CN109337903A - Long-chain non-coding RNA lncRNA-6585, antibody and application thereof - Google Patents

Long-chain non-coding RNA lncRNA-6585, antibody and application thereof Download PDF

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CN109337903A
CN109337903A CN201811239545.2A CN201811239545A CN109337903A CN 109337903 A CN109337903 A CN 109337903A CN 201811239545 A CN201811239545 A CN 201811239545A CN 109337903 A CN109337903 A CN 109337903A
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lncrna
cancer
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polypeptide
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CN109337903B (en
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李婉
卢春
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Nanjing Medical University
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Abstract

The invention discloses a long-chain non-coding RNA lncRNA-6585, an antibody and application thereof. According to the RNA lncRNA-6585P region contained in the long-chain non-coding RNA lncRNA-6585, the long-chain non-coding RNA lncRNA-6585 can encode and express polypeptides and participate in regulating and controlling the migration and invasion processes of tumor cells, so that the lncRNA-6585 and the encoded polypeptides thereof play an important role in the morbidity process of cervical cancer and can be used as a new molecular marker and a drug target for diagnosing and treating clinical tumor diseases.

Description

A kind of long-chain non-coding RNA lncRNA-6585 and its antibody and application
Technical field
The invention belongs to oncomolecularbiologies and antibody production techniques field, and in particular to a kind of long-chain non-coding RNA LncRNA-6585 and its antibody and application.
Background technique
In recent years, the tune that non-coding RNA plays in the biological processes such as tumor cell proliferation, differentiation, transfer and apoptosis Section effect is the focal issue that researchers are concerned about.Long-chain non-coding RNA (long noncoding RNA, lncRNA) is a kind of Transcript length is greater than 200 nt(nucleotide), it is initially considered as the functional RNA molecule for not translating into protein (Science, 2005,309 (5740): 1559-1563.).Recent study shows generation and the hair of lncRNA and many cancers Open up closely related, such as bladder cancer, breast cancer, liver cancer and prostate cancer (Urology, 2010,77 (2): 510.e1- 510.e5..Nature, 2010,464 (7291): 1071-1076.), also identifying in succession some has coding polypeptide ability LncRNA(Cell. 2015 Feb 12;160(4):595-606；Mol Cell. 2017 Oct 5;68(1):171- 184.e6；Nature. 2017 Jan 12;541 (7636): 228-232.).Therefore, the present invention is online by bioinformatics Software analysis finds the lncRNA with coding polypeptide ability and prepares antibody for the polypeptide of its coding, to swell for clinic New biomarker is excavated in the diagnosis of tumor disease, Index for diagnosis, provides break-through point for treatment.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of long-chain non-coding RNA lncRNA-6585 And its antibody and application.Closely related long-chain occurs for long-chain non-coding RNA lncRNA-6585 and human tumor of the invention Non-coding RNA.
A kind of long-chain non-coding RNA lncRNA-6585(Ensembl database login ENST00000426585, SEQ ID No:1), nucleic acid sequence encodes the nucleotide sequence such as SEQ ID No:2 of polypeptide as shown in SEQ ID No:1, and orders Entitled lncRNA-6585P.
It is the amino acid sequence of the polypeptide such as SEQ ID No:3 as improved.
The recombinant virus pCDH- of nucleotide sequence lncRNA-6585P containing coding polypeptide described in claim 1 The plasmid of lncRNA-6585P.
A kind of lncRNA-6585P is overexpressed the construction method of plasmid, includes the following steps: step 1, and synthesis obtains SEQ The target gene of ID No:2 sequence, finish up one Flag label of each addition；Step 2, restriction enzyme is usedXbo IWithBamH IDigestion, purifying, connection carried out to target gene and pCDH-CMV-MCS-EF1-copRFP carrier, conversion to Escherichia coli, i.e., Plasmid pCDH-lncRNA-6585P must be overexpressed.
The antibody that plasmid-encoded polypeptide is generated by immune response is overexpressed based on the lncRNA-6585P.
Above-mentioned antibody is in preparation prevention or treatment treatment cervical carcinoma, oophoroma, carcinoma of endometrium, breast cancer, colon cancer, liver Cancer, glioma, lung cancer, cancer of pancreas or gastric cancer product in application.
Above-mentioned lncRNA-6585P is in preparation prevention or treatment cervical carcinoma, oophoroma, carcinoma of endometrium, breast cancer, knot Intestinal cancer, liver cancer, glioma, lung cancer, cancer of pancreas or gastric cancer product in application.
The utility model has the advantages that
Long-chain non-coding RNA lncRNA-6585 provided by the invention can encode expression polypeptide, and participate in modulate tumor cell Invasive procedure is migrated, therefore, the polypeptide of lncRNA-6585 and its coding plays a significant role during tumor invasion, can be with New molecular labeling and drug target as diagnosis and treatment clinical tumor disease.
Detailed description of the invention
Fig. 1 is lncRNA chip results volcano schematic diagram, shows that differential expression is up to 2 times in 3 pairs of cervical carcinomas and cancer beside organism The distribution of above lncRNA；
Fig. 2 is that Western blot verifies lncRNA-6585P coding polypeptide；(A) endothelial cell line；(B) Cervical Cancer HeLa Cells System；
Fig. 3 is that Western blot detects anti-lncRNA-6585P antibody；(A) E7885 antibody test result；(B) E7886 antibody Testing result；
Fig. 4 is Transwell migration and Matrigel result after 12 h of endothelial cell inoculation for being overexpressed lncRNA-6585P； (A) migration experimental result statistics；(B) Matrigel result counts (* * *P < 0.001), the pCDH group that wherein ledgement is filled, Packless is lncRNA-6585P group；
Transwell migration and Matrigel result after Fig. 5 is 12 h of HeLa cell inoculation for being overexpressed lncRNA-6585P； (A) migration experimental result statistics；(B) Matrigel result counts (*P < 0.05, * * *P < 0.001), wherein ledgement is filled PCDH group, it is packless be lncRNA-6585P group.
Specific embodiment
Method of the invention is described in detail and is illustrated below with reference to specific example.Its content is to solution of the invention Release protection scope rather than limiting the invention.
Embodiment 1
The long-chain non-coding RNA lncRNA-6585P of low expression can encode polypeptide in cervical carcinoma
1, material
1.1 tissue
Clinical tissue sample of the invention is all from November, 2013 in June, 2017 in the attached Nanjing women and children of Nanjing Medical University The Ib1 phase cervical cancer patient of gynemetrics of health care institute treatment, each pair of sample include the cervical cancer tissues of operation excision and the cancer of pairing Side tissue.The use of all samples signs informed consent form by patient or its trustee, has obtained Nanjing Medical University's medicine Ethics Committee's approval.
1.2 materials and cell
Three plasmid packaging system of slow virus includes Lentiviral pCDH-CMV-MCS-EF1-copRFP, envelope plasmid PMD2.G and packaging plasmid are that this laboratory saves.293T cell needed for experiment and HeLa Cells are purchased From Cell Bank of Chinese Academy of Sciences, it is incubated at containing 10% inactivated fetal bovine serum (fetal bovine serum, FBS), gentamicin In the DMEM culture medium of (100 U/mL), streptomysin (100 μ g/mL) and penicillin (100 U/mL).Human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVECs) is the preservation of this laboratory, is incubated at containing 20% inactivation In fetal calf serum.Cell culture condition is 37 DEG C, 5% CO2。
1.3 reagent
Trizol reagent is purchased from U.S. Thermo Scientific company；Fluorescence real-time quantitative PCR (real-time Quantitative polymerase chain reaction, RT-qPCR) used in 2 × ChamQ SYBR qPCR Master Mix reagent is that biological Co., Ltd's product is only praised in Nanjing promise；The primer of RT-qPCR is the conjunction of Suzhou Jin Weizhi Science and Technology Ltd. At；The monoclonal antibody (monoclonal antibody, MAb) of anti-Flag is produced purchased from U.S. Thermo Scientific company Product；Elite ABC liquid and DAB colour reagent box are purchased from VECTOR company；The genetic chip product of ArrayStar company (Human LncRNA Microarray V3.0Service) is acted on behalf of by upper Haikang at bioengineering Co., Ltd.Used in PCR High fidelity enzyme (Phanta enzyme) is purchased from Nanjing Nuo Weizan Science and Technology Ltd.；Gel purification kit is purchased from U.S. Omega company； Restriction enzyme is Japan's TaKaRa Products；T4 DNA ligase, nucleic acid standard molecular weight Marker, plasmid transfection examination Agent LipofectamineTM2000, Trizol and protein standard molecular weight Marker is U.S. Thermo Scientific company Product；Competent E.coli DH5 α is purchased from Tiangeng biochemical technology Co., Ltd；RIPA cell pyrolysis liquid, protease inhibitors, Inhibitors of phosphatases and phenylmethyl sulfonylfluoride (phenylmethyl sulfonylfluoride, PMSF) are upper Haikang At biotech firm's product；Anti- reference gene α-tubulin MAb is purchased from U.S. Santa Cruz company；Horseradish peroxidase (HRP) goat anti-rabbit igg and sheep anti-mouse igg marked is the green skies Products of China.
2. method
The lncRNA chip technology of 2.1 cervical cancer tissues and cancer beside organism
Using the operation of the cervical cancer patient of 3 all ages and classes (39 years old, 54 years old, 65 years old) excision sample as object, Haikang in commission " ArrayStar Human LncRNA Microarray V3.0Service " service is carried out at bioengineering Co., Ltd, into Row difference lncRNA screening.
The ability of 2.2 analysis differential expression lncRNA coding polypeptides
Utilize online software RegRNA2.0(http: //regrna2.mbc.nctu.edu.tw/detection_ Output.php) and LncATLAS (http://lncatlas.crg.eu/) analysis differential expression lncRNA.Wherein transcribe Region is located at No. 22 chromosomes, and for nucleotide sequence as shown in SEQ ID NO:1, the lncRNA of 5 725 nt of overall length has potential volume The ability of code polypeptide, is named as lncRNA-6585.The possibility that prediction obtains encodes polypeptide region nucleotide sequence such as SEQ ID Shown in No:2, which is named as lncRNA-6585P.
The building of 2.3 recombinant plasmid pCDH-lncRNA-6585P
The SEQ ID No:2 sequence ending one Flag label of each addition predicted in 2.2, send company to synthesize.With restricted interior Enzyme cuttingXbo IWithBamH IDigestion, purifying, connection are carried out to synthesis gene and pCDH-CMV-MCS-EF1-copRFP carrier, turned Change to Escherichia coli, obtains the plasmid of pCDH-lncRNA-6585P.
The packaging of 2.4 recombinant viruses
The 293T cell of logarithmic growth phase, by 1.5 × 106A/plate cell number is seeded in 10 cm tissue culture plate of diameter, The not antibiotic DMEM culture medium of 10 ml is added, in 37 DEG C, 5% CO2Culture in incubator.Next day reaches when cell confluency degree When 70%~80%, liposome Lipofectamine is utilizedTM2000 will carry the plasmid pCDH- of lncRNA-6585 coding region LncRNA-6585P, packaging plasmid psPAX2, envelope plasmid pMD2.G according to 4:3:1 ratio cotransfection 293T cell, simultaneously Using pCDH-CMV-MCS-EF1-copRFP, psPAX2, pMD2.G plasmid co-transfection 293T cell as empty vector control.In turn 10 h replace fresh antibiotic complete medium after dye.Fluorescence microscope green fluorescence egg is utilized after transfecting 48 h The expression of white (green fluorescent protein, GFP).Cell culture is collected respectively after transfecting 48 h and 72 h PB to final concentration of 8 μ g/ml is added after mixing the supernatant collected twice in supernatant, then divides after 0.45 μm of filter filters Dress, saves backup in -70 DEG C.Slow virus obtained is respectively designated as pCDH(slow virus empty carrier) and lncRNA-6585P (recombinant slow virus of expression lncRNA-6585P).
Expression of 2.5 lncRNA-6585P in HUVECs and HeLa cell
Respectively by HUVECs and HeLa cell with 0.5 × 106/ hole is inoculated in 6 orifice plates, with plasma-free DMEM medium culture. Culture medium is removed when cell confluency degree reaches 70% ~ 80%, HUVECs is infected respectively with pCDH or lncRNA-6585 and HeLa is thin Born of the same parents inhale after 8 h and abandon supernatant fresh complete medium is added.Expression through fluorescence microscope GFP after 48 h；When When RFP is up to 90% or more, illustrate that stablizing expression pCDH-lncRNA-6585P and control cell constructs successfully.
2.6 Western blot verify protein expression
2.6.1 total protein of cell is extracted
It configures the every 100 μ L RIPA of cell pyrolysis liquid and 1 μ L protease inhibitors, 1 μ L inhibitors of phosphatases, 1 μ L is added PMSF, 25 4 × lauryl sodium sulfate of μ L (sodium dodecyl s μ lphate, SDS) buffer.By every 1 × 104It is a 100 μ L cell pyrolysis liquid lytic cells are added in cell, and be vortexed concussion 15 s, 8 min of ice bath, repeat concussion three times.Then 100 ° 5 min of C boiling water bath, 5 min of ice bath, as total protein of cell, albumen are placed in -70 °C of refrigerator-freezers after packing and save backup again.
2.6.2 Western blot
10% SDS-PAGE running gel is configured, swimming lane is added with every 20 μ L of hole in the protein sample of 2.5.1 preparation.Constant pressure 60 V electrophoresis, after band is placed in separation gel, adjustment 110 V of voltage continues electrophoresis to terminating.Use 100 mA100 min of constant current will afterwards Film is placed in after transferring film by protein delivery to (two) vinyl fluoride (polyvinylidene difluoride, PVDF) film partially is gathered 37 °C of 1 h of closing in 5% skim milk.Anti- Flag PAb, anti-GAPDH MAb is separately added into be incubated overnight in 4 °C of shaking tables.Next day Primary antibody is recycled, and with TBS-T detergent bar band (10 min × 3 time)；It is then respectively adding horseradish peroxidase The goat-anti rabbit of (horseradish peroxidase, HRP) label and sheep anti mouse secondary antibody, act on 1 h in 37 °C of insulating boxs, TBS-T detergent bar band (10 min × 3 time)；Finally detected using chemoluminescence method.
3, result
3.1 gene chip results
By Arraystar human lncRNA expression microarray V3.0 technology, 3 all ages and classes are obtained Cervical cancer patient tumor tissues and differential expression in cancer beside organism gene (Fig. 1).
3.2 lncRNA-6585 have coding polypeptide ability
Western blot result is as shown in Fig. 2, it can be seen from the figure that lncRNA-6585 can encode expression polypeptide.
Embodiment 2
The Antibody preparation of people long-chain non-coding RNA lncRNA-6585 coding polypeptide
1. Antibody preparation specific embodiment
The analysis and design of 1.1 polypeptide sequences
Characterization of antigenic epitopes is carried out using amino acid sequence of the DNAstar software to lncRNA-6585P coding polypeptide, mainly Hydrophily is conventionally assessed, antigenicity, surface possibility, the indexes such as flex region prepared the reality of antibody in conjunction with the past Border experience considers that amino acid structure complexity, oxidizable degree synthesize difficulty, and amino acid classification and distribution etc. are final to determine Select 56-70aa synthesis polypeptide C-TDSKLYIPSEYRSIS(19mg, SEQ ID No:4).
The preparation of 1.2 antigens
Peptide C-TDSKLYIPSEYRSIS is dissolved in PBS buffer solution, and sulfydryl is coupled on KLH, is immunized 2 Rabbit was delivered immune on May 22nd, 2018.
1.3 immune processes
The purifying of 1.4 antiserums
After antiserum C-TDSKLYIPSEYRSIS makees antigen affinity purification, the antibody after being concentrated.Obtain antibody E7885(concentration 2.71mg/ml) and E7886(concentration 2.31mg/ml).
1.5 Western blot detect antigen
Western blot testing conditions: antigen coat amount is 100ng, and antibody dilution ratio is 1:1000,1:5000,1: 10000,1:50000,1:100000,1:200000.
1.6 immunoblotting assay
Anti- lncRNA-6585P antibody is identified with Western blot by 2.6 the methods in example 1.
2. result
Western blot result is as shown in figure 3, the antibody as can be seen from the figure prepared can be used for detecting lncRNA- The expression of 6585P coding polypeptide.
Embodiment 3
Application of the long-chain non-coding RNA lncRNA-6585P in antitumor
1, material
1.1 cell
The used HUVECs and HeLa cell culture processes of experiment are the same as embodiment 2.
1.2 reagent
Slow virus pCDH and lncRNA-6585P are packed with embodiment 2.The cell Transwell (8 μm) is purchased from Millipore public affairs Department.
2, method
2.1 Transwell migration experiment
The DMEM culture medium that 500 μ L contain 10% inactivated fetal bovine serum is added into the every hole of 24 orifice plates, the cell Transwell is placed in In hole, it is put into cell incubator and balances 30 min.By pHAGE HUVEC, lncRNA-6585P HUVEC, pHAGE HeLa, Tetra- groups of cells of lncRNA-6585P HeLaa digest counting respectively, and cell density is adjusted to 5 × 10 with pure DMEM culture medium4 A/mL.200 μ L cell suspensions are added in each cell, i.e., every hole is 1 × 104A cell, 3 multiple holes of every group of setting.It is placed in training It supports and continues to cultivate in case, and take out cell after cultivating 12 h, formaldehyde fixes 15 min, 15 min of violet staining.After drying It takes pictures under the microscope, analysis of accounts is carried out to the cell quantity for passing through cell.
2.2 Matrigel Matrigels
Matrigel and DMEM culture medium are made into matrigel-DMEM mixed liquor by volume 1:7, are placed in spare on ice.It will The cell Transwell is placed in 24 orifice plates, and 500 μ L DMEM culture mediums are added in lower room, and 60 μ L matrigels-are added in each cell DMEM mixed liquor, 1 h of gel in 37 °C, 5% CO2 cell incubator.PHAGE HUVEC, lncRNA-6585P are digested respectively Tetra- groups of HUVEC, pHAGE HeLa, lncRNA-6585P HeLaa cells, after counting with DMEM culture medium be configured to 5 × 105/ The cell suspension of mL.200 μ L cell suspensions, i.e., every 1 × 105 cell in hole, 3 multiple holes of every group of setting are added to each cell. 24 orifice plates are put into cell incubator, cell dyeing is taken out in 12 h and fixes, counting of taking pictures under microscope, observation enters lower room Cell quantity is to judge cell invasion situation.
3, result
The migration and invasion of lncRNA-6585P inhibition endothelial cell and cervical cancer cell
LncRNA-6585P is verified in endothelial cell and cervical carcinoma by the migration of the cell Transwell and Matrigel Matrigel Effect in cell migration, statistical result show that after 12 h of plating cells, lncRNA-6585P group penetrates the thin of cell bottom Born of the same parents' quantity is less than pCDH group.Result above prompt, being overexpressed lncRNA-6585P can inhibit endothelial cell (Fig. 4 A and Fig. 4 B) With the migration and invasion of cervical cancer cell (Fig. 5 A and Fig. 5 B).
The above results show that the overexpression of lncRNA-6585P can obviously inhibit moving for endothelial cell and cervical cancer cell It moves and invasive ability, the polypeptide participation regulation for illustrating that the lncRNA-6585 of low expression level and its coding are expressed in cervical carcinoma is swollen Tumor occurs, and the potential become with anti-cervical cancer, provides new molecule for the diagnosing and treating of cervical carcinoma and other tumours Label and drug targets.
The foregoing is only a preferred embodiment of the present invention, the scope of protection of the present invention is not limited to this, it is any ripe Know those skilled in the art within the technical scope of the present disclosure, the letter for the technical solution that can be become apparent to Altered or equivalence replacement are fallen within the protection scope of the present invention.
Sequence table
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atagaatgtc aaaaggtgag actctctcat gtggctaaac accctcctac tgcagataca 840
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gttgcaccac tctcagggtc agcatcctcc ccagtgtggc atgggggagg ggaggggagg 960
gggcacgggg cctgggaggc tgtagcctaa aggcacgcag cgggcaagag actactgggc 1020
ggacccctgc cagccttcag ttccatgagc gtctggtgcc aggacgctcc ttgtgggctc 1080
tgccctggga gaagtgccct tgcccagtgc tccttctcgg tgtctgtgac gtgctgtctc 1140
cagtcctcct cattagcata ccttggccga cctttggctc atggaaaaac actttgctct 1200
tctcggaaca atttcttttt cacaactgag caaatgactg atgaggtgac cacactcatg 1260
accatactca tctgttggtg gtgggtgggg ggtggactgg atgaggacag cattgggcaa 1320
cactctttct ggcctcccca ctgcagggag ccacaattac tgtgggaggc cagggtagtc 1380
cagggtttcc ctttgggcca tcagcattcc aactgctcct cctacttctg gcctctggag 1440
aactaaatta ccaccatgca cttcccattc cacctgtgcc tcagcccgag ggtcctgctc 1500
ttcctgcata ttcaagctga tctttctagg gagtagcctg cactgtgcca gcactacccc 1560
cttgtcacag gcagcacctc cctgtgctgg gggagcctca gtctgccctt gggctcaggc 1620
ccctaggctc aggttagctg acattcccag gaaatcctac aacttctcct ttcctttcat 1680
ggctgtaaga cacagtccac tatcagtctc attgtaaaag acatttctaa agagtgtcct 1740
cctttctttc ccaacactag cagggacagt aagtgatgtt tcctgggaga tgctatggtg 1800
ttatttttgc actacaaggg ctctgaagac tgccaggctt gaaatgaaga acgatgtcac 1860
ctgcctatca cagccaggtg acaatggaca aggggcttca cctctgagct gttttccagc 1920
tgcactgggc acattcctcc cctcaccctg actcacaagg tgttagcaat aatggacact 1980
tctctacctt ctgccacact ctgaacaatg ccacacttcc atcaaatgga ttcatcccaa 2040
aggtaaagcc catcacttct agagggagaa aggtaatgtt ttcctaccac ttttatttga 2100
tctcatttag aaggtacttg aggaatagag aagactgggt tttctctggg attatactta 2160
ggcatttctt aacgactggg acatgttctg aaaaatctgt cattacacaa tttcatcatt 2220
gtgctatcat agagcataca cacccgaccc tagatggcat agcctactcc acacctaggc 2280
tgtacagtat ggcctgttgc tcaaggctac acacctgtac agcatgttac tgtactggat 2340
actgtaggca actgtaacac agtggtattt gtgtatctaa acgtatataa acacagaaaa 2400
ggtatagtaa aagatggtat aatttcatgg gaccaccatc cgatacacca tccatcacca 2460
acagaaacat ggttatgagg cacaggactg taactgcagc ccctgccatg aggtcaatgg 2520
cagcctggat agggggattg gacttcatgt ttgcccactc ttctggtctc atggaaggca 2580
gctctctgga gcttatgaca ggcaccacat atccctgggg ggaggcaaga gttcaccatt 2640
agtccaagtc tgcagcctcc cattctctag gcctgcagcc aaattactgg acctaaactt 2700
ctgggagcca tgaactgggc tctacattgt tccaaaggaa aggggctcaa ggaatcagtg 2760
cagcttagca attgtccctt agaaagctca cagatcaaaa acctcccctg cgtatcaatt 2820
ctacagccac cagcagcact tcaactgacc tttctgaaaa ctgaaaaaat gaacaccaaa 2880
taagtcaacc acagatgtac taacccacat caggcttgct tttttagagg cctgcgctga 2940
gccatcaact gaacaggctc acttctaaat cagaaagcag cctgtgctgc gtgcctaagg 3000
gaacgtcaca agacagtagg tctggaaaga agcacccagg cagccaaggg tgggagctgc 3060
accccaggat cacaaacgcc ttggtgaatg cctgctctac ctgcagtgtg gcccctttgg 3120
cctctacagc tggaagtctt catctgtaca aagtgaataa taaaaatatt ataaaacaaa 3180
ggcagtcaca aatagccaca ttggtgttga ggtgatcaaa cccaacacca ggccatgggg 3240
gctacaaagt ccggcagagt caaaggaatg agacaagcta atagtacaca aagtgggtcc 3300
agggggccaa tgctagtata gaggctgtga aggccctgaa ctctgggagc ccacaccatt 3360
tattggagat taaacaaaga agcaggtggt gaggacgtgc tggtcaaaag gaagcagtca 3420
catcaagtgt ttagtttata gctgtgtcgg tctagcattt tctttgaagc atatggaaca 3480
tgttctgcta ctcgagataa tgaacatttc cttctgcctc aaggtacaat cagtttatga 3540
tcctgggaga gcaagaagca aggagccagc aagtctggac acattccaga ggccacgagg 3600
ggttttatgt cctgagtcct ggattccatc caagccatga ggggttttat gccctaggct 3660
taggttgtag tgcggcgggg cagccttcca cccttaagca cagaacctgg tgttccatag 3720
gccacaagaa gttttaaact ctggacccag gacatgttcc aaggctcttt tcatattatg 3780
tcagactagc aagtcttgcc tcagcttttc tcccaacaat tggactgatg ggttgctcca 3840
ctgggcacaa gcatcatggg ttcttaaaac aaggccctga acaagcacca aatatgttcc 3900
tgtcaccaca ctccactagc ccttcaacta taaacatgca taggagtcac ctgggggcct 3960
tgctaaataa aatgcagctt ctgattcaat agtctcaaac aggaccagag attctgcgtc 4020
tcttgttgag ttcccgagtg aggcagacaa tgccagtcca cagactcaca ttttgagata 4080
cagcacctgg gccattgtgt tccaatgtgc ttgataacct ggagcaccta ttaaatatcc 4140
aagttgccag gactttcttc tggaaatctt aattcagtat gttttgtttg gagccttgga 4200
cgtttgggaa aactagaatt tctttctctc cctttagaca aaagtcaact actgctgagg 4260
catgggctta ataaatgttg actaaaatat ccaactcaac aaccaatcct gtataatttt 4320
caaactctgt caataacttg ctgggtccaa cctgcagacc ctggctgcgt gatggatgaa 4380
gaaatgcact cagacacagg tatcctgtga aagaatgggg taggggactg ggcagctccc 4440
agacactgag gagggtgcta taaagagtca gcaggtgcta taaagagtca ccaggcacag 4500
ccctgtcagg ctggggctcc aggcatttat tcagtacaga tttaatgaca aagttctcaa 4560
gtaaacacca caccactaga gggtaattaa cattgctgac ctcctgagta gagagcagtc 4620
atgcgcccgc aaatgatcaa aggtctgttt taggaccaca tgagtaaaca agctatttag 4680
ataaacttct ctacattcct atgtatctac gccctaagct tttaagagaa ttcagctgcc 4740
ttcagccaaa tcttttactg aaggtatgca aacctcccag ccttccaaga agttttgtgt 4800
ctatctccta caacttaatt tttataattt ctcccaccac cctgacagat cccctacaaa 4860
agaccatgtt tcttctcagc ttgctcagtc cagctgagcc agctttccac tggactcctg 4920
aatgtgagaa ggacacaggc tcccaggacc atccagaagc atctctcatc accttaaagc 4980
tctgaaacac tggctgggtg cagaggctca cacaagtaat cccaacacat taagaggctg 5040
aggtgggagg attgcttgag gccaggaatt caagaccagc ctaggcagca tagcaagacc 5100
ccatctctac aaaaagtaca ccccaaaatt acctgggtgt ggtggcacat gcctatagtc 5160
tcaattagga ggctgaggtg ggactattct ttgatcccag gagtttgagg ctgcagtggg 5220
gtatgatcgc accaccgcac tccagcctgg gagacagatt gagaccatct ctaaaacaac 5280
aacaggccag gtgctgtggc ttatgcctgt aatcccagca atttgggagg ccgaggcagg 5340
tggatcacct gagatcagga gttcgagacc agcctgggca acctggtgaa atcctgtctc 5400
tactaaaaat acaaaaattt agcagggcat ggtggcacac acctgtaatc ccagctactc 5460
gggaggccga ggcaggagca ttgcttgtac ccaggaggca gagattgcag tgagctgaga 5520
tcacgccatt gcactccagt ctgagcaaca agagcaaaac ttcgtctcaa aaaaataaaa 5580
aattaaaaaa taacaaaaaa tgagacagaa aacaaatatt ttgtttcatt attgcttgtt 5640
agttgattaa agtaattctg cttccacttt attttcaaag acactaattg ctacactgaa 5700
taaaacctta atggagtttc attat 5725
<210> 2
<211> 372
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atggattcac taccagattc tactgtatgc tctgacaact atgaccacaa tggcagcaag 60
attaagaaaa ttgtgcattc aattatatca tcctttgcat ttggactatt tggagttttc 120
ctggtcttac tggatgtcac tctcgtcctt gccgacctaa ttttcactga cagcaaactt 180
tatattcctt cggagtatcg ttctatttct ctagctattg ccttattttt tctcatggat 240
gttcttcttc gagtatttgt agaagggtgg attcttgctc tgtcacccag gctggagcgt 300
aatggcacaa tcttcgctca cttcaacctc tgccttctgg gttcgagcaa ttctcctgcc 360
tcagcctcct ga 372
<210> 3
<211> 123
<212> PRT
<213>polypeptide (Polypeptide)
<400> 3
Met Asp Ser Leu Pro Asp Ser Thr Val Cys Ser Asp Asn Tyr Asp His
1 5 10 15
Asn Gly Ser Lys Ile Lys Lys Ile Val His Ser Ile Ile Ser Ser Phe
20 25 30
Ala Phe Gly Leu Phe Gly Val Phe Leu Val Leu Leu Asp Val Thr Leu
35 40 45
Val Leu Ala Asp Leu Ile Phe Thr Asp Ser Lys Leu Tyr Ile Pro Ser
50 55 60
Glu Tyr Arg Ser Ile Ser Leu Ala Ile Ala Leu Phe Phe Leu Met Asp
65 70 75 80
Val Leu Leu Arg Val Phe Val Glu Gly Trp Ile Leu Ala Leu Ser Pro
85 90 95
Arg Leu Glu Arg Asn Gly Thr Ile Phe Ala His Phe Asn Leu Cys Leu
100 105 110
Leu Gly Ser Ser Asn Ser Pro Ala Ser Ala Ser
115 120
<210> 4
<211> 16
<212> PRT
<213>polypeptide (Polypeptide)
<400> 4
Cys Thr Asp Ser Lys Leu Tyr Ile Pro Ser Glu Tyr Arg Ser Ile Ser
1 5 10 15

Claims (7)

1. a kind of long-chain non-coding RNA lncRNA-6585, which is characterized in that its nucleic acid sequence is compiled as shown in SEQ ID No:1 The nucleotide sequence such as SEQ ID No:2 of code polypeptide, and it is named as lncRNA-6585P.
2. a kind of long-chain non-coding RNA lncRNA-6585 according to claim 1, which is characterized in that the polypeptide Amino acid sequence such as SEQ ID No:3.
3. the recombinant virus pCDH- of the nucleotide sequence lncRNA-6585P containing coding polypeptide described in claim 1 The overexpression plasmid of lncRNA-6585P.
4. being overexpressed the construction method of plasmid based on lncRNA-6585P as claimed in claim 3, which is characterized in that including as follows Step: step 1, synthesis obtains the target gene of SEQ ID No:2 sequence, and finish up one Flag label of each addition；Step 2, it uses Restriction enzymeXbo IWithBamH IDigestion, pure is carried out to target gene and pCDH-CMV-MCS-EF1-copRFP carrier Change, connection, conversion to Escherichia coli is to get overexpression plasmid pCDH-lncRNA-6585P.
5. being overexpressed plasmid-encoded polypeptide based on lncRNA-6585P as claimed in claim 3 to resist by what immune response generated Body.
6. antibody described in claim 5 is in preparation prevention or treatment cervical carcinoma, oophoroma, carcinoma of endometrium, breast cancer, colon Cancer, liver cancer, glioma, lung cancer, cancer of pancreas or gastric cancer product in application.
7. based on lncRNA-6585P described in claim 1 in preparation prevention or treatment cervical carcinoma, oophoroma, endometrium Cancer, breast cancer, colon cancer, liver cancer, glioma, lung cancer, cancer of pancreas or gastric cancer product in application.
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CN109735622A (en) * 2019-03-07 2019-05-10 天津市第三中心医院 LncRNA relevant to colorectal cancer and its application
CN109837276A (en) * 2019-02-22 2019-06-04 南京医科大学 A kind of long-chain non-coding RNA lncRNA-3608 and its antibody and application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ENST00000426585.5: "Transcript: TPTEP1204", 《ENSEMBL》 *
张咪: "长链非编码ＲNA525893 重组慢病毒载体的构建及其对宫颈癌细胞增殖的影响", 《江苏大学学报( 医学版)》 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109837276A (en) * 2019-02-22 2019-06-04 南京医科大学 A kind of long-chain non-coding RNA lncRNA-3608 and its antibody and application
CN109837276B (en) * 2019-02-22 2022-05-20 南京医科大学 Long-chain non-coding RNA lncRNA-3608 and antibody and application thereof
CN109735622A (en) * 2019-03-07 2019-05-10 天津市第三中心医院 LncRNA relevant to colorectal cancer and its application

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