gln
115 120 125
Arg?Gly?Arg?Lys?Gln?Arg?Arg?Gln?Pro?Cys?Thr?Val?Ser?Glu?Ile?Phe
130 135 140
His?Phe?Cys?Trp?Met?Leu?Phe?Ile?Tyr?Ala?Lys?Gly?Asn?Phe?Pro?Met
145 150 155 160
Ile?Ser?Asp?Asp?Leu?Val?Asn?Ser?Tyr?His?Leu?Leu?Leu?Cys?Ala?Leu
165 170 175
Asp?Leu?Val?Tyr?Gly?Asn?Ala?Leu?Gln?Cys?Ser?Asn?Arg?Lys?Glu?Leu
180 185 190
Val?Asn?Pro?Asn?Phe?Lys?Gly?Leu?Ser?Glu?Asp?Phe?His?Ala?Lys?Asp
195 200 205
Ser?Lys?Pro?Ser?Ser?Asp?Pro?Pro?Cys?Ile?Ile?Glu?Lys?Leu?Cys?Ser
210 215 220
Leu?His?Asp?Gly?Leu?Val?Leu?Glu?Ala?Lys?Gly?Ile?Lys?Glu?His?Phe
225 230 235 240
Trp?Lys?Pro?Tyr?Ile?Arg?Lys?Leu?Tyr?Glu?Lys?Lys?Leu?Leu?Lys?Gly
245 250 255
Lys?Glu?Glu?Asn?Leu?Thr?Gly?Phe?Leu?Glu?Pro?Gly?Asn?Phe?Gly?Glu
260 265 270
Ser?Phe?Lys?Ala?Ile?Asn?Lys?Ala?Tyr?Glu?Glu?Tyr?Val?Leu?Ser?Val
275 280 285
Gly?Asn?Leu?Asp?Glu?Arg?Ile?Phe?Leu?Gly?Glu?Asp?Ala?Glu?Glu?Glu
290 295 300
Ile?Gly?Thr?Leu?Ser?Arg?Cys?Leu?Asn?Ala?Gly?Ser?Gly?Thr?Glu?Thr
305 310 315 320
Ala?Glu?Arg?Val?Gln?Met?Lys?Asn?Ile?Leu?Gln?Gln?His?Phe?Asp?Lys
325 330 335
Ser?Lys?Ala?Leu?Arg?Ile?Ser?Thr?Pro?Leu?Thr?Gly?Val?Arg?Tyr?Ile
340 345 350
Lys?Glu?Asn?Ser?Pro?Cys?Val?Thr?Pro?Val?Ser?Thr?Ala?Thr?His?Ser
355 360 365
Leu?Ser?Arg?Leu?His?Thr?Met?Leu?Thr?Gly?Leu?Arg?Asn?Ala?Pro?Ser
370 375 380
Glu?Lys?Leu?Glu?Gln?Ile?Leu?Arg?Thr?Cys?Ser?Arg?Asp?Pro?Thr?Gln
385 390 395 400
Ala?Ile?Ala?Asn?Arg?Leu?Lys?Glu?Met?Phe?Glu?Ile?Tyr?Ser?Gln?His
405 410 415
Phe?Gln?Pro?Asp?Glu?Asp?Phe?Ser?Asn?Cys?Ala?Lys?Glu?Ile?Ala?Ser
420 425 430
Lys?His?Phe?Arg?Phe?Ala?Glu?Met?Leu?Tyr?Tyr?Lys?Val?Leu?Glu?Ser
435 440 445
Val?Ile?Glu?Gln?Glu?Gln?Lys?Arg?Leu?Gly?Asp?Met?Asp?Leu?Ser?Gly
450 455 460
Ile?Leu?Glu?Gln?Asp?Ala?Phe?His?Arg?Ser?Leu?Leu?Ala?Cys?Cys?Leu
465 470 475 480
Glu?Val?Val?Thr?Phe?Ser?Tyr?Lys?Pro?Pro?Gly?Asn?Phe?Pro?Phe?Ile
485 490 495
Thr?Glu?Ile?Phe?Asp?Val?Pro?Leu?Tyr?His?Phe?Tyr?Lys?Val?Ile?Glu
500 505 510
Val?Phe?Ile?Arg?Ala?Glu?Asp?Gly?Leu?Cys?Arg?Glu?Val?Val?Lys?His
515 520 525
Leu?Asn?Gln?Ile?Glu?Glu?Gln?Ile?Leu?Asp?His?Leu?Ala?Trp?Lys?Pro
530 535 540
Glu?Ser?Pro?Leu?Trp?Glu?Lys?Ile?Arg?Asp?Asn?Glu?Asn?Arg?Val?Pro
545 550 555 560
Thr?Cys?Glu?Glu?Val?Met?Pro?Pro?Gln?Asn?Leu?Glu?Arg?Ala?Asp?Glu
565 570 575
Ile?Cys?Ile?Ala?Gly?Ser?Pro?Leu?Thr?Pro?Arg?Arg?Val?Thr?Glu?Val
580 585 590
Arg?Ala?Asp?Thr?Gly?Gly?Leu?Gly?Arg?Ser?Ile?Thr?Ser?Pro?Thr?Thr
595 600 605
Leu?Tyr?Asp?Arg?Tyr?Ser?Ser?Pro?Pro?Ala?Ser?Thr?Thr?Arg?Arg?Arg
610 615 620
Leu?Phe?Val?Glu?Asn?Asp?Ser?Pro?Ser?Asp?Gly?Gly?Thr?Pro?Gly?Arg
625 630 635 640
Met?Pro?Pro?Gln?Pro?Leu?Val?Asn?Ala?Val?Pro?Val?Gln?Asn?Val?Ser
645 650 655
Gly?Glu?Thr?Val?Ser?Val?Thr?Pro?Val?Pro?Gly?Gln?Thr?Leu?Val?Thr
660 665 670
Met?Ala?Thr?Ala?Thr?Val?Thr?Ala?Asn?Asn?Gly?Gln?Thr?Val?Thr?Ile
675 680 685
Pro?Val?Gln?Gly?Ile?Ala?Asn?Glu?Asn?Gly?Gly?Ile?Thr?Phe?Phe?Pro
690 695 700
Val?Gln?Val?Asn?Val?Gly?Gly?Gln?Ala?Gln?Ala?Val?Thr?Gly?Ser?Ile
705 710 715 720
Gln?Pro?Leu?Ser?Ala?Gln?Ala?Leu?Ala?Gly?Ser?Leu?Ser?Ser?Gln?Gln
725 730 735
Val?Thr?Gly?Thr?Thr?Leu?Gln?Val?Pro?Gly?Gln?Val?Ala?Ile?Gln?Gln
740 745 750
Ile?Ser?Pro?Gly?Gly?Gln?Gln?Gln?Lys?Gln?Gly?Gln?Ser?Val?Thr?Ser
755 760 765
Ser?Ser?Asn?Arg?Pro?Arg?Lys?Thr?Ser?Ser?Leu?Ser?Leu?Phe?Phe?Arg
770 775 780
Lys?Val?Tyr?His?Leu?Ala?Ala?Val?Arg?Leu?Arg?Asp?Leu?Cys?Ala?Lys
785 790 795 800
Leu?Asp?Ile?Ser?Asp?Glu?Leu?Arg?Lys?Lys?Ile?Trp?Thr?Cys?Phe?Glu
805 810 815
Phe?Ser?Ile?Ile?Gln?Cys?Pro?Glu?Leu?Met?Met?Asp?Arg?His?Leu?Asp
820 825 830
Gln?Leu?Leu?Met?Cys?Ala?Ile?Tyr?Val?Met?Ala?Lys?Val?Thr?Lys?Glu
835 840 845
Asp?Lys?Ser?Phe?Gln?Asn?Ile?Met?Arg?Cys?Tyr?Arg?Thr?Gln?Pro?Gln
850 855 860
Ala?Arg?Ser?Gln?Val?Tyr?Arg?Ser?Val?Leu?Ile?Lys?Gly?Lys?Arg?Lys
865 870 875 880
Arg?Arg?Asn?Ser?Gly?Ser?Ser?Asp?Ser?Arg?Ser?His?Gln?Asn?Ser?Pro
885 890 895
Thr?Glu?Leu?Asn?Lys?Asp?Arg?Thr?Ser?Arg?Asp?Ser?Ser?Pro?Val?Met
900 905 910
Arg?Ser?Ser?Ser?Thr?Leu?Pro?Val?Pro?Gln?Pro?Ser?Ser?Ala?Pro?Pro
915 920 925
Thr?Pro?Thr?Arg?Leu?Thr?Gly?Ala?Asn?Ser?Asp?Met?Glu?Glu?Glu?Glu
930 935 940
Arg?Gly?Asp?Leu?Ile?Gln?Phe?Tyr?Asn?Asn?Ile?Tyr?Ile?Lys?Gln?Ile
945 950 955 960
Lys?Thr?Phe?Ala?Met?Lys?Tyr?Ser?Gln?Ala?Asn?Met?Asp?Ala?Pro?Pro
965 970 975
Leu?Ser?Pro?Tyr?Pro?Phe?Val?Arg?Thr?Gly?Ser?Pro?Arg?Arg?Ile?Gln
980 985 990
Leu?Ser?Gln?Asn?His?Pro?Val?Tyr?Ile?Ser?Pro?His?Lys?Asn?Glu?Thr
995 1000 1005
Met?Leu?Ser?Pro?Arg?Glu?Lys?Ile?Phe?Tyr?Tyr?Phe?Ser?Asn?Ser?Pro
1010 1015 1020
Ser?Lys?Arg?Leu?Arg?Glu?Ile?Asn?Ser?Met?Ile?Arg?Thr?Gly?Glu?Thr
1025 1030 1035 1040
Pro?Thr?Lys?Lys?Arg?Gly?Ile?Leu?Leu?Glu?Asp?Gly?Ser?Glu?Ser?Pro
1045 1050 1055
Ala?Lys?Arg?Ile?Cys?Pro?Glu?Asn?His?Ser?Ala?Leu?Leu?Arg?Arg?Leu
1060 1065 1070
Gln?Asp?Val?Ala?Asn?Asp?Arg?Gly?Ser?His
1075 1080 1082
<210> 5
<211> 26
<212> DNA
<213> artificial?sequence
<214〉artificial sequence
<223〉clone's primer
<400> 5
ggatccatgc?cgcccaaaac?cccccg 26
<210> 6
<211> 29
<212> DNA
<213> artificial?sequence
<214〉artificial sequence
<223〉clone's primer
<400> 6
cccgggtcat?ttctcttcct?tgtttgagg 29
<210> 7
<211> 26
<212> DNA
<213> artificial?sequence
<214〉artificial sequence
<223〉clone's primer
<400> 7
ggatccatgg?acgaggcggc?gcgggc 26
<210> 8
<211> 30
<212> DNA
<213> artificial?sequence
<214〉artificial sequence
<223〉clone's primer
<400> 8
cccgggtcag?tgggaaccac?ggtcattagc 30
<210> 9
<211> 20
<212> DNA
<213> artificial?sequence
<214〉artificial sequence
<223〉clone's primer
<400> 9
ttgaggttgt?aatggccaca 20
<210> 10
<211> 20
<212> DNA
<213> artificial?sequence
<214〉artificial sequence
<223〉clone's primer
<400> 10
cagacagaag?gcgttcacaa 20
<210> 11
<211> 20
<212> DNA
<213> artificial?sequence
<214〉artificial sequence
<223〉clone's primer
<400> 11
tgcaaggtat?tgccaatgaa 20
<210> 12
<211> 20
<212> DNA
<213> artificial?sequence
<214〉artificial sequence
<223〉clone's primer
<400> 12
gaatgaggtc?tcccctctcc 20

Claims (2)

1. contain the recombinant virus of tumor suppressor genes Rb 2, it is characterized in that, use following method structure and preparation:
A) molecular cloning of people's tumor suppressor genes Rb 2, its nucleotides sequence are classified Seq ID No.3 as, protein sequence is Seq ID.4;
B) people's tumor suppressor genes Rb 2 places CMV to start between enhanser and SV40PolyA signal terminating, forms a genetic expression assembly and stops by the expression of a messenger RNA(mRNA) that starts enhanser driving tumor suppressor gene and at the terminator place;
C) will be by method b) the expression assembly that obtains is inserted into viral metastasis transplanting physique grain or tumor suppressor genes Rb 2 is inserted in the viral metastasis transplanting physique grain that contains promotor and terminator, described viral transfer vector is an insect baculovirus: AcMNPV or LdMNPV, or adenovirus;
D) in insect cell, will be by c) the AcMNPV virus metastasis transplanting physique grain or the LdMNPV virus metastasis transplanting physique grain that obtain recombinate with the homologous sequence of virus; Or in mammalian cell 293 cells, will be by c) the adenovirus metastasis transplanting physique grain and the homologous sequence of virus that obtain recombinate; And
E) screening and purifying contain the recombinant virus of tumor suppressor genes Rb 2.
2. the described recombinant virus that contains tumor suppressor genes Rb 2 of claim 1 is used for suppressing the application of medicine of propagation, growth and the transfer of tumour cell in preparation.
CNB2004100622966A 2004-07-05 2004-07-05 Preparation of KTV series products for inhibiting tumor cell growth and use Expired - Lifetime CN100387716C (en)

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CN105986007B (en) * 2015-02-11 2020-03-17 深圳华大基因股份有限公司 Detection method of cancer tumor suppressor gene cluster (TSG)

Citations (2)

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CN1242051A (en) * 1996-12-31 2000-01-19 昂尼克斯药物公司 Cytopathic virus for the treatment and prevention of tumors
US6814963B2 (en) * 2001-03-23 2004-11-09 National Health Research Institutes Baculovirus-based expression system

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Publication number Priority date Publication date Assignee Title
CN1242051A (en) * 1996-12-31 2000-01-19 昂尼克斯药物公司 Cytopathic virus for the treatment and prevention of tumors
US6814963B2 (en) * 2001-03-23 2004-11-09 National Health Research Institutes Baculovirus-based expression system

Non-Patent Citations (4)

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Title
RB-mediated suppression of spontaneous multipleneuroendocrine neoplasia and lung metastases in Rb+/- mice. ALEXANDER YU. et al.Proc. Natl. Acad. Sci. USA,Vol.96 . 1999
RB-mediated suppression of spontaneous multipleneuroendocrine neoplasia and lung metastases in Rb+/- mice. ALEXANDER YU. et al.Proc. Natl. Acad. Sci. USA,Vol.96 . 1999 *
肿瘤抑制基因在恶性肿瘤基因治疗中的应用. 吴焱等.国外医学遗传学分册,第24卷第1期. 2001
肿瘤抑制基因在恶性肿瘤基因治疗中的应用. 吴焱等.国外医学遗传学分册,第24卷第1期. 2001 *

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