CN105541985A - Manta-angiogenesis-inhibitor-1 functional-area variant JG121 and application thereof - Google Patents
Manta-angiogenesis-inhibitor-1 functional-area variant JG121 and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Gastroenterology & Hepatology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a manta-angiogenesis-inhibitor-1 functional-area protein, a variant thereof and an application of the manta-angiogenesis-inhibitor-1 functional-area protein to preparing of tumor-growth-inhibiting medicine. Compared with an original protein, the cancer-cell inhibiting effect of the variant of the protein can be remarkably improved through eukaryotic expression.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to ray Angiostatin 1 functional zone variant.
Background technology
Ray Angiostatin 1 functional zone are hereinafter referred to as ray functional zone, and ray Angiostatin 1 is the protein (molecular weight 42KD) separated from a kind of ray tissue of South China Sea.Result of study shows: that ray Angiostatin 1 significantly suppresses chick chorioallantoic membrane itself and that KB cell is induced chick chorioallantoic membrane vasculogenesis; Be growth and the transfer that abdominal injection or gavage all significantly suppress nude mouse Lewis lung cancer, reduce tumor tissues microvessel density, lower the expression of angiogenic factors VEGF; Also high inhibition effect is had to the transfer of nude mouse Melanoma B16 cell; Lower the expression of short transfer factor CD44v6 and ErBb2; With more remarkable effect during 5 FU 5 fluorouracil (5-FU) combined utilization.The action target spot of ray Angiostatin 1 is different from the PTS Avastin that gene engineering institute of the U.S. develops.Avastin is gene engineering product, and be the antibody of VEGF, its action target spot is VEGF; Ray Angiostatin 1 is natural product, and it not only acts on VEGF, also acts on bFGF and PDGF.
In the prior art, in order to improve the activity of albumen, the specific site for albumen carries out specific sudden change and replacement, thus finds the way that active higher variant is routine.In order to improve the biological activity of this albumen further, applicant is by a large amount of experiments, carry out point mutation for site specific in the aminoacid sequence of ray Angiostatin 1 functional zone, thus obtain the variant than ray Angiostatin 1 functional zone itself with higher inhibit activities.
Summary of the invention
The present invention relates to the variant of the separation of Parent Protease, its position corresponding to position 9R, 17F, 42H, 57S, 62R, 71K, 80N, 89P, 100G, 101R, 129A, 134E, 151Q, 163P, 189K or 217R of the functional zone of SEQIDNO:1 at least one comprises modification.Specifically, variant of the present invention has the inhibit activities of improvement.
Preferably, being applied to method of the present invention, presenting the protein variant of the inhibit activities of improvement is following arbitrary modification: 9R/G, 17F/S, 42H/D, 57S/L, 62R/V, 71K/R, 80N/W, 89P/V, 100G/E, 101R/C, 129A/G, 134E/S, 151Q/N, 163P/K, 189K/R or 217R/T.
The invention still further relates to the method producing protein variant of the present invention, comprise (a) and cultivate host cell; (b) described protein variant is reclaimed.
In production method of the present invention, use method well known in the art culturing cell in the nutritional medium being suitable for producing described polypeptide.Such as; can by suitable culture medium and allow expression and/or the shake-flask culture that carries out under being separated the condition of described polypeptide, and small-scale in laboratory or industrial fermentation tank or large scale fermentation (comprise continuously, in batches, fed-batch or solid state fermentation) carry out culturing cell.Use methods known in the art to cultivate in suitable nutritional medium, described nutritional medium comprises Carbon and nitrogen sources and inorganic salt.Suitable substratum can obtain from commercial supplier or can according to disclosed composition preparation (such as, in the catalogue of American type culture collection).If polypeptide is secreted in nutritional medium, this polypeptide directly can reclaim from described substratum.If polypeptide is not secreted, it can reclaim from cell lysate (lysate).
Gained polypeptide can use methods known in the art to reclaim.Such as, polypeptide can be reclaimed from nutritional medium by ordinary method, and described ordinary method includes but not limited to centrifugal, filtration, extraction, spraying dry, evaporation or precipitation.
Polypeptide of the present invention can by multiple methods known in the art purifying, described method includes but not limited to that chromatography (such as, ion-exchange, affine, hydrophobic, chromatofocusing and size exclusion), electrophoresis method (such as, preparative (preparative) isoelectrofocusing), differential solubility (such as, ammonium sulfate precipitation), SDS-PAGE or extraction (see, such as, ProteinPurification, J.-C.Janson and LarsRyden compiles, VCHPublishers, NewYork, 1989).
Polypeptide of the present invention, for the preparation of corresponding medicine, goes for suppressing cancer.
Embodiment
Embodiment 1
1. the acquisition of ray Angiostatin 1 functional zone
Ray functional zone are made up of in the following order following amino-acid residue:
TLD1YKQLRDKETPSGFTLDDV1QTGVDNPGHPF1MTVGCVAGDEESYEVFKALFDPV1QDRHGGYKPTDKHKTDLNHENLKGGDDLDPNYVLSSRVRTGRS1KG1ALPPHCSRGERRLVEKLCLEGLATLTGEFQGKYYPLTTMSDAEQQQL1DDHFLFDKPVSPLLLASGMARDWPDARG1WHNNDKTFLVWVNEEDHLRV1SMQKGGNMKEVFRRFCVGLKK
2, the structure of ray Angiostatin 1 functional zone variant
The introducing of catastrophe point
(1), according to corresponding mutational site design corresponding mutagenic primer, adopt PCR fixed-point mutation method on the gene corresponding to ray Angiostatin 1 functional zone, amino acid sites corresponding for wild-type to be suddenlyd change;
(2), above-mentioned PCR primer reclaims after purifying through glue, carries out enzyme cut with restriction enzyme, after being connected with the plasmid pmD-18T carrier segments of cutting through same enzyme, and transformation of E. coli DH5 α competent cell;
(3), qualification recombinant plasmid: with enzyme cut, PCR method identifies recombinant plasmid, and order-checking inspection, obtains the nucleotide sequence after suddenling change thus.These nucleotide sequences say that corresponding aminoacid sequence is respectively on the basis of SEQIDNO:1, has carried out corresponding sudden change in 9R/G, 17F/S, 42H/D, 57S/L, 62R/V, 71K/R, 80N/W, 89P/V, 100G/E, 101R/C, 129A/G, 134E/S, 151Q/N, 163P/K, 189K/R or 217R/T position.
(4) expression of, pPIC6 α-ray Angiostatin 1 functional zone variant
Restriction enzyme site is introduced at two ends above-mentioned steps being built the ray Angiostatin 1 functional zone variant gene obtained, signal peptide sequence is added in upstream, structure obtains pPIC6 α-ray Angiostatin 1 functional zone variant vector, after qualification, by plasmid transfection Pichia pastoris, and carry out eukaryotic expression, adopt Blasticidin as selection markers.Induce its secreting, expressing target protein, find that the expressing quantity that methanol concentration is 0.5%, continuous induction obtains for 4 days is higher.The protein band obtained through SDS-PAGE electrophoretic separation can react with the antiserum(antisera) of the anti-hCG of rabbit in Westernblot detects.Experimental result shows that the expression amount average in pichia pastoris phaff of recombinant plasmid pPIC6 α-ray Angiostatin 1 functional zone variant that we build is that expression amount is about 140mg/L.
3, the growth of ray Angiostatin 1 functional zone variant Tumor suppression and transfer
BALB/c nude mice is divided into control group, the positive (endoxan) control group and experimental group at random, often organizes 8, male and female half and half.Every mouse dorsal sc inoculation 0.2mlLewis lung carcinoma cell suspension (4 × 106 cell) Lewis lung cancer cell.After tumor inoculation the 7th day, experimental group abdominal injection various dose ray functional zone solution, every day 1 time, totally 14 times; Control group abdominal injection equivalent solvent; Positive controls intraperitoneal injection of cyclophosphamide, 1 time weekly, totally 2 times.Within 21st day, sacrifice mouse, peel off tumour and weigh (being fixed for immunohistochemical experiment by 10% neutral formalin), dissect, observe and record hepatic metastases joint footing.Be calculated as follows inhibition rate of tumor growth and metastases inhibiting rate:
Concrete outcome is as follows:
4, the chick chorioallantoic membrane vasculogenesis that nasopharyngeal carcinoma cell (CNE-2Z) is induced is tested
Selection is clean, eggshell homogeneous, air chamber plant egg uniformly, and in 19200 types intelligence incubators, hatching 1 week, opens a diameter 1cm aperture at embryo head end under aseptic condition, form false air chamber.Inoculation CNE-2Z cell (1.9 × 106/ embryo), seals with scotch tape.Put in incubator and cultivate after 4 days, filter paper central authorities being made a call to an aperture is placed on the chorioallantoic membrane in air chamber, and add ray functional zone albumen and misfolded proteins liquid 100 μ l thereof respectively on filter paper, control group then adds equivalent solvent, every day 1 time, altogether administration 4 times.Then remove scotch tape, fix 12 minutes respectively with acetone and dehydrated alcohol.Cutting the chorioallantoic membrane being placed with filter paper puts on slide glass, abandons or adopts filter paper, and under microscope, the branch of random selecting 5 visual field counting visible vessels counts and takes a picture.Be calculated as follows vasculogenesis inductivity and Agiogenesis inhibition rate:
| Group | Chicken embryo number (only) | Dosage (a μ g/ * days) | Vessel branch is counted (X pulls out+S) | Inhibiting rate |
| Control group | 5 | — | 83.5 | — |
| SEQ ID NO:1 albumen | 5 | 100 | 42.2 | 49.5% |
| JG1 (9R/G) | 5 | 100 | 34.0 | 59.3% |
| JG2 (9R/W) | 5 | 100 | 71.2 | 14.7% |
| JG16 (17F/S) | 5 | 100 | 28.3 | 66.1% |
| JG20 (42H/R) | 5 | 100 | 83.5 | 0.0% |
| JG23 (42H/D) | 5 | 100 | 34.0 | 59.3% |
| JG30 (57S/P) | 5 | 100 | 68.1 | 18.4% |
| JG36 (57S/L) | 5 | 100 | 28.2 | 66.2% |
| JG42 (62R/V) | 5 | 100 | 39.4 | 52.8% |
| JG50 (71K/R) | 5 | 100 | 36.1 | 56.8% |
| JG55 (80N/W) | 5 | 100 | 29.7 | 64.4% |
| JG60 (89P/V) | 5 | 100 | 34.3 | 58.9% |
| JG63 (100G/E) | 5 | 100 | 35.3 | 57.7% |
| JG70 （101R/C) | 5 | 100 | 37.3 | 55.3% |
| JG80 （129A/S) | 5 | 100 | 63.2 | 24.3% |
| JG89 （129A/G) | 5 | 100 | 38.1 | 54.4% |
| JG121 （134E/S) | 5 | 100 | 26.7 | 68.0% |
| JG135 （151Q/N) | 5 | 100 | 36.0 | 56.9% |
| JG164 （163P/K) | 5 | 100 | 25.0 | 70.1% |
| JG175 （189K/R) | 5 | 100 | 37.1 | 55.6% |
| JG196 (134E/S and 80N/W) | 5 | 100 | 27.8 | 66.7% |
| JG217 （217R/ T） | 5 | 100 | 38.6 | 53.8% |
As can be seen from the experimental result of 3 and 4, the variant of 9R/G, 17F/S, 42H/D, 57S/L, 62R/V, 71K/R, 80N/W, 89P/V, 100G/E, 101R/C, 129A/G, 134E/S, 151Q/N, 163P/K, 189K/R or 217R/T is all significantly improved in inhibition of cancer cell rate and vascular study rate relative to original ray functional zone albumen, therefore achieves good effect.
Toxicological experiment
Acute toxicity test
Adopt healthy Kunming mouse, body weight 19 ~ 22 grams, male and female half and half.The front fasting 4h of administration (po), adopts multiple dosing method in 24h, takes the circumstances into consideration therebetween to supply feed, can't help water.Dosage is 100 times of (1p, 20mg/kg of the effective dose of ray functional zone original series and variant sequence thereof; Po, 40mg/kg), to observation post administration 7 days.Viewing duration records the toxic reaction situation of animal and the distribution of dead animal day by day.Because do not find dead mouse, do not measure LD50, then change and do " mtd test ", disposable 300 times of (1p, the 60mg/kg using the effective dose of ray functional zone and variant thereof; Po, 120mg/kg) 0.5ml and 0.8ml give the injection of 20 mouse peritoneals or gavage respectively, observe 14 days, result does not find mouse toxicity reaction or death yet.
Stability
Adopt Freeze Drying Technique to make ray functional zone and variant sample thereof, preserve 12 months in-20 DEG C of refrigerators, its biological activity (inhibiting angiogenesis, suppression vitro culture oncocyte and mice transplanted tumor growth) is without obviously reducing.
Ray functional zone and variant thereof can mix with pharmaceutically useful carrier or dissolve, the medicine of the various tumour of obtained treatment, comprise various formulation, this medicine can be used for leukemia, also can be used for various noumenal tumour, as the treatment of esophagus cancer, liver cancer, cancer of the stomach, carcinoma of the pancreas, colon and rectum carcinoma, cervical cancer, ovarian cancer, mammary cancer, nasopharyngeal carcinoma, oral carcinoma, lip cancer, skin carcinoma, bladder cancer, chorioepithelioma, carcinoma of parotid gland, lymphoma, myomata, melanoma and various intracranial tumorss etc., and the application in the people needing this treatment.In made various combination of oral medication, the content of ray functional zone and variant thereof is the about 0.001-100mg of every Kg body weight every day, preferably about 0.01-50mg, is more excellently about 0.05-30mg, and the best is 0.1-10mg about.Every bu 2-4 time uses, and within 10-30 days, is a course for the treatment of, general medication 3-6 course for the treatment of, each course for the treatment of interval 10-30 days.Made medicinal composition for injections, can supply intramuscular injection or intravenous drip, wherein the content of ray functional zone and variant thereof is the about 0.001-50mg of every Kg body weight every day, preferably about 0.01-25mg, is more excellently about 0.05-15mg, and the best is 0.1-3mg about.Every bu 1-3 time uses, and within 10-30 days, is a course for the treatment of, general medication 3-6 course for the treatment of, each course for the treatment of interval 10-30 days.But accurate dosage should be determined by doctor, depend primarily on the age of patient, body weight, the state of an illness, reaction etc.
Sequence table
< 110 > opens bravely
< 120 > ray Anti-angiogenesis factors 1 functional zone variant JG121 and application thereof
〈160〉1
〈210〉1
〈211〉225
〈212〉PRT
< 213 > ray
〈400〉1
TLDIYKQLRDKETPSGFTLDDVIQTGVDNPGHPFIMTVGCVAGDEESYEVFKALFDPVIQ60
DRHGGYKPTDKHKTDLNHENLKGGDDLDPNYVLSSRVRTGRSIKGIALPPHCSRGERRLV120
EKLCLEGLATLTGEFQGKYYPLTTMSDAEQQQLIDDHFLFDKPVSPLLLASGMARDWPDA180
RGIWHNNDKTFLVWVNEEDHLRVISMQKGGNMKEVFRRFCVGLKK225
Claims (4)
1. a ray Angiostatin 1 functional zone albumen, its aminoacid sequence is for shown in SEQIDNO:1.
2. a ray Angiostatin 1 functional zone protein variant, is characterized in that, on the amino acid whose basis shown in SEQIDNO:1, replaces accordingly at 134E/S.
3. protein variant as claimed in claim 2 is preparing the purposes in Tumor suppression medicine.
4. an anti-tumor medicinal preparation, it contains protein variant according to claim 2 and pharmaceutically suitable carrier.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610017459.1A CN105541985B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG121 of ray Angiostatin and its application |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610017459.1A CN105541985B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG121 of ray Angiostatin and its application |
| CN201410249736.2A CN104031136B (en) | 2014-06-06 | 2014-06-06 | Ray Angiostatin 1 functional areas variant and application thereof |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410249736.2A Division CN104031136B (en) | 2014-06-06 | 2014-06-06 | Ray Angiostatin 1 functional areas variant and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105541985A true CN105541985A (en) | 2016-05-04 |
| CN105541985B CN105541985B (en) | 2019-08-16 |
Family
ID=51462152
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|---|---|---|---|
| CN201610017478.4A Active CN105418753B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG217 of ray Angiostatin and its application |
| CN201610017475.0A Active CN105481966B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG175 of ray Angiostatin and its application |
| CN201610037005.0A Active CN105481967B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG16 of ray Angiostatin and its application |
| CN201610017409.3A Active CN105440117B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG55 of ray Angiostatin and its application |
| CN201610017404.0A Active CN105481962B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG42 of ray Angiostatin and its application |
| CN201610017401.7A Pending CN105541984A (en) | 2014-06-06 | 2014-06-06 | Manta-angiogenesis-inhibitor-1 functional-area variant JG36 and application thereof |
| CN201610017419.7A Active CN105440118B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG70 of ray Angiostatin and its application |
| CN201610017470.8A Expired - Fee Related CN105418742B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG135 of ray Angiostatin and its application |
| CN201610017414.4A Active CN105481965B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG63 of ray Angiostatin and its application |
| CN201610017442.6A Active CN105418752B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG89 of ray Angiostatin and its application |
| CN201610017473.1A Active CN105461792B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG164 of ray Angiostatin and its application |
| CN201610017410.6A Active CN105481964B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG60 of ray Angiostatin and its application |
| CN201610017393.6A Pending CN105481961A (en) | 2014-06-06 | 2014-06-06 | Ray angiogenesis inhibiting factor (1) functional domain variant JG23 and application thereof |
| CN201610017459.1A Active CN105541985B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG121 of ray Angiostatin and its application |
| CN201610017406.XA Expired - Fee Related CN105481963B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG50 of ray Angiostatin and its application |
| CN201410249736.2A Expired - Fee Related CN104031136B (en) | 2014-06-06 | 2014-06-06 | Ray Angiostatin 1 functional areas variant and application thereof |
Family Applications Before (13)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610017478.4A Active CN105418753B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG217 of ray Angiostatin and its application |
| CN201610017475.0A Active CN105481966B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG175 of ray Angiostatin and its application |
| CN201610037005.0A Active CN105481967B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG16 of ray Angiostatin and its application |
| CN201610017409.3A Active CN105440117B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG55 of ray Angiostatin and its application |
| CN201610017404.0A Active CN105481962B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG42 of ray Angiostatin and its application |
| CN201610017401.7A Pending CN105541984A (en) | 2014-06-06 | 2014-06-06 | Manta-angiogenesis-inhibitor-1 functional-area variant JG36 and application thereof |
| CN201610017419.7A Active CN105440118B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG70 of ray Angiostatin and its application |
| CN201610017470.8A Expired - Fee Related CN105418742B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG135 of ray Angiostatin and its application |
| CN201610017414.4A Active CN105481965B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG63 of ray Angiostatin and its application |
| CN201610017442.6A Active CN105418752B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG89 of ray Angiostatin and its application |
| CN201610017473.1A Active CN105461792B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG164 of ray Angiostatin and its application |
| CN201610017410.6A Active CN105481964B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG60 of ray Angiostatin and its application |
| CN201610017393.6A Pending CN105481961A (en) | 2014-06-06 | 2014-06-06 | Ray angiogenesis inhibiting factor (1) functional domain variant JG23 and application thereof |
Family Applications After (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610017406.XA Expired - Fee Related CN105481963B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG50 of ray Angiostatin and its application |
| CN201410249736.2A Expired - Fee Related CN104031136B (en) | 2014-06-06 | 2014-06-06 | Ray Angiostatin 1 functional areas variant and application thereof |
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| CN (16) | CN105418753B (en) |
Families Citing this family (1)
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| CN104774898B (en) * | 2015-04-24 | 2020-07-14 | 浙江海洋学院 | Application of tuna dark meat protein anti-cervical cancer polypeptide |
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- 2014-06-06 CN CN201610017478.4A patent/CN105418753B/en active Active
- 2014-06-06 CN CN201610017475.0A patent/CN105481966B/en active Active
- 2014-06-06 CN CN201610037005.0A patent/CN105481967B/en active Active
- 2014-06-06 CN CN201610017409.3A patent/CN105440117B/en active Active
- 2014-06-06 CN CN201610017404.0A patent/CN105481962B/en active Active
- 2014-06-06 CN CN201610017401.7A patent/CN105541984A/en active Pending
- 2014-06-06 CN CN201610017419.7A patent/CN105440118B/en active Active
- 2014-06-06 CN CN201610017470.8A patent/CN105418742B/en not_active Expired - Fee Related
- 2014-06-06 CN CN201610017414.4A patent/CN105481965B/en active Active
- 2014-06-06 CN CN201610017442.6A patent/CN105418752B/en active Active
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Also Published As
| Publication number | Publication date |
|---|---|
| CN105481967A (en) | 2016-04-13 |
| CN105481964A (en) | 2016-04-13 |
| CN105461792A (en) | 2016-04-06 |
| CN105418753A (en) | 2016-03-23 |
| CN105481961A (en) | 2016-04-13 |
| CN105418753B (en) | 2018-12-21 |
| CN105481966B (en) | 2019-05-28 |
| CN105440117B (en) | 2018-10-26 |
| CN105481965B (en) | 2019-04-26 |
| CN105418742A (en) | 2016-03-23 |
| CN105541985B (en) | 2019-08-16 |
| CN105461792B (en) | 2019-08-20 |
| CN105440118B (en) | 2018-10-16 |
| CN105418742B (en) | 2018-12-25 |
| CN105481962B (en) | 2019-08-20 |
| CN105481964B (en) | 2018-10-09 |
| CN105418752A (en) | 2016-03-23 |
| CN105481962A (en) | 2016-04-13 |
| CN104031136B (en) | 2016-08-17 |
| CN105440118A (en) | 2016-03-30 |
| CN105541984A (en) | 2016-05-04 |
| CN105481966A (en) | 2016-04-13 |
| CN105418752B (en) | 2018-12-25 |
| CN105440117A (en) | 2016-03-30 |
| CN105481963A (en) | 2016-04-13 |
| CN105481967B (en) | 2019-08-13 |
| CN105481963B (en) | 2019-08-16 |
| CN105481965A (en) | 2016-04-13 |
| CN104031136A (en) | 2014-09-10 |
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