CN101372707B - T lymphocyte for screening and activating dormant infection HIV-1 compound and preparation thereof - Google Patents
T lymphocyte for screening and activating dormant infection HIV-1 compound and preparation thereof Download PDFInfo
- Publication number
- CN101372707B CN101372707B CN200810038851XA CN200810038851A CN101372707B CN 101372707 B CN101372707 B CN 101372707B CN 200810038851X A CN200810038851X A CN 200810038851XA CN 200810038851 A CN200810038851 A CN 200810038851A CN 101372707 B CN101372707 B CN 101372707B
- Authority
- CN
- China
- Prior art keywords
- hiv
- cell
- lymphocyte
- preparation
- plasmid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 241000713772 Human immunodeficiency virus 1 Species 0.000 title claims abstract description 21
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 230000003213 activating effect Effects 0.000 title claims abstract description 13
- 150000001875 compounds Chemical class 0.000 title claims abstract description 12
- 238000012216 screening Methods 0.000 title claims abstract description 9
- 208000015181 infectious disease Diseases 0.000 title claims description 16
- 210000004027 cell Anatomy 0.000 claims abstract description 74
- 241000725303 Human immunodeficiency virus Species 0.000 claims abstract description 20
- 241000700605 Viruses Species 0.000 claims abstract description 17
- 239000003814 drug Substances 0.000 claims abstract description 12
- 108700008625 Reporter Genes Proteins 0.000 claims abstract description 10
- 108010048367 enhanced green fluorescent protein Proteins 0.000 claims abstract 4
- 239000013612 plasmid Substances 0.000 claims description 22
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 10
- 102100040247 Tumor necrosis factor Human genes 0.000 claims description 10
- 206010041047 Slow virus infection Diseases 0.000 claims description 8
- 230000004913 activation Effects 0.000 claims description 7
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 5
- 238000010367 cloning Methods 0.000 claims description 5
- 238000004806 packaging method and process Methods 0.000 claims description 5
- 108090000848 Ubiquitin Proteins 0.000 claims description 4
- 102000044159 Ubiquitin Human genes 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000003960 Ligases Human genes 0.000 claims description 3
- 108090000364 Ligases Proteins 0.000 claims description 3
- 230000001939 inductive effect Effects 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 238000001890 transfection Methods 0.000 claims description 3
- 230000008034 disappearance Effects 0.000 claims description 2
- 238000005199 ultracentrifugation Methods 0.000 claims description 2
- 238000004321 preservation Methods 0.000 abstract description 4
- 208000032420 Latent Infection Diseases 0.000 abstract description 3
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 3
- 230000010354 integration Effects 0.000 abstract description 2
- 238000007877 drug screening Methods 0.000 abstract 1
- 238000010353 genetic engineering Methods 0.000 abstract 1
- 230000003612 virological effect Effects 0.000 description 14
- 208000030507 AIDS Diseases 0.000 description 8
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 108010002586 Interleukin-7 Proteins 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 208000031886 HIV Infections Diseases 0.000 description 3
- 241000713340 Human immunodeficiency virus 2 Species 0.000 description 3
- 108060001084 Luciferase Proteins 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- 208000011580 syndromic disease Diseases 0.000 description 3
- 102100034343 Integrase Human genes 0.000 description 2
- 108010061833 Integrases Proteins 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 210000004970 cd4 cell Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000003018 immunosuppressive agent Substances 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 230000000527 lymphocytic effect Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000004088 simulation Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 241001483952 Peach chlorotic mottle virus Species 0.000 description 1
- 239000004697 Polyetherimide Substances 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 238000011225 antiretroviral therapy Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 230000010307 cell transformation Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000013326 plasmid cotransfection Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the fields of genetic engineering and cell engineering, pertaining to a human T-lymphocytic series model for screening and reactivating latent infection HIV-1 compound and a preparation method thereof. The invention discloses a T lymphocyte used for screening and activating latent infection HIV-1 drug, and the T lymphocyte is a Jurkat stable strain that is infected by butnot expresses HIV slow virus carrying reporter gene. The preservation number of the T lymphocyte is CCTCC NO.C200821. The invention also provides a preparation method for the T lymphocyte, including infecting human T-lymphocytic series Jurkat with the HIV-1 slow virus carrying EGFO reporter gene, and carrying out cell sorting and HIV integration test so as to obtain the clone with HIV integrationwhile not expressing EGFP. The invention is the cell model for the virus preservation library to control drug screening.
Description
Technical field
The invention belongs to the genetic and cell engineering field, relating to a kind of human T lymphocyte who is used to screen once more activating dormant infection HIV-1-1 compound is model and preparation method thereof.
Background technology
Acquired immune deficiency syndrome (AIDS) (AIDS) is the communicable disease that is infected a kind of serious harm people life and health that causes by HIV.Add up global AIDS patient according to WHO and surpass 4,000 ten thousand, annual newly-increased patient 5,000,000, and annual death is about 3,000,000.At present; acquired immune deficiency syndrome (AIDS) clinical treatment method mainly is efficient degeneration-resistant biography record virus therapy (Highly active antiretroviral therapy; HAART); because it can effectively reduce virus load (virus load) and protection immunologic function, thereby has correspondingly prolonged HIV the infected's survival time.Yet this therapy can only suppress virus replication, can not thoroughly remove the infection of HIV, and most patient the virus bounce-back can occur after interrupting HAART, and its major cause is that HIV can be incorporated in the genome of silent oscillation memory cell CD4 positive T cell.These cell transformation period are longer, and according to estimates, the cell that body will be removed these viral storage vaults needs 40-60.Recently, the strategy of immunostimulation treatment can pass through the activation once more to cells infected, thereby promotes necrocytosis to quicken the removing of virus base with this, thereby this strategy is that new thinking has been opened up in the control of viral storage vault.Although to the existing several schemes of the elimination of viral storage, do not obtain satisfied effect clinically yet, therefore, researching and developing novel activating dormant infection cell drug has been extremely urgent, is a key link and set up the relative medicine screening model.
Summary of the invention
The purpose of this invention is to provide a kind of human T lymphocyte who is used to screen once more activating dormant infection HIV-1-1 compound.
Another object of the present invention provides the preparation method of above-mentioned cell.
A further object of the present invention provides the application of above-mentioned cell.
HIV is divided into HIV-1 and two types of HIV-2.Being widely current in the strain in the whole world at present is the HIV-1 type.Though the HIV-2 strain is found in the area, West Africa, As time goes on, this strain also is detected in Europe, the U.S. and South America, some the infecteds of Asia, and especially HIV-2 the infected's quantity of Asia India increases sharply, and China also finds successively in areas such as Xinjiang, Shanghai.No matter certainly from the whole world and even China, HIV-1 is current main acquired immune deficiency syndrome (AIDS) epidemic isolates.If will obtain activating dormant infection HIV-1 once more-1 compound of effective and safe, just must be the enterprising row filter of cell model that HIV-1 hides on a large scale.Because the silent oscillation that HIV-1 hides memory cd4 cell comparatively small amt, and the same on its form with normal cd4 cell, the cell of separation and purification and identifying virus storage vault is difficult, thereby, be difficulty of medicaments sifting model with patient HIV-1 latent infection cell.For this reason, the present invention will carry the slow virus infection human lymphocyte system of the HIV-1 of EGFP reporter gene, simulation HIV-1 infects the lymphocytic situation of normal people T, pass through cell sorting, obtain the cell of not expressing and carry out clonal expansion, these clones are carried out HIV integrate to detect, then, obtain to have that HIV integrates but the clone that do not express EGFP.In order to verify the operability of this cell model, we utilize TNF-α to induce, obtain epigamic EGFP cloning by expression at last, this genetically modified cell system is human T lymphocyte's model of dormant infection HIV-1-1, this model will lay the foundation for Mechanism Study, the especially screening for viral storage control medicine that HIV-1 hides.
The invention provides the T lymphocyte of a kind of screening and activating dormant infection HIV-1-1 medicine, this cell is that the HIV slow virus infection that is carried reporter gene is not simultaneously expressed Jurkat again and stablized strain.
Jurkat of the present invention stablize strain submitted to Chinese typical culture collection center (Chinese Wuhan. Wuhan University) preservation, preserving number is CCTCC NO.C200821, the name be called Jurkat-LTRGT clone, preservation date is on May 19th, 2008.
The invention provides a kind ofly, form (its structural representation is respectively shown in Fig. 1,2 and 3) by FGW plasmid, CMV Δ 8.9 and coating plasmid VSVG and form .. based on HIV-1 slow virus package carrier system
FGW plasmid of the present invention is on FUGW lentiviral vectors basis, and reconstruction forms through genetically deficient ubiquitin control region.FUGW, CMV Δ 8.9 and coating plasmid VSVG derive from U.S. Carlos doctor Lois and are so kind as to give.FUGW derives from article: Lois C, et al.Germline transmission and tissue-specificexpression of transgenes delivered by lentiviral vectors.Science, 2002,295:868-72.The FGW plasmid is by the FUGW deutero-, removes HIV long terminal repeat (LTR) behind the HIV-1 deleterious gene and FLAP as skeleton, only expresses the green fluorescence protein gene (EGFP) by the LTR regulation and control; PCMV. Δ 8.9 is the packaging structure plasmid, mainly plays viral packaging function, expresses zymoprotein and forms viral capsid structure and intergrase (IN); VSVG is the coating plasmid.
Medicine of the present invention can be a synthetic, also can be to extract from plant.
On the other hand, the invention provides the preparation method of above-mentioned cell, the slow virus infection human T lymphocyte who is about to carry the HIV-1 of EGFP reporter gene is Jurkat, integrates by cell sorting and HIV and detects, and obtains to have that HIV integrates but the clone that do not express EGFP.
Among the present invention, can further utilize TNF-a to carry out the activation of inducing of different concns, analysis of fluorescence intensity and fluorescence positive cells number screen and obtain the EGFP cloning by expression of inducibility at last.
The present invention also provides the preparation method of slow virus, and this method may further comprise the steps:
This preparation method may further comprise the steps:
(1) the FUGW carrier is cut through the PacI+BamhI enzyme, reclaims 7KB fragment and T4 ligase enzyme and connects and the FGW plasmid of acquisition ubiquitin control region disappearance;
(2) FGW plasmid, coating plasmid VSVG and packaging structure plasmid CMV Δ 8.9 plasmids mix, and transfection people 293T cell after 48 hours, is collected supernatant, obtains FGW virus through ultracentrifugation, and its titre should be at 10E5-10E8, and-80 degree store.
Among the preparation method of the present invention, adopting the mixing quality ratio in (2) is 5: 4: 3-4: 3: 2.Plasmid FGW, pCMV. Δ 8.9, when reaching VSVG three part cotransfections, three's mass ratio can be 2: 1.5: 1; As three plasmid co-transfection 100CM culture dish, FGW, pCMV. Δ 8.9 and VSVG aequum can be 10ug, 7.5ug and 5ug.The cotransfection method can be passed through liposome, calcium phosphate, PEI etc.
(3) (2) are obtained FGW virus infection human T lymphocyte Jurkat, infect 72 hours, collect the cell of luciferase expression not and on 96 orifice plates, carry out the individual cells clonal expansion after flow cytometer carries out cell sorting.Each cell clone genomic dna of extracting carries out viral integrase and detects.
(4) it is as follows that viral integrase detects the primer: EGFP (4021)-F, 5-TAGCAATACAGCAGCTACCAATGCTGATTG-3; EGFP (4371)-R, 5-GTGTTCTCTCCTTCATTGGCTTCTTCTACC-3.PCR reaction conditions are 95 degree 1 minute; 95 degree 30 seconds, 58 degree 40 seconds, 72 degree 45 seconds; 30 circulations. extend 10 fens .PCR products electrophoresis on 1.5 gels, the purpose band should be at the single band at 350BP place.
(5) (4) being integrated the positive cells clone increases, part cell cryopreservation, part cell is induced activation (0ng/mL, 0.3ng/mL, 0.6ng/mL with what TNF-carried out different concns, 1.2ng/mL, 2.4ng/mL, 4.8ng/mL9.6ng/mL), analysis of fluorescence intensity and fluorescence positive cells number screen at last and the EGFP cloning by expression that obtains inducibility is the cell model that viral storage control medicine screens.
(6) FGW virus infection target cell is except that human T lymphocyte Jurkat, and can be that people's monokaryon is huge has a liking for cell, star spongiocyte.
The present invention also provides the application of above-mentioned cell, being about to the above-mentioned human T lymphocyte who is used to screen once more activating dormant infection HIV-1-1 compound of compound adding is model cell, and the compound that can make cell green fluorescence occur is candidate's activating dormant infection HIV-1-1 medicine.
The slow virus infection human T lymphocyte that the present invention will carry the HIV-1 of EGFP reporter gene is Jurkat, and simulation HIV-1 infects the lymphocytic situation of normal people T, integrates by cell sorting and HIV and detects, and obtains to have that HIV integrates but the clone that do not express EGFP.Then, utilize TNF-a to carry out the activation of inducing of different concns, analysis of fluorescence intensity and fluorescence positive cells number screen and obtain the EGFP cloning by expression of inducibility at last.The purpose of this invention is to provide a kind of HIV-1 Study on Mechanism of hiding that is, especially the cell model that screens for viral storage control medicine.
Description of drawings
Fig. 1 .FGW carrier structure synoptic diagram.
Fig. 2 .VSVG carrier structure synoptic diagram
Fig. 3 .pCMV. Δ 8.9 structural representations.
Fig. 4 .TNF-α induces the result schematic diagram that activates observation of cell fluorescence under the fluorescent microscope of back.
Fig. 5 .TNF-α induces the result schematic diagram that activates back cell green fluorescence intensity.
Fig. 6. the TNF-α of different concns induces the result schematic diagram that activates back green fluorescence cell count.
Embodiment
Lentiviral vectors FUGW cuts with the PacI+BamhI enzyme, reclaims the 8691BP fragment, and the T4 ligase enzyme connects, and transform bacteria clone extracting dna sequence analysis obtains the FGW plasmid that the ubiquitin control region lacks at last.
The preparation of embodiment 2 lentiviral vectorss
With the FGW carrier, mix with 2: 1: 1 mass ratios with packaging structure plasmid CMV Δ 8.9 and coating plasmid (VSVG).Utilize liposome LipofectamineTM on the 293T cell, to carry out transfection, under fluorescent microscope, observe behind 24～48h, occur collecting viral supernatant behind a large amount of fluorescence, the viral supernatant of collecting concentrates the standby or use immediately of back packing. during the recombinant slow virus determination of activity, viral stock solution after concentrating is done the different ratios dilution, under fluorescent microscope, carry out the fluorescence counting behind the cells infected 48h, determine titre.The FGW carrier in contrast.The result shows that the site-specific integration retroviral titre is 6.8X10
-7TU/ML.
The above-mentioned 1ul slow virus FGW-of system (by plasmid CMV Δ 8.9 packings) is infected Jurkat clone on 6 orifice plates.Observe under fluorescent microscope behind the 72h, the result shows that Jurkat is a cell visible green fluorescence; Efficiency of infection is 82.1%.Collect the cell of luciferase expression not and on 96 orifice plates, carry out the individual cells clonal expansion.
Confluence analysis behind the embodiment 4 FGW slow virus infection cells
The above-mentioned infected cell genomic dna of extracting identifies with PCR and sequence analysis method whether viral genome is integrated: EGFP (4021)-F, 5-TAGCAATACAGCAGCTACCAATGCTGATTG-3; EGFP (4371)-R, 5-GTGTTCTCTCCTTCATTGGCTTCTTCTACC-3.PCR reaction conditions are 95 degree 1 minute; 95 degree 30 seconds, 58 degree 40 seconds, 72 degree 45 seconds; 30 circulations. extend 10 fens .PCR products electrophoresis on 1.5 gels.The result shows, FGW-slow virus infection Jurkat cell, the year do not see luciferase expression, but can amplify the purpose band of 350bp.
The TNF-α of embodiment 5. different concns induces Jurkat cell reporter gene to activate
In order to verify whether integrate the positive cells clone can be activated by immunosuppressor, cell is induced activation (0ng/mL with what TNF-α carried out different concns on 24 orifice plates, 0.3ng/mL, 0.6ng/mL, 1.2ng/mL, 2.4ng/mL, 4.8ng/mL, 9.6ng/mL), after 24 hours, with flow cytometry analysis fluorescence intensity and fluorescence positive cells number, the result shows that along with TNF-α concentration raises, Jurkat cell expressing fluorescence intensity also increases, fluorescence positive cells number reaches maximum when 2.4ng/mLTNF-α concentration, is in plateau later on.
In order to verify whether integrate the positive cells clone can be activated by immunosuppressor, cell is induced activation (0ng/mL with what IL-7 carried out different concns on 24 orifice plates, 0.3ng/mL, 0.6ng/mL, 1.2ng/mL, 2.4ng/mL, 4.8ng/mL, 9.6ng/mL), after 24 hours, with flow cytometry analysis fluorescence intensity and fluorescence positive cells number, the result shows, along with IL-7 concentration raises, Jurkat cell expressing fluorescence intensity also increases, and fluorescence positive cells number tends towards stability more than or equal to 2.ng/mL IL-7 concentration the time.
Sequence table
＜110〉Fudan University
＜120〉T lymphocyte of a kind of screening and activating dormant infection HIV-1-1 compound and preparation method thereof
<130>31
<160>3
<170>PatentIn?version?3.1
<210>1
<211>30
<212>DNA
<213>Artificial
<400>1
tagcaataca?gcagctacca?atgctgattg 30
<210>2
<211>30
<212>DNA
<213>Artificial
<400>2
gtgttctctc?cttcattggc?ttcttctacc 30
<210>3
<211>8691
<212>DNA
<213>Artificial
<220>
<221>cmv?promoter
<222>(1)..(817)
<223>
<220>
<221>LTR
<222>(818)..(1117)
<223>
<220>
<221>RRE
<222>(1118)..(1895)
<223>
<220>
<221>FLP
<222>(1896)..(2600)
<223>
<220>
<221>EGFP
<222>(2601)..(3434)
<223>
<220>
<221>WHV
<222>(3435)..(4002)
<223>
<220>
<221>LTR
<222>(4003)..(4831)
<223>
<400>3
gtcgacggat?cgggagatct?cccgatcccc?tatggtgcac?tctcagtaca?atctgctctg 60
atgccgcata?gttaagccag?tatctgctcc?ctgcttgtgt?gttggaggtc?gctgagtagt 120
gcgcgagcaa?aatttaagct?acaacaaggc?aaggcttgac?cgacaattgc?atgaagaatc 180
tgcttagggt?taggcgtttt?gcgctgcttc?gcgatgtacg?ggccagatat?acgcgttgac 240
attgattatt?gactagttat?taatagtaat?caattacggg?gtcattagtt?catagcccat 300
atatggagtt?ccgcgttaca?taacttacgg?taaatggccc?gcctggctga?ccgcccaacg 360
acccccgccc?attgacgtca?ataatgacgt?atgttcccat?agtaacgcca?atagggactt 420
tccattgacg?tcaatgggtg?gagtatttac?ggtaaactgc?ccacttggca?gtacatcaag 480
tgtatcatat?gccaagtacg?ccccctattg?acgtcaatga?cggtaaatgg?cccgcctggc 540
attatgccca?gtacatgacc?ttatgggact?ttcctacttg?gcagtacatc?tacgtattag 600
tcatcgctat?taccatggtg?atgcggtttt?ggcagtacat?caatgggcgt?ggatagcggt 660
ttgactcacg?gggatttcca?agtctccacc?ccattgacgt?caatgggagt?ttgttttggc 720
accaaaatca?acgggacttt?ccaaaatgtc?gtaacaactc?cgccccattg?acgcaaatgg 780
gcggtaggcg?tgtacggtgg?gaggtctata?taagcagcgc?gttttgcctg?tactgggtct 840
ctctggttag?accagatctg?agcctgggag?ctctctggct?aactagggaa?cccactgctt 900
aagcctcaat?aaagcttgcc?ttgagtgctt?caagtagtgt?gtgcccgtct?gttgtgtgac 960
tctggtaact?agagatccct?cagacccttt?tagtcagtgt?ggaaaatctc?tagcagtggc 1020
gcccgaacag?ggacttgaaa?gcgaaaggga?aaccagagga?gctctctcga?cgcaggactc 1080
ggcttgctga?agcgcgcacg?gcaagaggcg?aggggcggcg?actggtgagt?acgccaaaaa 1140
ttttgactag?cggaggctag?aaggagagag?atgggtgcga?gagcgtcagt?attaagcggg 1200
ggagaattag?atcgcgatgg?gaaaaaattc?ggttaaggcc?agggggaaag?aaaaaatata 1260
aattaaaaca?tatagtatgg?gcaagcaggg?agctagaacg?attcgcagtt?aatcctggcc 1320
tgttagaaac?atcagaaggc?tgtagacaaa?tactgggaca?gctacaacca?tcccttcaga 1380
caggatcaga?agaacttaga?tcattatata?atacagtagc?aaccctctat?tgtgtgcatc 1440
aaaggataga?gataaaagac?accaaggaag?ctttagacaa?gatagaggaa?gagcaaaaca 1500
aaagtaagac?caccgcacag?caagcggccg?ctgatcttca?gacctggagg?aggagatatg 1560
agggacaatt?ggagaagtga?attatataaa?tataaagtag?taaaaattga?accattagga 1620
gtagcaccca?ccaaggcaaa?gagaagagtg?gtgcagagag?aaaaaagagc?agtgggaata 1680
ggagctttgt?tccttgggtt?cttgggagca?gcaggaagca?ctatgggcgc?agcgtcaatg 1740
acgctgacgg?tacaggccag?acaattattg?tctggtatag?tgcagcagca?gaacaatttg 1800
ctgagggcta?ttgaggcgca?acagcatctg?ttgcaactca?cagtctgggg?catcaagcag 1860
ctccaggcaa?gaatcctggc?tgtggaaaga?tacctaaagg?atcaacagct?cctggggatt 1920
tggggttgct?ctggaaaact?catttgcacc?actgctgtgc?cttggaatgc?tagttggagt 1980
aataaatctc?tggaacagat?ttggaatcac?acgacctgga?tggagtggga?cagagaaatt 2040
aacaattaca?caagcttaat?acactcctta?attgaagaat?cgcaaaacca?gcaagaaaag 2100
aatgaacaag?aattattgga?attagataaa?tgggcaagtt?tgtggaattg?gtttaacata 2160
acaaattggc?tgtggtatat?aaaattattc?ataatgatag?taggaggctt?ggtaggttta 2220
agaatagttt?ttgctgtact?ttctatagtg?aatagagtta?ggcagggata?ttcaccatta 2280
tcgtttcaga?cccacctccc?aaccccgagg?ggacccgaca?ggcccgaagg?aatagaagaa 2340
gaaggtggag?agagagacag?agacagatcc?attcgattag?tgaacggatc?ggcactgcgt 2400
gcgccaattc?tgcagacaaa?tggcagtatt?catccacaat?tttaaaagaa?aaggggggat 2460
tggggggtac?agtgcagggg?aaagaatagt?agacataata?gcaacagaca?tacaaactaa 2520
agaattacaa?aaacaaatta?caaaaattca?aaattttcgg?gtttattaca?gggacagcag 2580
agatccagtt?tggttaatga?tccccgggta?ccggtcgcca?ccatggtgag?caagggcgag 2640
gagctgttca?ccggggtggt?gcccatcctg?gtcgagctgg?acggcgacgt?aaacggccac 2700
aagttcagcg?tgtccggcga?gggcgagggc?gatgccacct?acggcaagct?gaccctgaag 2760
ttcatctgca?ccaccggcaa?gctgcccgtg?ccctggccca?ccctcgtgac?caccctgacc 2820
tacggcgtgc?agtgcttcag?ccgctacccc?gaccacatga?agcagcacga?cttcttcaag 2880
tccgccatgc?ccgaaggcta?cgtccaggag?cgcaccatct?tcttcaagga?cgacggcaac 2940
tacaagaccc?gcgccgaggt?gaagttcgag?ggcgacaccc?tggtgaaccg?catcgagctg 3000
aagggcatcg?acttcaagga?ggacggcaac?atcctggggc?acaagctgga?gtacaactac 3060
aacagccaca?acgtctatat?catggccgac?aagcagaaga?acggcatcaa?ggtgaacttc 3120
aagatccgcc?acaacatcga?ggacggcagc?gtgcagctcg?ccgaccacta?ccagcagaac 3180
acccccatcg?gcgacggccc?cgtgctgctg?cccgacaacc?actacctgag?cacccagtcc 3240
gccctgagca?aagaccccaa?cgagaagcgc?gatcacatgg?tcctgctgga?gttcgtgacc 3300
gccgccggga?tcactctcgg?catggacgag?ctgtacaagt?aaagcggccg?cgactctaga 3360
attcgatatc?aagcttatcg?ataatcaacc?tctggattac?aaaatttgtg?aaagattgac 3420
tggtattctt?aactatgttg?ctccttttac?gctatgtgga?tacgctgctt?taatgccttt 3480
gtatcatgct?attgcttccc?gtatggcttt?cattttctcc?tccttgtata?aatcctggtt 3540
gctgtctctt?tatgaggagt?tgtggcccgt?tgtcaggcaa?cgtggcgtgg?tgtgcactgt 3600
gtttgctgac?gcaaccccca?ctggttgggg?cattgccacc?acctgtcagc?tcctttccgg 3660
gactttcgct?ttccccctcc?ctattgccac?ggcggaactc?atcgccgcct?gccttgcccg 3720
ctgctggaca?ggggctcggc?tgttgggcac?tgacaattcc?gtggtgttgt?cggggaaatc 3780
atcgtccttt?ccttggctgc?tcgcctgtgt?tgccacctgg?attctgcgcg?ggacgtcctt 3840
ctgctacgtc?ccttcggccc?tcaatccagc?ggaccttcct?tcccgcggcc?tgctgccggc 3900
tctgcggcct?cttccgcgtc?ttcgccttcg?ccctcagacg?agtcggatct?ccctttgggc 3960
cgcctccccg?catcgatacc?gtcgacctcg?agacctagaa?aaacatggag?caatcacaag 4020
tagcaataca?gcagctacca?atgctgattg?tgcctggcta?gaagcacaag?aggaggagga 4080
ggtgggtttt?ccagtcacac?ctcaggtacc?tttaagacca?atgacttaca?aggcagctgt 4140
agatcttagc?cactttttaa?aagaaaaggg?gggactggaa?gggctaattc?actcccaacg 4200
aagacaagat?atccttgatc?tgtggatcta?ccacacacaa?ggctacttcc?ctgattggca 4260
gaactacaca?ccagggccag?ggatcagata?tccactgacc?tttggatggt?gctacaagct 4320
agtaccagtt?gagcaagaga?aggtagaaga?agccaatgaa?ggagagaaca?cccgcttgtt 4380
acaccctgtg?agcctgcatg?ggatggatga?cccggagaga?gaagtattag?agtggaggtt 4440
tgacagccgc?ctagcatttc?atcacatggc?ccgagagctg?catccggact?gtactgggtc 4500
tctctggtta?gaccagatct?gagcctggga?gctctctggc?taactaggga?acccactgct 4560
taagcctcaa?taaagcttgc?cttgagtgct?tcaagtagtg?tgtgcccgtc?tgttgtgtga 4620
ctctggtaac?tagagatccc?tcagaccctt?ttagtcagtg?tggaaaatct?ctagcagggc 4680
ccgtttaaac?ccgctgatca?gcctcgactg?tgccttctag?ttgccagcca?tctgttgttt 4740
gcccctcccc?cgtgccttcc?ttgaccctgg?aaggtgccac?tcccactgtc?ctttcctaat 4800
aaaatgagga?aattgcatcg?cattgtctga?gtaggtgtca?ttctattctg?gggggtgggg 4860
tggggcagga?cagcaagggg?gaggattggg?aagacaatag?caggcatgct?ggggatgcgg 4920
tgggctctat?ggcttctgag?gcggaaagaa?ccagctgggg?ctctaggggg?tatccccacg 4980
cgccctgtag?cggcgcatta?agcgcggcgg?gtgtggtggt?tacgcgcagc?gtgaccgcta 5040
cacttgccag?cgccctagcg?cccgctcctt?tcgctttctt?cccttccttt?ctcgccacgt 5100
tcgccggctt?tccccgtcaa?gctctaaatc?gggggctccc?tttagggttc?cgatttagtg 5160
ctttacggca?cctcgacccc?aaaaaacttg?attagggtga?tggttcacgt?agtgggccat 5220
cgccctgata?gacggttttt?cgccctttga?cgttggagtc?cacgttcttt?aatagtggac 5280
tcttgttcca?aactggaaca?acactcaacc?ctatctcggt?ctattctttt?gatttataag 5340
ggattttgcc?gatttcggcc?tattggttaa?aaaatgagct?gatttaacaa?aaatttaacg 5400
cgaattaatt?ctgtggaatg?tgtgtcagtt?agggtgtgga?aagtccccag?gctccccagc 5460
aggcagaagt?atgcaaagca?tgcatctcaa?ttagtcagca?accaggtgtg?gaaagtcccc 5520
aggctcccca?gcaggcagaa?gtatgcaaag?catgcatctc?aattagtcag?caaccatagt 5580
cccgccccta?actccgccca?tcccgcccct?aactccgccc?agttccgccc?attctccgcc 5640
ccatggctga?ctaatttttt?ttatttatgc?agaggccgag?gccgcctctg?cctctgagct 5700
attccagaag?tagtgaggag?gcttttttgg?aggcctaggc?ttttgcaaaa?agctcccggg 5760
agcttgtata?tccattttcg?gatctgatca?gcacgtgttg?acaattaatc?atcggcatag 5820
tatatcggca?tagtataata?cgacaaggtg?aggaactaaa?ccatggccaa?gttgaccagt 5880
gccgttccgg?tgctcaccgc?gcgcgacgtc?gccggagcgg?tcgagttctg?gaccgaccgg 5940
ctcgggttct?cccgggactt?cgtggaggac?gacttcgccg?gtgtggtccg?ggacgacgtg 6000
accctgttca?tcagcgcggt?ccaggaccag?gtggtgccgg?acaacaccct?ggcctgggtg 6060
tgggtgcgcg?gcctggacga?gctgtacgcc?gagtggtcgg?aggtcgtgtc?cacgaacttc 6120
cgggacgcct?ccgggccggc?catgaccgag?atcggcgagc?agccgtgggg?gcgggagttc 6180
gccctgcgcg?acccggccgg?caactgcgtg?cacttcgtgg?ccgaggagca?ggactgacac 6240
gtgctacgag?atttcgattc?caccgccgcc?ttctatgaaa?ggttgggctt?cggaatcgtt 6300
ttccgggacg?ccggctggat?gatcctccag?cgcggggatc?tcatgctgga?gttcttcgcc 6360
caccccaact?tgtttattgc?agcttataat?ggttacaaat?aaagcaatag?catcacaaat 6420
ttcacaaata?aagcattttt?ttcactgcat?tctagttgtg?gtttgtccaa?actcatcaat 6480
gtatcttatc?atgtctgtat?accgtcgacc?tctagctaga?gcttggcgta?atcatggtca 6540
tagctgtttc?ctgtgtgaaa?ttgttatccg?ctcacaattc?cacacaacat?acgagccgga 6600
agcataaagt?gtaaagcctg?gggtgcctaa?tgagtgagct?aactcacatt?aattgcgttg 6660
cgctcactgc?ccgctttcca?gtcgggaaac?ctgtcgtgcc?agctgcatta?atgaatcggc 6720
caacgcgcgg?ggagaggcgg?tttgcgtatt?gggcgctctt?ccgcttcctc?gctcactgac 6780
tcgctgcgct?cggtcgttcg?gctgcggcga?gcggtatcag?ctcactcaaa?ggcggtaata 6840
cggttatcca?cagaatcagg?ggataacgca?ggaaagaaca?tgtgagcaaa?aggccagcaa 6900
aaggccagga?accgtaaaaa?ggccgcgttg?ctggcgtttt?tccataggct?ccgcccccct 6960
gacgagcatc?acaaaaatcg?acgctcaagt?cagaggtggc?gaaacccgac?aggactataa 7020
agataccagg?cgtttccccc?tggaagctcc?ctcgtgcgct?ctcctgttcc?gaccctgccg 7080
cttaccggat?acctgtccgc?ctttctccct?tcgggaagcg?tggcgctttc?tcatagctca 7140
cgctgtaggt?atctcagttc?ggtgtaggtc?gttcgctcca?agctgggctg?tgtgcacgaa 7200
ccccccgttc?agcccgaccg?ctgcgcctta?tccggtaact?atcgtcttga?gtccaacccg 7260
gtaagacacg?acttatcgcc?actggcagca?gccactggta?acaggattag?cagagcgagg 7320
tatgtaggcg?gtgctacaga?gttcttgaag?tggtggccta?actacggcta?cactagaaga 7380
acagtatttg?gtatctgcgc?tctgctgaag?ccagttacct?tcggaaaaag?agttggtagc 7440
tcttgatccg?gcaaacaaac?caccgctggt?agcggtggtt?tttttgtttg?caagcagcag 7500
attacgcgca?gaaaaaaagg?atctcaagaa?gatcctttga?tcttttctac?ggggtctgac 7560
gctcagtgga?acgaaaactc?acgttaaggg?attttggtca?tgagattatc?aaaaaggatc 7620
ttcacctaga?tccttttaaa?ttaaaaatga?agttttaaat?caatctaaag?tatatatgag 7680
taaacttggt?ctgacagtta?ccaatgctta?atcagtgagg?cacctatctc?agcgatctgt 7740
ctatttcgtt?catccatagt?tgcctgactc?cccgtcgtgt?agataactac?gatacgggag 7800
ggcttaccat?ctggccccag?tgctgcaatg?ataccgcgag?acccacgctc?accggctcca 7860
gatttatcag?caataaacca?gccagccgga?agggccgagc?gcagaagtgg?tcctgcaact 7920
ttatccgcct?ccatccagtc?tattaattgt?tgccgggaag?ctagagtaag?tagttcgcca 7980
gttaatagtt?tgcgcaacgt?tgttgccatt?gctacaggca?tcgtggtgtc?acgctcgtcg 8040
tttggtatgg?cttcattcag?ctccggttcc?caacgatcaa?ggcgagttac?atgatccccc 8100
atgttgtgca?aaaaagcggt?tagctccttc?ggtcctccga?tcgttgtcag?aagtaagttg 8160
gccgcagtgt?tatcactcat?ggttatggca?gcactgcata?attctcttac?tgtcatgcca 8220
tccgtaagat?gcttttctgt?gactggtgag?tactcaacca?agtcattctg?agaatagtgt 8280
atgcggcgac?cgagttgctc?ttgcccggcg?tcaatacggg?ataataccgc?gccacatagc 8340
agaactttaa?aagtgctcat?cattggaaaa?cgttcttcgg?ggcgaaaact?ctcaaggatc 8400
ttaccgctgt?tgagatccag?ttcgatgtaa?cccactcgtg?cacccaactg?atcttcagca 8460
tcttttactt?tcaccagcgt?ttctgggtga?gcaaaaacag?gaaggcaaaa?tgccgcaaaa 8520
aagggaataa?gggcgacacg?gaaatgttga?atactcatac?tcttcctttt?tcaatattat 8580
tgaagcattt?atcagggtta?ttgtctcatg?agcggataca?tatttgaatg?tatttagaaa 8640
aataaacaaa?taggggttcc?gcgcacattt?ccccgaaaag?tgccacctga?c 8691
Claims (5)
1. the T lymphocyte of screening and activating dormant infection HIV-1-1 medicine is characterized in that, this cell is that the Jurkat that this report gene is not expressed simultaneously in the HIV slow virus infection that is carried reporter gene again stablizes strain; The preparation method of described HIV slow virus may further comprise the steps: (1) FUGW carrier is cut through the PacI+BamhI enzyme, reclaims the 7KB fragment and the FGW plasmid of acquisition ubiquitin control region disappearance with the connection of T4 ligase enzyme; (2) FGW plasmid, coating plasmid VSVG and packaging structure plasmid CMV Δ 8.9 plasmids mix, and transfection people 293T cell after 48 hours, is collected supernatant, obtains FGW virus through ultracentrifugation, and its titre is at 10E5-10E8, and-80 degree store.
2. cell as claimed in claim 1 is characterized in that, this cell is that preserving number is the cell of CCTCCNO.C200821.
3. the preparation method of the described cell of claim 1, it is characterized in that, with the slow virus infection human T lymphocyte who carries the HIV-1 of EGFP reporter gene is Jurkat, integrates by cell sorting and HIV and detects, and obtains to have that HIV integrates but the clone that do not express EGFP.
4. preparation method as claimed in claim 3 is characterized in that, further utilizes TNF-a to carry out the activation of inducing of different concns, and analysis of fluorescence intensity and fluorescence positive cells number screen and obtain the EGFP cloning by expression of inducibility at last.
5. the application of the described cell of claim 1 is characterized in that, compound is added the described cell of claim 1, and the compound that can make cell green fluorescence occur is candidate's activating dormant infection HIV-1-1 medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810038851XA CN101372707B (en) | 2008-06-12 | 2008-06-12 | T lymphocyte for screening and activating dormant infection HIV-1 compound and preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810038851XA CN101372707B (en) | 2008-06-12 | 2008-06-12 | T lymphocyte for screening and activating dormant infection HIV-1 compound and preparation thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101372707A CN101372707A (en) | 2009-02-25 |
| CN101372707B true CN101372707B (en) | 2011-02-16 |
Family
ID=40447084
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810038851XA Expired - Fee Related CN101372707B (en) | 2008-06-12 | 2008-06-12 | T lymphocyte for screening and activating dormant infection HIV-1 compound and preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101372707B (en) |
-
2008
- 2008-06-12 CN CN200810038851XA patent/CN101372707B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| 修文琼，等.抗艾滋病细胞株的交叉耐性及抑制细胞凋亡3.《中国公共卫生》.2003,第19卷(第12期), |
| 修文琼等.抗艾滋病细胞株的交叉耐性及抑制细胞凋亡3.《中国公共卫生》.2003,第19卷(第12期), * |
| 倪 崖，等.HIV - 1VN Jurkat细胞株HIV - 1病毒分泌动力学及培养优化的研究.《中国卫生检验杂志》.2006,第16卷(第4期), |
| 倪崖等.HIV-1VN Jurkat细胞株HIV-1病毒分泌动力学及培养优化的研究.《中国卫生检验杂志》.2006,第16卷(第4期), * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101372707A (en) | 2009-02-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2457282C (en) | Method for expression of small antiviral rna molecules within a cell | |
| CA2680129C (en) | Method for expression of small antiviral rna molecules with reduced cytotoxicity within a cell | |
| US20030157691A1 (en) | Method for expression of small antiviral RNA molecules within a cell | |
| US20030101471A1 (en) | Method for producing transgenic birds and fish | |
| CN110656123B (en) | Method for screening sgRNA high-efficiency action target based on CRISPR-Cas13d system and application | |
| CN107557388B (en) | Lentiviral vector for CAR-T preparation and construction method and application thereof | |
| KR101522217B1 (en) | Fsh producing cell clone | |
| CN112011574B (en) | Lentiviral vector, construction method and application thereof | |
| CN108949693A (en) | A kind of pair of T cell immune detection point access carries out the method and application of gene knockout | |
| CN114196705B (en) | Recombinant adeno-associated virus packaging plasmid, recombinant adeno-associated virus and application thereof | |
| CN113584062B (en) | Fusion imaging gene, lentivirus expression plasmid, lentivirus and cell thereof, and preparation method and application thereof | |
| CN113652451B (en) | Lentiviral vector, construction method and application thereof | |
| CN107267556B (en) | anti-HBV drug screening cell model of targeted HBV core promoter and construction and application thereof | |
| IL198421A (en) | Adipocyte-specific constructs for specifically inhibiting platelet-type 12 lipoxygenase expression and uses thereof | |
| CN101899465A (en) | Recombinant J subgroup avian leucosis virus infective cloned plasmids and preparation method and application thereof | |
| CN101372707B (en) | T lymphocyte for screening and activating dormant infection HIV-1 compound and preparation thereof | |
| KR102009273B1 (en) | Recombinant foot-and-mouth disease virus expressing protective antigen of type O-TAW97 | |
| CN106701806B (en) | Assess serial transcription factor Reporter System and its application of Chinese medicine and/or natural drug efficiency | |
| KR101535070B1 (en) | Recomnication expression vector of vascular growth factor and the vascular growth factor expressing stem cell line thereof | |
| KR20170017115A (en) | Method of testing genotype and phenotype for simultaneously predicting drug resistance against protease inhibitor, reverse transcriptase inhibitor and integrase inhibitor | |
| CN112481285A (en) | Synthesis method of target gene fragment | |
| CN116672442B (en) | Preparation of medicine for treating osteosarcoma by combining ligustilide and HER2-CAR-T cells | |
| CN112626029B (en) | Transgenic modified Daudi cell and preparation method and application thereof | |
| KR20230075800A (en) | Rheumatoid arthritis disease simulation model and screening platform with coronavirus infection | |
| CN110499314B (en) | Protein eukaryotic expression promoter, protein expression vector, construction method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: BEIJING AIKANG RUIHAO BIOLOGICAL TECHNOLOGY LLC Free format text: FORMER OWNER: FUDAN UNIVERSITY Effective date: 20150114 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 200433 YANGPU, SHANGHAI TO: 101300 HAIDIAN, BEIJING |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20150114 Address after: 101300, room 2, floor 4, building 5, 218 East Road, Haidian District, Beijing Patentee after: Hao Rui Beijing Kang'ai biological technology limited liability company Address before: 220 Handan Road, Shanghai, No. 200433 Patentee before: Fudan University |
|
| C56 | Change in the name or address of the patentee |
Owner name: BEIJING BIOHEALTHCARE BIOTECHNOLOGY CO., LTD. Free format text: FORMER NAME: BEIJING AIKANG RUIHAO BIOLOGICAL TECHNOLOGY LLC |
|
| CP03 | Change of name, title or address |
Address after: 101300, Beijing, Haidian District information industry base on the third floor, No. 1, building four, paragraph 417, C Patentee after: The great biotech inc of Beijing Kang Airui Address before: 101300, room 2, floor 4, building 5, 218 East Road, Haidian District, Beijing Patentee before: Hao Rui Beijing Kang'ai biological technology limited liability company |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110216 Termination date: 20180612 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |