CN105440117B - 1 function region variants JG55 of ray Angiostatin and its application - Google Patents
1 function region variants JG55 of ray Angiostatin and its application Download PDFInfo
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- CN105440117B CN105440117B CN201610017409.3A CN201610017409A CN105440117B CN 105440117 B CN105440117 B CN 105440117B CN 201610017409 A CN201610017409 A CN 201610017409A CN 105440117 B CN105440117 B CN 105440117B
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Classifications
-
- 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
-
- 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
-
- 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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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- 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 kind of 1 functional areas albumen of ray Angiostatin and its variants, and its application in preparing the drug for inhibiting tumour growth.So that the protein variant of the present invention is passed through eukaryotic expression, the inhibition of cancer cell can be significantly improved relative to original protein.
Description
Technical field
The invention belongs to biotechnologies, specifically, the present invention relates to the changes of 1 functional areas of ray Angiostatin
Body.
Background technology
1 functional areas of ray Angiostatin hereinafter referred to as ray functional areas, ray Angiostatin 1 are from the South Sea
The protein (molecular weight 42KD) separated in a kind of ray tissue in marine site.Result of study is shown：Ray Angiostatin 1
Significantly inhibit chick chorioallantoic membrane itself and KB cell's induction chick chorioallantoic membrane angiogenesis；Either
Intraperitoneal injection or gavage all significantly inhibit the growth and transfer of nude mouse Lewis lung cancer, reduce tumor tissues microvessel density,
Lower the expression of angiogenic factors VEGF；Also there is high inhibition effect to the transfer of nude mouse Melanoma B16 cell；It lowers
Promote the expression of transfer factor CD44v6 and ErBb2；It is more notable with effect when 5 FU 5 fluorouracil (5-FU) use in conjunction.Ray blood vessel is given birth to
It is different from the anti-cancer drugs Avastin that gene technology research institute of the U.S. develops at the action target spot of inhibiting factor 1.Avastin
It is gene engineering product, is the antibody of VEGF, action target spot is VEGF；Ray Angiostatin 1 is natural products, it
VEGF is acted not only on, bFGF and PDGF are also acted on.
In the prior art, in order to improve the activity of albumen, for albumen specific site carry out specificity mutation and
Substitution, to find the way that the higher variant of activity is conventional.In order to further increase the bioactivity of the albumen, applicant
Through a large number of experiments, for specific site in 1 function region amino acid sequence of ray Angiostatin a little dash forward
Become, to obtain the variant that there is higher inhibitory activity than 1 functional areas of ray Angiostatin itself.
Invention content
The present invention relates to the variants of the separation of Parent Protease, correspond to SEQ ID NO at least one:1 functional areas
Position 9R, 17F, 42H, 57S, 62R, 71K, 80N, 89P, 100G, 101R, 129A, 134E, 151Q, 163P, 189K or 217R
Position include modification.Specifically, the variant of the present invention has improved inhibitory activity.
Preferably, the method for being applied to the present invention, the protein variant that improved inhibitory activity is presented are following any 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 further relates to the method for the protein variant for generating the present invention, include (a) cultivating host cell；(b) it recycles
The protein variant.
In the production method of the present invention, trained in the nutrition for being suitable for generating the polypeptide using method well known in the art
It supports in base and cultivates cell.For example, can be by suitable culture medium and under conditions of allowing to express and/or detach the polypeptide
In the shaking flask culture of progress and laboratory or industrial fermentation tank small-scale or large scale fermentation (including it is continuous, in batches, feed supplement
In batches or solid state fermentation) cultivate cell.It is cultivated in suitable nutrient medium using methods known in the art, institute
It includes carbon source and nitrogen source and inorganic salts to state nutrient medium.Suitable culture medium can obtain from commercial supplier or can basis
Disclosed composition is prepared (for example, in catalogue of American type culture collection).If polypeptide is secreted into nutrition culture
In base, which can directly recycle from the culture medium.It, can be from cell lysate if polypeptide is not secreted
(lysate) it recycles.
Gained polypeptide can be recycled using methods known in the art.For example, polypeptide can be by conventional method from nutrition
It is recycled in culture medium, the conventional method includes but not limited to centrifugation, filtering, extraction, spray drying, evaporation or precipitates.
The present invention polypeptide can be purified by a variety of methods known in the art, the method includes but be not limited to chromatograph
(for example, ion exchange, affine, hydrophobic, chromatofocusing and size exclusion), electrophoresis method are (for example, preparative
(preparative) isoelectric focusing), differential solubility (for example, ammonium sulfate precipitation), SDS-PAGE or extraction (see, e.g.,
Protein Purification, J.-C.Janson and Lars Ryden is compiled, VCH Publishers, New York, and 1989).
The polypeptide of the present invention is used to prepare corresponding drug, goes for inhibiting cancer.
Specific implementation mode
Embodiment 1
1. the acquisition of 1 functional areas of ray Angiostatin
Ray functional areas are made of in the following order following amino acid residue：
TLD1YKQLRDKETPSGFTLDDV1QTGVDNPGHPF1MTVGCVAGDEESYEVFKALFDPV1QDRHGGYKPTDKHKTDLN
HENLKGGDDLDPNYVLSSRVRTGRS1KG1ALPPHCSRGERRLVEKLCLEGLATLTGEFQGKYYPLTTMSDAEQQQL1
DDHFLFDKPVSPLLLASGMARDWPDARG1WHNNDKTFLVWVNEEDHLRV1SMQKGGNMKEVFRRFCVGLKK
2, the structure of 1 function region variants of ray Angiostatin
The introducing of catastrophe point
(1), corresponding mutagenic primer is designed according to corresponding mutational site, is given birth in ray blood vessel using PCR fixed-point mutation methods
The corresponding amino acid sites of wild type are mutated on the gene corresponding to 1 functional areas of inhibiting factor；
(2), above-mentioned PCR product carries out digestion after glue recovery purifying with restriction enzyme, digestion identical as process
After the connection of plasmid pmD-18T carrier segments, bacillus coli DH 5 alpha competent cell is converted；
（3）, identification recombinant plasmid：Recombinant plasmid is identified with digestion, PCR method, and inspection is sequenced, is thus obtained
Nucleotide sequence after mutation.These nucleotide sequences say corresponding amino acid sequence respectively in SEQ ID NO：1 basis
On, 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 location have carried out corresponding mutation.
（4）, 1 function region variants of pPIC6 α-ray Angiostatin expression
The both ends for the 1 functional areas variant gene of ray Angiostatin that above-mentioned steps structure obtains are introduced into digestion position
Signal peptide sequence is added in upstream in point, and structure obtains 1 functional areas variant vector of pPIC6 α-ray Angiostatin, identifies
Afterwards, by plasmid transfection Pichia pastoris, and eukaryotic expression is carried out, using Blasticidin as selection markers.Induce it
Secreting, expressing destination protein finds that methanol concentration is that 0.5%, 4 days obtained expressing quantities of continuous induction are higher.Through SDS-
The protein band that PAGE is separated by electrophoresis can react in Westernblot detections with the antiserum of rabbit-anti hCG.Experiment
The result shows that recombinant plasmid pPIC6 α -1 function region variants of ray Angiostatin that we build finish red ferment in Pasteur
It is about 140 mg/ L that average expression quantity, which is expression quantity, in mother.
3,1 function region variants of ray Angiostatin inhibit the growth and transfer of tumour
BALB/c nude mices are randomly divided into control group, the positive (cyclophosphamide) control group and experimental group, and every group 8, male and female are each
Half.It is inoculated with 0.2ml Lewis lung carcinoma cells suspension (4 × 106 cell) Lewis lung carcinoma cells per mouse dorsal sc.Tumor inoculation
The 7th day afterwards, various dose ray functional areas solution, 1 time a day, totally 14 times was injected intraperitoneally in experimental group；Equivalent is injected intraperitoneally in control group
Solvent；Positive controls intraperitoneal injection of cyclophosphamide, 1 times a week, totally 2 times.Mouse is sacrificed within 21st day, stripping tumour, which is weighed, (to be used
10% neutral formalin is fixed for immunohistochemical experiment), it dissects, observe and record hepatic metastases nodule number.It is calculated as follows
Inhibition rate of tumor growth and metastases inhibiting rate：
Concrete outcome is as follows：
4, the chick chorioallantoic membrane angiogenesis experiment of nasopharyngeal carcinoma cell (CNE-2Z) induction
Uniform kind of cleaning, eggshell homogeneous, gas chamber egg are selected, is hatched 1 week in 19200 type intelligence incubators, it is sterile
Under the conditions of in embryo head end open a diameter 1cm apertures, form false gas chamber.CNE-2Z cells (1.9 × 106/ embryo) are inoculated with, transparent adhesive tape is used
Band sealing.It sets after being cultivated 4 days in incubator, the filter paper that center is made a call to an aperture is placed on the indoor chorioallantoic membrane of gas, adds ray respectively
In on filter paper, control group then adds equivalent solvent by functional areas albumen and its 100 μ l of misfolded proteins liquid, 1 time a day, is administered 4 times altogether.
Then remove adhesive tape, 12 minutes are fixed respectively with acetone and absolute ethyl alcohol.It cuts and is placed with the chorioallantoic membrane of filter paper and sets glass slide
On, filter paper is abandoned or adopted, the branch that 5 visuals field counting visible vessels are randomly selected under microscope counts and takes a picture.Blood is calculated as follows
Pipe generates inductivity and Agiogenesis inhibition rate：
| Group | Chicken embryo number（Only） | Dosage（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% |
From 3 and 4 experimental result can be seen that 9R/G, 17F/S, 42H/D, 57S/L, 62R/V, 71K/R, 80N/W,
The variant of 89P/V, 100G/E, 101R/C, 129A/G, 134E/S, 151Q/N, 163P/K, 189K/R or 217R/ T both with respect to
Original ray functional areas albumen is all significantly improved in cancer inhibition rate and vascular study rate, therefore achieves very well
Effect.
Toxicological experiment
Acute toxicity test
Using healthy Kunming mouse, 19~22 grams of weight, half male and half female.Fasting 4h before administration (po), use are interior for 24 hours more
Secondary dose regimen takes the circumstances into consideration supply feed, can't help water therebetween.Dosage is the effective agent of ray functional areas original series and variant sequence thereof
100 times of (1p, 20mg/kg of amount；Po, 40mg/kg), give observation post administration 7 days.Day by day the toxic reaction of animal is recorded during observation
The distribution of situation and dead animal.Because not finding dead mouse, LD50 is not measured, changes then and does " mtd test ", one
300 times of (1p, 60mg/kg of secondary property ray functional areas and its effective dose of variant；Po, 120mg/kg) 0.5ml and 0.8ml points
Not Gei the injection of 20 mouse peritoneals or gavage, observe 14 days, as a result find mouse toxicity reaction or dead yet.
Stability
Ray functional areas and its variant sample are made using Freeze Drying Technique, are preserved 12 months in -20 DEG C of refrigerators, are given birth to
Object activity (inhibits angiogenesis, inhibits in vitro culture oncocyte and mice transplanted tumor growth) nothing to be substantially reduced.
Ray functional areas and its variant can mix or dissolve with pharmaceutical carrier, and the drug of the various tumours for the treatment of, packet is made
Include various dosage forms, which can be used for leukaemia, also can be used for various entity tumors, as cancer of the esophagus, liver cancer, gastric cancer, cancer of pancreas,
Colon and rectum carcinoma, cervical carcinoma, oophoroma, breast cancer, nasopharyngeal carcinoma, carcinoma of mouth, lip cancer, cutaneum carcinoma, carcinoma of urinary bladder, on chorion
The treatment of skin cancer, carcinoma of parotid gland, lymthoma, myomata, melanoma and various intracranial tumors etc., and its in the people for needing this treatment
In application.In made various combination of oral medication, the content of ray functional areas and its variant is daily big per Kg weight
About 0.001-100mg, preferably from about 0.01-50mg, more excellent about 0.05-30mg, most preferably about 0.1-10mg.Make for 2-4 times per bu
With, 10-30 days as a treatment course, 3-6 course for the treatment of of general medication, each course for the treatment of interval 10-30 days.Made injectable drug group
Close object, for intramuscular injection or intravenous drip, wherein the content of ray functional areas and its variant be it is daily per Kg weight about
0.001-50mg, preferably from about 0.01-25mg, more excellent about 0.05-15mg, most preferably about 0.1-3mg.Per 1-3 use of bu,
10-30 days as a treatment course, 3-6 course for the treatment of of general medication, each course for the treatment of interval 10-30 days.However, accurate dosage should be by doctor
It determines, depends primarily on age, weight, the state of an illness, reaction of patient etc..
Sequence table
< 110 > is brave
120 > rays Anti-angiogenesis factors of <, 1 function region variants JG55 and its application
〈160〉1
〈210〉1
〈211〉225
〈212〉PRT
213 > rays of <
〈400〉1
TLDIYKQLRD KETPSGFTLD DVIQTGVDNP GHPFIMTVGC VAGDEESYEV FKALFDPVIQ 60
DRHGGYKPTD KHKTDLNHEN LKGGDDLDPN YVLSSRVRTG RSIKGIALPP HCSRGERRLV 120
EKLCLEGLAT LTGEFQGKYY PLTTMSDAEQ QQLIDDHFLF DKPVSPLLLA SGMARDWPDA 180
RGIWHNNDKT FLVWVNEEDH LRVISMQKGG NMKEVFRRFC VGLKK 225
Claims (3)
1. a kind of 1 functional areas protein variant of ray Angiostatin, which is characterized in that in SEQ ID NO：Ammonia shown in 1
On the basis of base acid, replaced accordingly in 80N/W.
2. protein variant as described in claim 1 is lung cancer or nose preparing the purposes in inhibiting tumour medicine, the tumour
Pharynx cancer.
3. a kind of anti-tumor medicinal preparation, it contains protein variant described in claim 1 and pharmaceutically suitable carrier.
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| CN201610017409.3A CN105440117B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG55 of ray Angiostatin and its application |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610017409.3A CN105440117B (en) | 2014-06-06 | 2014-06-06 | 1 function region variants JG55 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 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201410249736.2A Division CN104031136B (en) | 2014-06-06 | 2014-06-06 | Ray Angiostatin 1 functional areas variant and application thereof |
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| Publication Number | Publication Date |
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| CN105440117A CN105440117A (en) | 2016-03-30 |
| CN105440117B true CN105440117B (en) | 2018-10-26 |
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 |
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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 |
Family Applications After (12)
| Application Number | Title | Priority Date | Filing Date |
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| 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 |
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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 CN201610017409.3A patent/CN105440117B/en active Active
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- 2014-06-06 CN CN201610017401.7A patent/CN105541984A/en active Pending
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- 2014-06-06 CN CN201610017470.8A patent/CN105418742B/en not_active Expired - Fee Related
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Also Published As
| Publication number | Publication date |
|---|---|
| CN105481967A (en) | 2016-04-13 |
| CN105481964A (en) | 2016-04-13 |
| CN105541985A (en) | 2016-05-04 |
| 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 |
| 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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