CN1304573C - NDRG2 and NDRG4 protein and their use for diagnosing and suppressing tumour - Google Patents
NDRG2 and NDRG4 protein and their use for diagnosing and suppressing tumour Download PDFInfo
- Publication number
- CN1304573C CN1304573C CNB2004100007384A CN200410000738A CN1304573C CN 1304573 C CN1304573 C CN 1304573C CN B2004100007384 A CNB2004100007384 A CN B2004100007384A CN 200410000738 A CN200410000738 A CN 200410000738A CN 1304573 C CN1304573 C CN 1304573C
- Authority
- CN
- China
- Prior art keywords
- ndrg2
- ndrg4
- period
- protein
- albumen
- 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
Images
Landscapes
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention largely expresses NDRG2 protein and NDRG4 protein encoded by N-myc downstream regulator gene subtype ndrg2 and ndrg4 by using a genetic engineering method, and researches physicochemical properties of the NDRG2 protein and the NDRG4 protein and biological functions of tumour cells. The flow cytometry result indicates that the influence of the NDRG2 protein on gastric cancer 7901 cells is revealed in extended G1 period and shortened G2 period and S period; the influence on liver cancer HHCC cells is revealed in shortened G1 period and G2 period and extended S period; the influence of the NDRG4 protein on the gastric cancer 7901 cells and the liver cancer HHCC cells is revealed in extended G1 period and shortened G2 period and S period. The nude mouse tumorigenesis inhibiting animal experiment and other animal experiments prove that the NDRG2 protein and the NDRG4 protein have obvious inhibiting effect on the growth of tumour cells and the formation of tumours for nude mice, wherein the tumour inhibiting effect of the high-dose group is obvious, and the effect of the low-dose group is correspondingly lower.
Description
Technical field
The present invention relates to N-myc downstream regulatory gene hypotype 2 and/or 4 albumen and the application in diagnosis and inhibition tumour thereof.
Background technology
Cancer is activation and/or the disappearance of cancer suppressor gene or the product of deactivation of proto-oncogene.Oncogene is to be present in cell or the virus, can induce the normal cell conversion and make it obtain the gene of one or more true tumor features.This gene is relevant with the generation development of tumour.Oncogene is high conservative and limited expression in normal cell, and the differentiation of pair cell, propagation play an important role.Can cause cell carcinogenesis after oncogene is activated, the inherent oncogene claims cellular oncogene (c-onc) in the cell usually, and the c-onc that is in the disactivation state is called proto-oncogene (proto-onc).
The item key of oncogene activation is gene amplification, and it is that some genes are copied into a plurality of copy numbers by not clear mechanism in the phalangeal cell, thereby causes the increase of its product, the cell normal function is interfered and canceration takes place.Oncogene amplification is often relevant with biological behaviours such as metastases, infiltration and resistance, so oncogene amplification can be used as the development that molecule marker is followed the trail of the state of an illness, monitors and shifts and recurrence, observes prognosis.
As far back as nineteen forty-two, people such as Charles have just at first proposed the notion of cancer suppressor gene, still the independent characteristics of none can define cancer suppressor gene up to now, but cancer suppressor gene should meet 3 primary conditions: (1) this gene is necessary normal expression in the related normal tissue of malignant tumour; (2) this gene functionally inactive or structural modification occur or expresses defective in malignant tumour; (3) wild-type with this gene imports in the tumour cell of this gene unconventionality, can partly or entirely change its malignant phenotype.
People's N-myc downstream regulatory gene (human N-myc downstream regulated gene) is divided into following hypotype, that is: ndrg1, ndrg2, ndrg3, ndrg4, ndrg-like and brain development associated molecule (bdrm), they have higher homology, but expression level obvious difference in tissue development stage and distribution, not clear (the Zhang Wenhong etc. of concrete function, The Fourth Military Medical University's journal, 2002,23 volumes, 8 phases, the 716-720 page or leaf), the sudden change of ndrg gene may be inducement (Kalaydjieva L etc., the Am J Hum Genet of heredity motion and sensory nerve disease (HMSNL), 2002,67 volumes, 1 phase, 47-58 page or leaf).
The ndrg4 gene is positioned at karyomit(e) 16q21-q22.3, mainly in brain and heart, express, its mRNA has 3 isomer: NDRG4-B, NDRG4-B (var) and NDRG4-H, Northern blot and Western blot confirm that NDRG4-B only expresses in brain, NDRG4-H had then not only expressed (Zhou RH etc., Genomics in brain but also in heart, calendar year 2001,73 volumes, 1 phase, 86-97 page or leaf).
The ndrg2 gene is that China The Fourth Military Medical University finds first, is included by GenBank, and the number of landing is AFI59092 (Deng Yanchun etc., biological chemistry and biophysics progress, calendar year 2001,28 volumes, 1 phase, the 72-76 page or leaf), this gene is positioned at karyomit(e) 14q11.2 (Qu X etc., Mol Cell Biochem, 2002,229 volumes, 1-2 phase, 35-44 page or leaf).Through immunohistochemical study, NDRG2 albumen is present in the tenuigenin, is a kind of cytoplasmic protein (Zhang Wenhong etc., The Fourth Military Medical University's journal, 2002,23 volumes, 8 phases, the 716-719 page or leaf), confirm ndrg2 mRNA wide expression in normal cerebral tissue through hybridization in situ experiment, expression level in normal brain activity and lung tissue is significantly higher than glioma and cancerous lung tissue (Li Jian etc. respectively, biological chemistry and biophysics progress, 2002,29 volumes, 2 phases, the 223-227 page or leaf), the expression in stomach and stomach organization does not have significant difference (Liu Xin equality, biological chemistry and biophysics progress, 2003,30 volumes, 1 phase, 116-121 page or leaf).
At present, oneself has launched the preliminary study of function and mechanism of aspect to the ndrg2 gene both at home and abroad, as adopt RT-PCR to carry out expression study (Li Jian etc., biological chemistry and biophysics progress, 2002 of mRNA level, 29 volumes, 2 phases, the 223-227 page or leaf), adopt flow cytometry, gene transfection, sense-rna or RNAi carry out research (the Liu Xin equality of the mechanism of action, biological chemistry and biophysics progress, 2003,30 volumes, 1 phase, the 116-121 page or leaf), adopt immunohistochemistry technique and immunoblotting to carry out research (Li Jian etc., biological chemistry and biophysics progress, 2002 of protein expression level, 29 volumes, 2 phases, the 223-227 page or leaf) or the like, up-to-date studies confirm that is in HGC-27 and the SGC-cancer of the stomach 7901 at stomach cancer cell at least, relevant (the Liu Xin equality of ndrg2 expression of gene with the proliferation and differentiation of cell, biological chemistry and biophysics progress, 2003,30 volumes, 1 phase, the 116-121 page or leaf).
Although downstream regulatory gene (the human N-mycdownstream regulated gene in people's the myc family gene has been thought in present research, hNDRG) N-myc may be relevant with the generation of tumour, and it is classified as press down cancer candidate gene (Deng YC etc., Shengwu Huaxue Yu Shengwu Wuli Jinzhan, calendar year 2001,28 volumes, 1 phase, the 72-76 page or leaf), relevant with the generation development of neural system and tumor in respiratory system, but above-mentioned research is just carried out at isolated cells system, up to now the report that does not form about growth of tumour cell and tumour in NDGR2 and NDRG4 albumen inhibition animal body or the human body.
Summary of the invention
The present invention uses gene engineering method, NDRG2 that great expression N-myc downstream regulatory gene hypotype ndrg2 and ndrg4 are coded and NDRG4 albumen, studied the proteic physico-chemical property of NDRG2 and NDRG4 and they biological function, and confirmed that by animal experiments such as flow cytometry, the inhibition of nude mice tumorigenesis NDRG2 and NDRG4 albumen form to the growth of tumour cell and to the tumour of nude mice and all has significant inhibitory effect tumour cell.
Therefore, one aspect of the present invention provides isolating people N-myc downstream regulatory gene hypotype ndrg2 and ndrg4, has the nucleotide sequence shown in SEQ ID NO:1 and the SEQ ID NO:3.
Another aspect of the present invention provides isolating people NDRG2 and NDRG4 protein polypeptide, has the aminoacid sequence shown in SEQ ID NO:2 and the SEQ ID NO:4.
One side more of the present invention provides NDRG2 and the application of NDRG4 albumen in the medicine of preparation diagnosis and/or inhibition digestive system tumor, particularly in preparation diagnosis and/or suppress application in the medicine of cancer of the stomach and liver cancer, protein polypeptide wherein can make up with the diluent or carrier of allowing on the pharmacopedics.
Description of drawings
The BamHI+HindIII double digestion electrophoresis result of Fig. 1 NDRG2 expression vector.
1.pQE30/NDRG2; 2.pQE30/NDRG2 the BamHI+HindIII double digestion;
3.DL15000Marker；4.DL2000Marker
The BamHI+HindIII double digestion electrophoresis result of Fig. 2 NDRG4 expression vector.
1.DL15000Marker；2.DL2000Marker；3.pQE30/NDRG4
4.pQE30/NDRG4 the BamHI+HindIII double digestion
The protein induced expression of results of Fig. 3 NDRG2.
1. molecular weight Marker (LMW); 2.IPTG after inducing; 3.IPTG before inducing
The protein induced expression of results of Fig. 4 NDRG4.
1.IPTG after inducing; 2.IPTG before inducing; 3. molecular weight Marker (LMW)
Fig. 5 NDRG2 and NDRG4 albumen are to the influence of cancer of the stomach 7901 and the growth of liver cancer HHCC cell.
Cancer of the stomach 7901 cells that do not add sample during Fig. 5-1 40h;
NDRG2 albumen is to the effect of cancer of the stomach 7901 cells during Fig. 5-2 40h;
NDRG4 albumen is to the effect of cancer of the stomach 7901 cells during Fig. 5-3 40h;
The liver cancer HHCC cell that does not add sample during Fig. 5-4 30h;
NDRG2 albumen is to the effect of liver cancer HHCC cell during Fig. 5-5 30h;
NDRG4 albumen is to the effect of liver cancer HHCC cell during Fig. 5-6 30h.
Fig. 6 different concns NDRG2 albumen is to the restraining effect of tumour cell cancer of the stomach 7901.
A: protein denaturation contrast b: blank; C:NDRG2 albumen
Fig. 7 different concns NDRG2 albumen is to the restraining effect of tumour cell liver cancer HHCC.
A: protein denaturation contrast b: blank; C:NDRG2 albumen
Fig. 8 different concns NDRG4 albumen is to the restraining effect of cancer of the stomach 7901 tumour cells.
A: blank; B:NDRG4 albumen
Fig. 9 different concns NDRG4 albumen is to the restraining effect of liver cancer HHCC tumour cell.
A: blank; B:NDRG4 albumen
Figure 10 flow cytometry is investigated NDRG2 albumen to the cancer of the stomach influence of 7901 tumour cell cell cycles.
Figure 11 flow cytometry is investigated NDRG2 albumen to the liver cancer HHCC influence of tumour cell cell cycle.
Figure 12 flow cytometry is investigated NDRG4 albumen to the cancer of the stomach influence of 7901 tumour cell cell cycles.
Figure 13 flow cytometry is investigated NDRG4 albumen to the liver cancer HHCC influence of tumour cell cell cycle.
Figure 14 NDRG2 and NDRG4 albumen suppress experimental result to the tumor growth of nude mice.
Figure 14-1 positive controls (endoxan);
The blank model control group of Figure 14-2;
Figure 14-3 low dose group (20 μ g/ml);
Figure 14-4 high dose group (100 μ g/ml).
Figure 15 NDRG2 and NDRG4 albumen are to nude mice tumor growth restraining effect column diagram.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The clone of embodiment 1:ndrg2 and ndrg4 gene and order-checking
1, the transformation of purpose fragment double enzyme site
To contain the segmental plasmid pMD18-T of purpose (the precious biotech firm in Dalian product) and transform host bacterium JF1125 (Shanghai Medical Univ provides), the segmental double enzyme site of purpose is 5 ' end BamHI and 3 ' end EcoRI, if use expression vector pQE30 (QIAGEN company product), need to transform its restriction enzyme site as 5 ' end BamHI and 3 ' end HindIII, design PCR primer is:
Forward: 5 '-CG
GGATCCGCGGAGCTGCAGGAGGT-3 '
Oppositely: 5 '-CC
AAGCTTTTAACAGGAGACCTCCATGGTG-3 '
The pcr amplification reaction system is:
10 * pfuDNA polymeric enzyme reaction buffer 5ul
Forward primer (25pmol/ul) 1ul
Reverse primer (25pmol/ul) 1ul
dNTPs(10Mm) 1ul
Dna profiling 3ul
PfuDNA polysaccharase (promega company product) 0.5ul
Add ddH
2O to 50ul
The PCR condition is:
94 ℃ of sex change 2 minutes; 94 ℃ of 30S, 59 ℃ of 55S, 74 ℃ 2 minutes, totally 30 circulations; 74 ℃ were extended 5 minutes.
2, BamHI and HindIII double digestion
PCR product and pQE30 are carried out double digestion, and the enzyme tangent condition is:
BamHI (the precious biotech firm in Dalian product) 3ul
HindIII (the precious biotech firm in Dalian product) 3ul
10×Kbuffer 6ul
DNA 3ug
Add ddH
2O to 60ul
After 30 ℃ of enzymes are cut 2h, after enzyme cut product and separate with 1.5% agarose electrophoresis, take respective segments, carry out glue with reference to glass milk test kit specification sheets (precious Imtech product) and reclaim, respectively get 5ul and reclaim fragment and identify with agarose electrophoresis.
3, directed cloning
Connect and do directed cloning with dna ligation kit (the precious biotech firm in Dalian product), condition of contact is:
pQE30 1ul
Insert DNA 10ul
Solution I 11ul
16 ℃ were reacted 30 minutes.
4, the preparation of M15 competent cell (QIAGEN company product)
Get 250ml and be cultured to OD
600Be 0.6 M15 cell, the centrifugal 10min of 2500g gets TB buffer (10mM Pipes, 1,4-piperazine two ethyl sulfonic acids that precipitation adds 4 ℃ of precoolings of 80ml; 55mM, MnCl
215mM, CaCl
2250mM, KCl.Add MnCl
2Preceding is 6.7 with 5N KOH adjust pH, autoclaving), resuspended gently, 4 ℃ of 10min, the centrifugal 10min of 2500g is resuspended in precipitation among the 20ml TB buffer of 4 ℃ of precoolings, adds 1.5ml DMSO, and behind 4 ℃ of 10min, packing is preserved.
5, transform the M15 host cell
To connect product 10ul and transform into M15 competent cell 100ul, add LB substratum 200ul, 37 ℃ of 30min, coating ammonia benzyl resistance LB solid medium flat board was cultivated 16 hours for 37 ℃.
6, the evaluation of positive colony
Select some positive colonies and have in the ammonia benzyl resistance LB liquid nutrient medium at 5ml, 37 ℃ of following 200rpm cultivated 16 hours, extracted plasmid and identified.The plasmid extracting method is referring to the little extraction reagent kit specification sheets of plasmid (promega company product).Above-mentioned plasmid is carried out BamHI and HindIII double digestion, and carry out agarose gel electrophoresis simultaneously with DL1500 and DL2000 molecular weight maker and the plasmid that do not carry out double digestion.
The result of agarose gel electrophoresis shows that plasmid construction is entirely true, and the clip size of ndrg2 and ndrg4 is respectively (attached Fig. 1 and 2) about 1000bp.
7, the dna sequencing of positive colony
To identify that correct positive colony carries out dna sequencing, examining order is finished in plug Parkson, Beijing gene engineering company limited.
Sequencing result shows that ndrg2 and ndrg4 have the nucleotide sequence shown in SEQ ID NO:1 and the SEQ ID NO:3 respectively, and the size of ndrg2 and ndrg4 is respectively 1,074bp and 1,020bp.
Proteic preparation of embodiment 2:NDRG2 and NDRG4 and purifying
1, the investigation of expression amount
The pQE30/M15 positive colony is inserted 5ml ammonia benzyl resistance LB liquid nutrient medium, 37 ℃ of following 200rpm cultivated after 16 hours, getting 5ml imports in the 300ml ammonia benzyl resistance LB liquid nutrient medium, 37 ℃ of shake-flask culture are about 3 hours in the 1000ml triangular flask, to nutrient solution OD value be 0.5 o'clock, take out 1ml at the centrifugal 10min of 5000rpm, get precipitation as blank; Adding IPTG in triangular flask is 1.5mmol/L to final concentration, and NDRG2 continues to cultivate 5 hours in 37 ℃, and NDRG4 continues to cultivate 4 hours in 39 ℃, takes out the centrifugal 10min of 5000rpm, gets precipitation, investigates expression amount.
The result of abduction delivering shows that the expression amount of NDRG2 is 42.85%, and molecular weight is 39977Da; The expression amount of NDRG4 is 20.98%, and molecular weight is 38856Da (accompanying drawing 3 and 4).
2, the optimization of abduction delivering condition
Adopt orthogonal experiment design [L
9(3
4)] investigation NDRG2 and suitable inductor IPTG concentration, expression culture temperature, abduction delivering time and the initial bacterial concentration of inducing of NDRG4 albumen.
Table 1 orthogonal experiment level of factor design table
The result shows: inductor IPTG concentration is 1.5mmol/L, express culture temperature is that 37 ℃, abduction delivering time are that 5hr, the initial bacterial concentration OD value of inducing are 0.5; The inducing culture condition suitable to NDRG4 albumen is: inductor IPTG concentration is 1.5mmol/L, express culture temperature is that 39 ℃, abduction delivering time are that 4hr, the initial bacterial concentration OD value of inducing are 0.5.Adopt above-mentioned inducing culture condition that NDRG2 and NDRG4 albumen are prepared in a large number respectively.
3, the investigation of best carrying out ultrasonic bacteria breaking time
With the nutrient solution 100ml of abduction delivering centrifugal 10 minutes at 5000rpm, keep supernatant, get the broken bacterium liquid that precipitation adds 20 times of volumes (Tris damping fluid 30mmol/L, pH8.5), carrying out ultrasonic bacteria breaking, power is 200W, and ultrasonic time 5,10,15,20 minutes respectively all keeps supernatant at every turn, get the same method of precipitation and add broken bacterium liquid processing, all draw 1ml at every turn and go up cleer and peaceful resolution of precipitate liquid, at 5000rpm centrifugal 10 minutes, get precipitation and carry out the SDS-PAGE gel electrophoresis to investigate best ultrasonic time.
The result shows and contained tropina when ultrasonic time is 5 minutes in the supernatant, therefore the enough broken bacterium of 5 minutes ultrasonic time.
4, solubilization of inclusion bodies liquid determines
(Tris damping fluid 30mmol/L+1%Triton+1M urea+1M NaCl pH8.5), fully stirs evenly back concussion washing, and the centrifugal 15min of 5000rpm gets and precipitates 5 times of volume lysates of adding to get the washings that precipitates 10 times of volumes of adding.Lysate is Tris damping fluid, phosphate buffered saline buffer, citrate buffer, the sodium-acetate buffer that contains the pH value difference 8.5,7.5,5.0,4.0 of 6M Guanidinium hydrochloride or 6M urea respectively.The final suitable lysate of determining is the 30mM Tris damping fluid (pH8.5) that contains 6M urea.
5, NDRG2 and the research of NDRG4 albumen inclusion body redissolution method
(1) dialysis redissolution method research
Get NDRG2, NDRG4 inclusion body solution 5ml after the dissolving, pack in the dialysis tubing, insert 500ml extracellular fluid dialysis (promptly reducing the lysate that corresponding urea concentration is 2M), every stirring is 2 hours on magnetic stirring apparatus, changing extracellular fluid dialysis makes its urea concentration reduce 2M, when urea concentration is reduced to 2M, be replaced by not urea-containing extracellular fluid dialysis, 4 ℃ of dialysed overnight.
(2) dilution redissolution method research
Method one: get NDRG2, NDRG4 inclusion body solution 5ml after the dissolving, slowly drip redissolve liquid 1 (30mM Tris damping fluid, pH8.5), redissolve liquid 2 (30mM Tris damping fluid, pH8.0) and redissolve liquid 3 (30mM Tris damping fluid, pH7.5).
Method two: get NDRG2, NDRG4 inclusion body solution 5ml after the dissolving, add rapidly 10 times of volumes redissolve liquid 1 (30mM Tris damping fluid, pH8.5), redissolve liquid 2 (30mM Tris damping fluid, pH8.0) and redissolve liquid 3 (30mM Tris damping fluid, pH7.5).
Because when adopting dialysis redissolution method and first kind of dilution redissolution method, flocks in various degree all appears in the proteic lysate of NDRG2 and NDRG4, and when adopting second kind of dilution redissolution method, when adopting redissolution liquid 1 and 2, the proteic lysate of NDRG2 and NDRG4 still keeps clarification, therefore adopt second kind of dilution redissolution method that NDRG2 and NDRG4 albumen are carried out the inclusion body redissolution, selected redissolution liquid is for redissolving liquid 1 and 2, and further calculates NDRG2 and the proteic redissolution rate of NDRG4 is respectively 69% and 78%.
6, redissolution back inclusion body solution filters
With the NDRG2 after redissolving, NDRG4 inclusion body solution centrifugal 20 minutes, get supernatant and filter with 0.45um filter membrane (millipore company product) at 5000rpm.
7, the concentration method research of redissolution back inclusion body solution
Learnt from else's experience filtering redissolution inclusion body solution respectively in order to method is concentrated down, investigated yield.
(1) ultrafiltration: get ultrafiltration cup (the millipore company product) ultrafiltration of 50ml redissolution inclusion body solution, ultra-filtration membrane molecular retention amount is 75000, final simmer down to 5ml.
(2) PEG concentrates: getting 50ml, to redissolve the inclusion body solution molecular retention amount of packing into be 75000 dialysis tubing, buries with PEG 20000 powder, is concentrated into 5ml.
(3) ammonium sulfate precipitation: get 50ml redissolution inclusion body solution, add 25g (NH
4)
2SO
4, centrifugal 20 minutes of 5000rpm abandons supernatant, gets precipitation and adds redissolution liquid 5ml dissolving.
(4) lyophilize: get 10ml and redissolve inclusion body solution, divide and put in 5 freeze-drying bottles ,-70 ℃ freezing after, freeze-drying in Freeze Drying Equipment is to the about 2ml of cumulative volume.
The result shows, containing more salt in the concentrated solution of ammonium sulfate precipitation need dialyse and remove, the rate of recovery is low and consuming time, the ultrafiltration cost is higher, salt in lyophilize complex operation and the concentrated solution also needs dialysis to remove, therefore consider that yield takes into account accessibility, finally adopt PEG to concentrate mode the inclusion body solution after redissolving is concentrated.
8, purification process research
(1) desalination:
Desalting column (Hitrap Desalting, 26 * 20), flow velocity: 8ml/min, moving phase: Tris damping fluid 30mmol/L, detect wavelength 280nm.
(2) ion-exchange purification research:
Adopt Q Sepharose FF, DEAE Sepharose FF, QXL Sepharose FF, ANX Sepharose FF, Source 30Q as filler respectively, investigate appropriate filler.
Balance liquid adopts the 30mmol/L Tris damping fluid of pH7.5, pH8.0, pH8.5, and elutriant adopts 30mmol/L Tris damping fluid+2M NaCl.
Flow velocity: 1ml/min detects wavelength: 280nm, applied sample amount: 500ul, last sample loading mode: injection annulus, chromatographic condition: go up 5 post beds of sample back balance, 20 post beds of 0～50% elutriant gradient elution.
(3) affinity chromatography
Get the protein solution employing CHELATING SEPHAROSE FAST FLOW affinity chromatography filler purifying that ion-exchange purification goes out, sample solution is: the imidazoles of the Tris damping fluid 30mmol/L+30mmol/L of pH8.5, elutriant is: the imidazoles of the Tris damping fluid 30mmol/L+500mmol/L of pH8.5.
Flow velocity: 1ml/min detects wavelength: 280nm, applied sample amount: 500ul, last sample loading mode: injection annulus, chromatographic condition: go up 3 post beds of sample back balance, 2 post beds of elutriant wash-out.
(4) molecular sieve
Adopt Sephacryl S-100HR chromatographic stuffing.Last sample and elution buffer are: the Tris damping fluid 30mmol/L of pH8.5.The post bed: 16 * 100, flow velocity: 1ml/min detects wavelength: 280nm, applied sample amount: 500ul, last sample loading mode: injection annulus.
The result shows that Source 30Q is suitable to filler when adopting the ion-exchange purification method; Affinity chromatography and molecular sieve are suitable purification process, and when adopting above-mentioned two kinds of methods respectively, the proteic purity of NDRG2 is 94% and 96%, and the proteic purity of NDRG4 is respectively 92% and 97%.
9, isoelectric point determination
Get the protein solution after the above-mentioned renaturation,, investigate its iso-electric point with doing isoelectrofocusing behind the dialysis tubing dialysis desalination.The proteic iso-electric point of NDRG2 and NDRG4 is respectively 6.3 and 6.2.
10, the N terminal amino acid sequence is measured
Measurement result through centralab of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences shows that NDRG2 and NDRG4 albumen n end aminoacid sequence are respectively:
NDRG2：HHHHHH GSAELQEVQITEEKPL
NDRG4：HHHHHH GSPECWDGEHDIETPY。
Embodiment 3NDRG2 and NDRG4 albumen are to the restraining effect of tumour cell cancer of the stomach 7901 and liver cancer HHCC
Get cancer of the stomach 7901 cells and liver cancer HHCC cell and be diluted to 7 * 10 with 1640 substratum that contain 10% serum
4/ ml adds NDRG2 and NDRG4 albumen respectively, cultivate and observe after 30 and 40 hours, simultaneously with do not add NDRG2 and NDRG4 proteic, cultivate 30 and 40 hours, be diluted to 7 * 10 with 1640 substratum that contain 10% serum
4Cancer of the stomach 7901 cells of/ml and liver cancer HHCC cell are investigated NDRG2 and the NDRG4 albumen restraining effect to tumour cell cancer of the stomach 7901 and liver cancer HHCC in contrast.
The result shows and not to add the not influence of growth to cancer of the stomach 7901 and liver cancer HHCC tumour cell of NDRG2 and NDRG4 albumen, and add after NDRG2 and NDRG4 albumen cultivates 30 and 40 hours respectively, the growth of cancer of the stomach 7901 and liver cancer HHCC tumour cell is subjected to obvious suppression, and along with the prolongation of incubation time, restraining effect obvious more (Fig. 5).
1640 substratum of getting 1% serum dilute cancer of the stomach 7901 cells respectively and liver cancer HHCC cell is 7 * 10
4/ ml adds 96 orifice plate 1-12 holes respectively, and the multiple every hole 100ul in hole is cultured to cell attachment.1640 substratum with 1% serum mix with 5: 1 with NDRG2 and NDRG4 albumen (2mg/ml), the multiple hole of 100ul adds the 1st hole of 96 orifice plates, two-fold dilution NDRG2 albumen to the 8 holes, NDRG4 albumen to the 12 holes are simultaneously with liquid in damping fluid Tris (pH8.5), sex change NDRG2 and NDRG4 albumen (Tris damping fluid mixing), NDRG2 and the ultrafiltration of NDRG4 albumen and liquid and do contrast with liquid under the NDRG2 of 37 ℃ of processing of Proteinase K 4h and the proteic ultrafiltration of NDRG4 down.Cultivated 72 hours.
Table 2NDRG2 is to the restraining effect result of tumour cell cancer of the stomach 7901
| Sex change contrast (a) | 0.474 | 0.420 | 0.428 | 0.478 | 0.476 | 0.476 | 0.453 | 0.502 |
| 0.464 | 0.469 | 0.498 | 0.465 | 0.467 | 0.497 | 0.459 | 0.470 | |
| Blank (b) | 0.396 | 0.410 | 0.410 | 0.434 | 0.444 | 0.446 | 0.431 | 0.451 |
| 0.388 | 0.405 | 0.432 | 0.411 | 0.437 | 0.429 | 0.446 | 0.491 | |
| Sample (c) | 0.139 | 0.216 | 0.260 | 0.352 | 0.409 | 0.417 | 0.398 | 0.440 |
| 0.161 | 0.240 | 0.269 | 0.377 | 0.427 | 0.423 | 0.413 | 0.454 |
Table 3NDRG2 is to the restraining effect result of tumour cell liver cancer HHCC
| Sex change contrast (a) | 0.636 | 0.685 | 0.692 | 0.649 | 0.767 | 0.731 | 0.718 | 0.593 |
| 0.639 | 0.720 | 0.736 | 0.752 | 0.678 | 0.646 | 0.629 | 0.675 | |
| Blank (b) | 0.496 | 0.458 | 0.493 | 0.540 | 0.540 | 0.584 | 0.542 | 0.536 |
| 0.409 | 0.482 | 0.529 | 0.569 | 0.558 | 0.563 | 0.545 | 0.541 | |
| Sample (c) | 0.114 | 0.352 | 0.544 | 0.559 | 0.655 | 0.618 | 0.566 | 0.524 |
| 0.111 | 0.342 | 0.565 | 0.607 | 0.608 | 0.592 | 0.577 | 0.609 |
Table 4NDRG4 is to the restraining effect result of tumour cell cancer of the stomach 7901
| Blank (a) | 0.795 | 0.739 | 0.698 | 0.792 | 0.758 | 0.668 | 0.847 | 0.716 | 0.726 | 0.719 | 0.734 | 0.694 |
| 0.760 | 0.706 | 0.669 | 0.758 | 0.723 | 0.639 | 0.816 | 0.664 | 0.680 | 0.681 | 0.691 | 0.725 | |
| Sample (b) | 0.334 | 0.378 | 0.302 | 0.313 | 0.411 | 0.401 | 0.546 | 0.633 | 0.596 | 0.714 | 0.685 | 0.706 |
| 0.363 | 0.332 | 0.333 | 0.364 | 0.459 | 0.449 | 0.516 | 0.581 | 0.559 | 0.662 | 0.669 | 0.694 |
Table 5NDRG4 is to the restraining effect of tumour cell liver cancer HHCC
| Blank (a) | 0.755 | 0.706 | 0.685 | 0.769 | 0.750 | 0.699 | 0.810 | 0.670 | 0.722 | 0.723 | 0.690 | 0.711 |
| 0.704 | 0.732 | 0.621 | 0.717 | 0.704 | 0.672 | 0.775 | 0.696 | 0.690 | 0.688 | 0.664 | 0.743 | |
| Sample (b) | 0.280 | 0.333 | 0.319 | 0.330 | 0.487 | 0.441 | 0.543 | 0.607 | 0.577 | 0.670 | 0.695 | 0.658 |
| 0.255 | 0.293 | 0.278 | 0.352 | 0.458 | 0.479 | 0.517 | 0.615 | 0.616 | 0.714 | 0.642 | 0.649 |
The result of table 2-table 5 shows when not adding NDRG2 and NDRG4 albumen, to cancer of the stomach 7901 and the not influence of liver cancer HHCC growth of tumour cell, the growth of cancer of the stomach 7901 and liver cancer HHCC tumour cell is subjected to obvious inhibition behind NDRG2 and the NDRG4 albumen and add, and along with the raising of protein concentration, restraining effect also strengthens, but no longer brings into play restraining effect (accompanying drawing 6-9) behind the protein denaturation.
In 3 culturing bottles, add respectively cancer of the stomach 7901 cells and liver cancer HHCC cell each 60 * 10
4Be total to 5ml, headpin and No. 2 bottles add NDRG2 and each 300ul of NDRG4 albumen (2mg/ml) respectively, and No. 3 bottle adds blank damping fluid 300ul, cultivate taking-up in 36 hours, according to cycletest plus DNA reagent kit (BD company product) operation instructions, measure the cell cycle.
Table 6NDRG2 albumen is to the cancer of the stomach influence of 7901 cell cycles
| Blank group | Sample sets | |
| G1 | 66.97％ | 75.4％ |
| G2 | 3.65％ | 1.23％ |
| S | 29.37％ | 23.37％ |
Table 7NDRG2 albumen is to the influence of liver cancer HHCC cell cycle
| Blank group | Sample sets | |
| G1 | 60.29％ | 54.01％ |
| G2 | 5.76％ | 3.05％ |
| S | 33.94％ | 42.94％ |
Table 6 and table 7 result show that NDRG2 albumen shows that to the influence of cancer of the stomach 7901 cells G1 phase prolongation, G2 and S phase shorten.And NDRG2 albumen to the influence of liver cancer HHCC cell show G1 and G2 phase shorten, the S phase prolongs.Although NDRG2 albumen is described cancer of the stomach 7901 and liver cancer HHCC cell all being shown restraining effect, act on the different steps of cell cycle, is to be arrested in the G1 phase and to be to rest on the S phase (accompanying drawing 10 and 11) to liver cancer HHCC cell to cancer of the stomach 7901 cells.
Table 8NDRG4 albumen is to the cancer of the stomach influence of 7901 cell cycles
| Blank group | Sample sets | |
| G1 | 60.99％ | 68.35％ |
| G2 | 4.75％ | 1.39％ |
| S | 34.26％ | 30.27％ |
Table 9NDRG4 albumen is to the influence of liver cancer HHCC cell cycle
| Blank group | Sample sets | |
| G1 | 58.73％ | 68.47％ |
| G2 | 11.12％ | 7.95％ |
| S | 30.15％ | 23.59％ |
Table 8 and table 9 result show, NDRG4 albumen shows that to cancer of the stomach 7901 and liver cancer HHCC impact cell G1 phase prolongation, G2 and S phase shorten, and is the G1 phase that is arrested in (accompanying drawing 12 and 13).
Embodiment 5 nude mice tumorigenesis suppress experiment
Get liver cancer HHCC tumour cell, usefulness P.B.S. (1 *, 1% serum) be diluted to 6 * 10
6/ ml, 6ml altogether.Get 1.5ml and add NDRG2 and NDRG4 albumen stoste 1ml, getting 2.5ml is high dose group.Get 1.5ml and add NDRG2 and NDRG4 albumen stoste 0.125ml, adding 0.875ml P.B.S., to get 2.5ml be low dose group.Get 1.5ml and add 1ml P.B.S. and get negative group of 2.5ml, get 1.5ml and add endoxan solution 1ml and get positive group of 2.5ml (endoxan adds P.B.S. and makes 21mg/ml).Get 24 nude mices, be divided into 4 groups, 6 every group, inject 0.3ml respectively, observed for 4 weeks.Every nude mice of 4 weeks back measurement produces the size of tumour, draws NDRG2 and NDRG4 albumen to nude mice tumor growth restraining effect column diagram (accompanying drawing 15).
The result shows that the positive drug group has certain tumor inhibition effect, and the high dose group tumor inhibition effect is obvious, a little less than the low dose group effect relatively, relatively confirms NDRG2 and the proteic tumor inhibition effect of NDRG4 obviously (accompanying drawing 14 and 15) with the blank group.
Sequence table
(1) general information:
(i) applicant: Nat'l Pharmaceutical ﹠ Biological Products Control Institute
(ii) denomination of invention: NDRG2 and NDRG4 albumen and the application in diagnosis and inhibition tumour thereof
(iii) sequence number: 4
(2) information of SEQ ID NO:1
(i) sequence signature
(A) length: 1,074bp
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(iii) sequence description: SEQ ID NO:1:
ATGGCGGAGCTGCAGGAGGTGCAGATCACAGAGGAGAAGCCACTGTTGCCAGGACAGACGCCTG
AGGCGGCCAAGACTCACTCTGTGGAGACACCATACGGCTCTGTCACTTTCACTGTCTATGGCACCCC
CAAACCCAAACGCCCAGCGATCCTTACCTACCACGATGTGGGACTCAACTATAAATCTTGCTTCCAG
CCACTGTTTCAGTTCGAGGACATGCAGGAAATCATTCAGAACTTTGTGCGGGTTCATGTGGATGCCC
CTGGAATGGAAGAGGGAGCCCCTGTGTTCCCTTTGGGATATCAGTACCCATCTCTGGACCAGCTTGC
AGACATGATCCCTTGCGTCCTGCAGTACCTAAATTTCTCTACAATAATTGGAGTTGGTGTTGGAGCTG
GAGCCTACATCCTGGCGAGATATGCTCTTAACCACCCGGACACTGTTGAAGGTCTTGTCCTCATCAA
CATTGATCCCAATGCCAAGGGTTGGATGGATTGGGCAGCCCACAAGCTAACAGGCCTCACCTCTTCC
ATTCCGGAGATGATCCTTGGACATCTTTTCAGCCAGGAAGAGCTCTCTGGAAATTCTGAGTTGATAC
AAAAGTACAGAAATATCATTACACATGCACCCAACCTGGATAACATTGAATTGTACTGGAACAGCTA
CAACAACCGCCGAGACCTGAACTTTGAGCGTGGAGGTGATATCACCCTCAGGTGTCCTGTGATGCT
GGTGGTAGGAGACCAAGCACCTCATGAAGATGCAGTGGTGGAATGTAACTCAAAACTGGACCCCA
CCCAGACCTCGTTCCTCAAGATGGCTGACTCCGGAGGTCAGCCCCAGCTGACTCAGCCAGGCAAGC
TGACCGAGGCCTTCAAGTACTTCCTGCAAGGCATGGGCTACATGGCCTCATCCTGCATGACTCGCCT
GTCCCGGTCTCGTACAGCCTCTCTGACCAGTGCAGCATCCGTTGATGGCAACCGGTCCCGCTCTCGC
ACCCTGTCCCAGAGCAGCGAGTCTGGAACTCTTTCTTCGGGGCCCCCGGGGCACACCATGGAGGTC
TCCTGTTAA
(3) information of SEQ ID NO:2
(i) sequence signature
(A) length: 356 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: aminoacid sequence
(iii) sequence description: SEQ ID NO:2
AELQEVQITEEKPLLPGQTPEAAKTHSVETPYGSVTFTVYGTPKPKRPAILTYHDVGLNYKSCFQPLFQF
EDMQEIIQNFVRVHVDAPGMEEGAPVFPLGYQYPSLDQLADMIPCVLQYLNFSTIIGVGVGAGAYILAR
YALNHPDTVEGLVLINIDPNAKGWMDWAAHKLTGLTSSIPEMILGHLFSQEELSGNSELIQKYRNIITHA
PNLDNIELYWNSYNNRRDLNFERGGDITLRCPVMLVVGDQAPHEDAVVECNSKLDPTQTSFLKMADSG
GQPQLTQPGKLTEAFKYFLQGMGYMASSCMTRLSRSRTASLTSAASVDGNRSRSRTLSQSSESGTLSSG
PPGHTMEVSC
(4) information of SEQ ID NO:3
(i) sequence signature
(A) length: 1,020bp
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(iii) sequence description: SEQ ID NO:3
ATGCCGGAGTGCTGGGATGGGGAACATGACATCGAGACACCCTACGGCCTTCTGCATGTAGTGATC
CGGGGCTCCCCCAAGGGGAACCGCCCAGCCATCCTCACCTACCATGATGTGGGCCTCAACCACAAA
CTATGCTTCAACACCTTCTTCAACTTCGAGGACATGCAGGAGATCACCAAGCACTTTGTGGTGTGTC
ACGTGGATGCCCCTGGACAACAGGTGGGGGCGTCGCAGTTTCCTCAGGGGTACCAGTTCCCCTCCA
TGGAGCAGCTGGCTGCCATGCTCCCCAGCGTGGTGCAGCATTTCGGGTTCAAGTATGTGATTGGCAT
CGGAGTGGGCGCCGGAGCCTATGTGCTGGCCAAGTTTGCACTCATCTTCCCCGACCTGGTGGAGGG
GCTGGTGCTGGTGAACATCGACCCCAATGGCAAAGGCTGGATAGACTGGGCTGCCACCAAGCTCTC
CGGCCTAACTAGCACTTTACCCGACACGGTGCTCTCCCACCTCTTCAGCCAGGAGGAGCTGGTGAA
CAACACAGAGTTGGTGCAGAGCTACCGGCAGCAGATTGGGAACGTGGTGAACCAGGCCAACCTGC
AGCTCTTCTGGAACATGTACAACAGCCGCAGAGACCTGGACATTAACCGGCCTGGAACGGTGCCCA
ATGCCAAGACGCTCCGCTGCCCCGTGATGCTGGTGGTTGGGGATAATGCACCCGCTGAGGACGGGG
TGGTGGAGTGCAACTCCAAACTGGACCCGACCACTACGACCTTCCTGAAGATGGCAGACTCTGGA
GGGCTGCCCCAGGTCACACAGCCAGGGAAGCTGACTGAAGCCTTCAAATACTTCCTGCAAGGCATG
GGCTACATGCCCTCAGCCAGCATGACCCGCCTGGCACGCTCCCGCACTGCATCCCTCACCAGTGCC
AGCTCGGTGGATGGCAGCCGCCCACAGGCCTGCACCCACTCAGAGAGCAGCGAGGGGCTGGGCCA
GGTCAACCACACCATGGAGGTGTCCTGTTAA
(5) information of SEQ ID NO:4
(i) sequence signature
(A) length: 338 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: aminoacid sequence
(iii) sequence description: SEQ ID NO:2
PECWDGEHDIETPYGLLHVVIRGSPKGNRPAILTYHDVGLNHKLCFNTFFNFEDMQEITKHFVVCHV
DAPGQQVGASQFPQGYQFPSMEQLAAMLPSVVQHFGFKYVIGIGVGAGAYVLAKFALIFPDLVEGLV
LVNIDPNGKGWIDWAATKLSGLTSTLPDTVLSHLFSQEELVNNTELVQSYRQQIGNVVNQANLQLFW
NMYNSRRDLDINRPGTVPNAKTLRCPVMLVVGDNAPAEDGVVECNSKLDPTTTTFLKMADSGGLPQ
VTQPGKLTEAFKYFLQGMGYMPSASMTRLARSRTASLTSASSVDGSRPQACTHSESSEGLGQVNHTM
EVSC
Claims (2)
1, contain the application of protein polypeptide in the medicine of preparation diagnosis and/or inhibition digestive system tumor of aminoacid sequence shown in SEQ ID NO.2 or the SEQ ID NO.4, the diluent or carrier of allowing on protein polypeptide wherein and the pharmacopedics makes up.
2, application as claimed in claim 1 is characterized in that described digestive system tumor is cancer of the stomach and liver cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100007384A CN1304573C (en) | 2004-01-16 | 2004-01-16 | NDRG2 and NDRG4 protein and their use for diagnosing and suppressing tumour |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100007384A CN1304573C (en) | 2004-01-16 | 2004-01-16 | NDRG2 and NDRG4 protein and their use for diagnosing and suppressing tumour |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1641021A CN1641021A (en) | 2005-07-20 |
| CN1304573C true CN1304573C (en) | 2007-03-14 |
Family
ID=34866875
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100007384A Expired - Fee Related CN1304573C (en) | 2004-01-16 | 2004-01-16 | NDRG2 and NDRG4 protein and their use for diagnosing and suppressing tumour |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1304573C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101632833B (en) * | 2008-07-25 | 2013-11-06 | 上海市计划生育科学研究所 | Prostatic cancer related gene and application thereof |
| CN101570762B (en) * | 2009-05-05 | 2011-06-15 | 中国人民解放军第四军医大学 | Vector for lowering expression of cell-specific ndrg2 gene |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5595902A (en) * | 1993-02-06 | 1997-01-21 | Garvan Institute Of Medical Research | DNA encoding human protein kinase C (iota) |
| CN1331170A (en) * | 2000-06-28 | 2002-01-16 | 上海博德基因开发有限公司 | Polypeptide-human Pura protein 13.31 and polynucleotide for coding it |
| CN1335397A (en) * | 2000-07-21 | 2002-02-13 | 中国人民解放军第四军医大学 | Novel gene P40 containing acyl group carrying protein-like structural domain |
-
2004
- 2004-01-16 CN CNB2004100007384A patent/CN1304573C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5595902A (en) * | 1993-02-06 | 1997-01-21 | Garvan Institute Of Medical Research | DNA encoding human protein kinase C (iota) |
| CN1331170A (en) * | 2000-06-28 | 2002-01-16 | 上海博德基因开发有限公司 | Polypeptide-human Pura protein 13.31 and polynucleotide for coding it |
| CN1335397A (en) * | 2000-07-21 | 2002-02-13 | 中国人民解放军第四军医大学 | Novel gene P40 containing acyl group carrying protein-like structural domain |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1641021A (en) | 2005-07-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1304573C (en) | NDRG2 and NDRG4 protein and their use for diagnosing and suppressing tumour | |
| CN100335632C (en) | Seven kinds of yak milk protein gene sequence | |
| CN1225481C (en) | Recombinant fusion resisting tomur attack and transfer | |
| CN1869064A (en) | Tumour antigen protein and tumour antigen peptide | |
| CN1274827C (en) | Cancer gene and its coded protein and special cell line | |
| CN100340656C (en) | High-efficiency expression in Pichia yeast, fermentation and purification of human gamma-interferon | |
| CN1712536A (en) | Expression of interleukin 24 from yeast cell | |
| CN1244700C (en) | Human acetylcholinesterase isomer protein (AR-ACHE) and its gene coding sequence | |
| CN1876681A (en) | Cancer-suppressing protein and its gene and uses | |
| CN1506375A (en) | Azurin as bacterial protein with wide-spectrum antitumor function and its use and medicinal composition | |
| CN1233831C (en) | SiRNA and expression plasmid for inhibiting human bc1-2 gene expression and their use for preparing medicine | |
| CN1904041A (en) | Human recombination phospholipase D2, its preparation method and application in preparation medicine | |
| CN1840541A (en) | Helicobacter pylori protein and its preparing method and use | |
| CN1923276A (en) | Important regulation protein PAK7 of NF[kappa]B signal path | |
| CN1300315C (en) | Prothoracic gland hormone gene of calf of milk cow and its cloning method and use | |
| CN1305901C (en) | Tumor transfer related protein and coding gene thereof | |
| CN100335137C (en) | Use of human homology cassette gene LHX4 in preparing medicine for treating phaeochromocytoma | |
| CN1587406A (en) | SiRNA double chain for inhibiting bc 1-2 gen expression and use | |
| CN1861190A (en) | Application of anion exchange protein 1 and gene used as target for preparing medicine to treat alimentary canal tumor | |
| CN1869066A (en) | Tumour antigen protein and tumour antigen peptide | |
| CN1661053A (en) | Relativity between gene of growth hormone 1 and essential hypertension | |
| CN1532202A (en) | ANtisense oligonucleotides of targeting human cell death inhibiting protein family member survival protein mRNA, medicinal composition containing them and its use for treating cancer | |
| CN1458160A (en) | Gene recombination tumour blood vessel death factor PFK and its derivative fusion protein | |
| CN1661051A (en) | Relativity between gene of growth hormone 1 and essential hypertension | |
| CN1611603A (en) | Novel DNA fragment and its inhibition from tumour |
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 | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |