CN105177032B - New tumor necrosin receptor-Fc fusion gene and its product albumen - Google Patents
New tumor necrosin receptor-Fc fusion gene and its product albumen Download PDFInfo
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Abstract
The invention discloses a kind of methods for improving recombined human TNFR75-Fc track fusion amount, and the method includes improving TNFR75-Fc fusion described in the new SEQ ID NO:3 of one kind；Providing a kind of carrier simultaneously is pCI-gs-TNFR75-Fc；During Fusion gene construction, 18 base complementrities that 18 bases and human IgG1 Fc genetic fragment 5 ' are held are introduced in 3 ' the end primers of TNFR75；The preparation method of Fc sections of genetic fragments of human soluble TNFRcDNA and human IgG1's heavy chain constant region in the screening technique and MSX concentration of cell strain, SEQ ID NO:3 sequence is improved simultaneously, finally improves fusion protein expression.
Description
The application is application No. is 201010268409.3, applying date 2010.09.01, and entitled novel people is swollen
The divisional application of tumor necrosis factor receptor-Fc fusion gene and its product albumen.
Technical field
The invention belongs to genetic engineering pharmaceutical fields, and in particular to a kind of novel TNFR-Fc fusion and its expression produce
Object.
Background technique
Rheumatoid arthritis (Rheumatoid Arthritis, RA) be the most common autoimmune disease of the mankind it
One, illness rate about 1% is obviously related to human leukocyte antigen DR4/DR1, has certain genetic predisposition.The clinic of RA is special
Sign is mostly the synovium of joint inflammation of hand, foot, wrist, knee, stern etc., eventually leads to joint injury.Synovitis causes big amount lymphocyte
Infiltration mainly includes macrophage, T lymphocyte and thick liquid cell, while blood vessel hyperplasia.Joint injury take place mostly in synovial membrane with
Cartilage and bone adjacent place.
RA is a kind of to involve the systemic autoimmune disease based on joint.Recent study shows neoplasm necrosis
The factor-α (TNF-α) is key factor (the Feldmann M 2001 in RA pathogenesis；RavinderN Maini,2001；
RavinderN Maini, 199), body can be stimulated to generate a series of factors, such as interleukins (IL) -1, IL-6, IL-8
With the generation of granulocyte colony stimulating factor etc., and inducing endothelial cell expresses adhesion molecule, attracts leucocyte to affected joints.
TNF-α also stimulates synovial macrophages, fibroblast, osteoclast and cartilage cell to generate matrix metalloproteinase, and presses down
The synthesis of chondroproteoglycan processed, and can lead to the damage of synovial membrane inflammation and articular cartilage.Block TNF-α can be in RA morbidity machine
The upstream of system blocks inflammatory reaction, achievees the purpose that treat RA.
Tumor necrosis factor α (Tumor Necrosis Factor Alpha, TNF α) and lymphotoxin
(Lymphotoxin, LT) be by receptor TNFR75 and TNFR55 together in conjunction with and play biological action.Human tumour
Mecrosis factor receptors 75 (TNFR75) are made of 461 amino acid, and the amino acid residue of the end N- 235, is TNFR75 albumen
The cell membrane outskirt segment that enzyme hydrolysis is got off, also referred to as soluble TNF R75 (sTNFR75).
Many experiments evidence show soluble TNF R75 and TNFR55 (i.e. the film outer portion of receptor) can by with TNF α
With LT effectively in conjunction with and block TNF α and LT in vivo biological action (Hunns-Martin Lorenz, 2002；
Smith, C.A. etc. (1990), science, 248:1019-1023), and the wider hair of TNFR75 distribution, affinity are stronger.But
TNFR half-life period is shorter.
Human IgG immunoglobulin like protein half-life period may be up to 21 days, they are the most abundant protein in human blood, be divided into
IgA, IgG, IgM, IgE and IgD, wherein IgG has tetra- hypotypes of IgG1, IgG2, IgG3 and IgG4.IgG1Fc sections by 232 ammonia
Base acid residue composition, including hinge area, the area CH2 and the area CH3.It has been reported that the cysteine in the Fc hinge region of IgG is residual
Base and other protein (such as various cell factors and soluble recepter) in conjunction with and form fusion protein, make protein-based be similar to
IgG molecule but without the region CH1 and light chain.Due to homologous in structure, Fc fusion protein is shown and the human IgG of similar isotype
Comparable interior medicine dynamics characteristic.
External someone constructs merging for people TNFRII receptor part and the Fc segment of IgG by genetic engineering means
Albumen, this albumen have neutralization (United States Patent (USP) 5,605,590) to TNF in vitro.The country also has building is such to melt
Hop protein, horse cyanines etc. CN1417334A soluble part in tumor necrosis factor acceptor recombination and antigen-4 fusion protein gene with
A kind of fusion protein containing linker is constructed in product；Liu Chang gate of a village etc. is in CN1502632A Novel TNFR-FC fusion protein
In construct a kind of truncated fusion protein；Xu Bin etc. constructs a kind of human tumour using bicistronic mRNA in CN101003575
FC sections of fusion proteins of necrosin soluble receptor II-antibody.
This kind of drug humanization degree is high, and caused immunogenic response is low, and the treatment works well, than depend merely on anodyne and
Anti-inflammatory agent more effectively controls the state of an illness.But the generally existing preparation method of this kind of therapeutic agent is complicated at present, and expression quantity is low, expensive
Equal assistant officers' problem to be solved.
Summary of the invention
There is provided a kind of novel recombined human TNFR75-Fc fusions and its table for the problem that the present invention solves
The protein product reached.
The Second Problem that the present invention solves is to construct a kind of weight containing recombined human TNFR75-Fc fusion segment
Group carrier pCI-gs-TNFR75-Fc, after transfection mammalian cell, the amplification of eukaryocyte is marked, and can greatly improve purpose
Copy number of the gene in host cell, to improve the expression quantity of destination protein.
The third problem that the present invention solves has been to provide a kind of lactation containing recombinant vector pCI-gs-TNFR75-Fc
Zooblast can stablize expression TNFR75-Fc.The mammalian cell can be the common food in one's mouths such as Chinese hamster ovary celI, NSO cell
Newborn System in Animal Cell Biotechnology cell strain.
The 4th problem that the present invention solves is amplification screening of being pressurizeed by sulphur ammonia methionine (MSX), makes to express purpose
Albuminous cell strain expression quantity improves at least one order of magnitude, when carrying out highly-expressing cell strain screening, finally proposes MSX concentration
For height to 400~500 μm, screening monoclonal expression quantity reaches 20~30pg/cell24hr.
To solve the above problems, the present invention is implemented by following technical solution:
The total serum IgE of HL-60 cell strain is extracted, RT-PCR expands people sTNFR75 genetic fragment；PCR amplification human IgG1's Fc base
Because of segment.Two genetic fragments are spliced using Overlap extention round pcr to obtain coding rhTNFR:Fc fusion
The full length cDNA sequence of albumen.By the cDNA sequence insertion clone intermediate vector T easy of acquisition, confirmed just through sequencing
The building of carrier for expression of eukaryon is carried out after really.
RhTNFR:Fc fusion protein cDNA segment is cloned in expression vector, using liposomal transfection teclmiques, by sterile extracting
Recombinant plasmid rhTNFR:Fc/pCI-gs import Chinese hamster ovary celI in, through MSX pressurize screen, obtained the rhTNFR of high expression quantity:
The gene engineering monoclonal cell strain of Fc fusion protein, when MSX concentration is increased to 400~500 μm, screening monoclonal expression
Amount reaches 20~30pg/cell24hr.
The present invention has synthesized a kind of human tumor necrosis factor receptor fusion by the method for OVERLAP PCR
TNFR75-Fc, using the eukaryocyte efficient expression vector containing GS system, in mammalian cells by MSX pressurization sieve
So that the expression quantity of the fusion protein improves 20 percentage points, fusion protein activity improves 8 percentage points for choosing.
Specific embodiment
Invention is further described in detail With reference to embodiment, but the present invention is not limited to following implementations
Example.
The preparation of 1 human soluble TNFR75cDNA of embodiment
1, PCR amplification TNFR75cDNA
Design primer A:5 ' TCAAG CTT3 ' primer B:5 ' GTC ACA of ATG GCT CCC GTC GCC GTC TGG
AGA TTT GGG CTC GTC GCC AGT GCT CCC TTC AGC 3 ',
Another set primer is designed, linker sequence, the sequence (Gly4Ser) 3, for a coding will be introduced in primer B
The hydrophobic peptides of 15 amino acid, their mobility is preferable, does not influence the native three dimensional structure of TNFR and Fc, linker
DNA sequence dna be 5 '-GGT GGC GGT GGA AGC GGC GGT GGC GGA AGC GGC GGT GGC GGC AGC-
3’。
Design introduces the primer B of linker sequence.Primer Linker-B:5 ' GCT GCC GCC ACC GCC GCT TCC
GCC ACC GCC GCT TCC ACC GCC ACC GTC。
Using people in loop total serum IgE as template, it is as template, with A and Linker-B using primer A and B respectively
Template carries out RT-PCR reaction, and 94 DEG C of denaturation 5min are started the cycle over, 94 DEG C of 30sec, 58 DEG C of 30sec, 72 DEG C of 1min, and totally 32
Circulation, last 72 DEG C of extensions 8min.It obtains coding sTNFR75 and compares the genetic fragment of sTNFR75-linker.
2, the identification of PCR product and gel recycling
Above-mentioned condition is subjected to PCR, is identified on agarose gel electrophoresis, finds destination protein molecular weight in product
The band of about 800bp recycles the segment.
The clone of 2 Fc sections of genetic fragments of human IgG1's heavy chain constant region of embodiment
Design primer C:5 ' GAG CCC AAA TCT TGT GAC GAG CCC AAA TCT TGT GAC AAA 3 ', draws
Object D:5 ' AGTGAA TTCTCA TTT ACC CGG AGA CAG GGA 3'；
Design primer linker-C:GGT GGC GGT GGA AGC GGC GGT GGC GGA AGC GGC GGT GGC
GGC AGC GAG。
Using IgG1 Fc/T easy vector plasmid as template, respectively using C and D as primer, using Linker-C and D as primer,
RT-PCR reaction is carried out, 94 DEG C of denaturation 5min are started the cycle over, 94 DEG C of 20sec, 62 DEG C of 30sec, 72 DEG C of 2min, totally 35 circulations,
Last 72 DEG C of extensions 10min.Amplification obtains encoding human IgG1 Fc and compares the genetic fragment of linker-IgG1 Fc.
The acquisition of 3 TNFR75-Fc fusion of embodiment
1, PCR amplification encodes rhTNFR-Fc fusion protein/rhTNFR-linker-Fc full-length gene segment
Using Fc sections of pcr amplification products of sTNFR75PCR amplified production and human IgG1 as template, with A:5 ' TCAAG CTT
ATG GCT CCC GTC GCC GTC TGG 3 ' and D:5 ' AGTGAA TTC TCA TTT ACC CGG AGA CAG GGA
3 ' be primer, carries out RT-PCR reaction.
Simultaneously using Fc sections of pcr amplification products of sTNFR75-linker pcr amplification product and people linker-IgG1 as mould
Plate, with A:5 ' TCAAG CTTATG GCT CCC GTC GCC GTC TGG 3 ' and D:5 ' AGTGAA TTC TCA TTT
ACC CGG AGA CAG GGA 3 ' is primer, carries out RT-PCR reaction.
RT-PCR amplified reaction step: 94 DEG C of denaturation 5min are started the cycle over, 94 DEG C of 40sec, 62 DEG C of 30sec, 70 DEG C of 1min,
Totally 30 circulations, last 72 DEG C of extensions 10min.
Amplification obtains the full-length cDNA segment of rhTNFR-Fc and rhTNFR-linker-Fc fusion protein, genetic fragment
Long is respectively 1470bp and 1485bp.
2, rhTNFR-Fc/rhTNFR-linker-Fc fusion protein cDNA clone is in intermediate vector
RhTNFR-Fc/rhTNFR-linker-Fc fusion protein cDNA pcr amplification product and linear T easy vector
(Promega) it is attached reaction.Connection product Transformed E .coli E. coli competent host cell, is coated on and is previously added
Blue hickie screening is carried out on the LB plate (containing 100 μ g/ml ampicillins and 15 μ g/ml tetracyclines) of X-gal and IPTG.From turn
Change and select white single colonie at random on plate, respectively inoculation proliferation, carries out plasmid Rapid identification, obtain TNFR75-Fc and control
RhTNFR-linker-Fc fusion.
The expression of 4 TNFR75-Fc/rhTNFR-linker-Fc fusion of embodiment
1, rhTNFR-Fc/rhTNFR-linker-Fc fusion segment is cloned in expression vector
The building of rhTNFR-Fc/rhTNFR-linker-Fc expressing fusion protein type recombinant plasmid is using through business table
The EcoR I single endonuclease digestion site for the pCI-gs being transformed up to carrier pCI-neo, is inserted into correct through determined dna sequence result
RhTNFR-Fc/rhTNFR-linker-Fc genetic fragment, with the both forward and reverse directions of Spe1 single endonuclease digestion identification Insert Fragment, connection is produced
Object Transformed E .coli competent cell obtains rhTNFR-Fc/pCI-gs/E.coli Top10F ' and control after screening and identification
RhTNFR-linker-Fc/pCI-gs/E.coli Top10F ' recombinant bacterium.
2, a large amount of preparations of expression type recombinant plasmid
The strain Escherichia coli of recombinant plasmid rhTNFR-Fc/pCI-gs/rhTNFR-linker-Fc/pCI-gs will be carried
Be inoculated in 200ml fluid nutrient medium, after being incubated overnight use Qiagen Plasmid Maxi Kits, to specifications into
A large amount of preparations of row recombinant plasmid.Gained Plasmid DNA, through 70% ethanol washing, it is sterile air-dry after, be dissolved in 500 μ l sterile waters
In, it takes 2 μ l plasmids to dilute 100 times and detects A260 value and A280 value respectively, when the ratio of A260/A280 is between 1.75-1.85,
The plasmid is then used for cell transfecting.
3, cell strain transfects
After the Chinese hamster ovary celI cultivated in culture bottle two days is digested with 0.25% trypsin solution, appropriate progress trypan blue is taken
Dyeing counting, with 3 × 105The single hole of a cell number inoculation 6 orifice plates；It, can be into up to 90% full layer after cell inoculation 6 orifice plates 24 hours
Row transfection；250 μ l OPTI-MEM I (Gibco) are respectively taken, are added in two sterile centrifugation tubes, 10 μ l are separately added into two pipes
Lipofectamine (Invitrogen) and 4 μ g dilutes plasmid, and the liquid in two pipes is mixed into same pipe, is stored at room temperature
Form it into DNA within 20 minutes: liposome complex；At the same time, the cell culture medium abandoned in 6 orifice plates is inhaled, is grown and is trained with cell
Supporting base (DMEM/F12/10%FCS), gently 2ml cell growth medium is added after twice in washing cell in each hole；By 500 μ l
DNA: liposome complex solution is added in the corresponding aperture in 6 orifice plates, is placed in 37 DEG C, 5%CO2Overnight incubation in incubator.
The screening of 5 TNFR75-Fc/TNFR75-linker-Fc fusion protein overexpression cell line of embodiment
After recombinant cell strain obtained by embodiment 4 is screened is digested with 0.25% trypsin solution, passed with the ratio of 1:3
Enter overnight incubation in three 100mm culture dishes；After 24 hours, the culture medium in 100mm culture dish is changed to Selective agar medium,
Final concentration of 25 μm are added in DMEM (no glutamine) culture solution containing 10% dialysis fetal calf serum (Gibco BRL)
MSX (thiamines methionine, Sigma) is used as selective agent；According to cell death situation every 2-4 days replacement Selective agar mediums, about
7-10 days cells start mortality, and cell clone starts to occur after about 14 days, clone when cell clone diameter reaches 1-2mm
Ring picks from the plate monoclonal and transfers in 24 orifice plates, and every batch of transfects about 50 single cell clones of picking.
Single cell clone is grown in 24 orifice plates when expiring layer to 50-70%, and the culture supernatant of each clone is taken to carry out ELISA inspection
It surveys, according to cell growth status and expression quantity, chooses cell clone progress drug of the expression quantity greater than 1pg/cell24hr and add
Companding increases screening；The concentration for stepping up selective agent MSX expands the recombinant plasmid being integrated in chromosome, dense to drug
When degree is improved to 500 μm, screening monoclonal expression quantity reaches 20~30pg/cell24hr.The culture supernatant for taking each clone, is answered
Its fusion protein expression is detected with ELISA method, the 50 progress expression quantity measurements of each picking monoclonal, the results are shown in Table 1 in total.
Table 1, expression quantity contrast and experiment
It is calculated by table 1 it is found that producing novel recombination TNFR75-Fc expression quantity through the invention and improves 20 than control experiment
Percentage point, it is more advantageous to the expanding production of the albumen.
6 TNFR75-Fc/TNFR75-linker-Fc fusion protein activity comparative test of embodiment
The fusion protein TNFR75-Fc/TNFR75-linker-Fc expressed with the cell strain filtered out is carried out
Sepharose-A affinity chromatography and hydrophobic chromatography.The specific steps are (1) by protein A affinity chromatography purifying protein, in albumin A
In affinity chromatography, 1mol/L is also added in the elution process of albumen in the urea that 1mol/L is added when albumen loading in eluent
The urea of urea.Destination protein under being eluted at about 3.5 low pH；(2) sample obtained by (1) is directly passed through into hydrophobic chromatography loading,
Salting liquid used is ammonium sulfate, and filler used is the filler containing butyl or phenyl.Obtain the fusion that purity is greater than 90%
Albumen.
Then active determination test is carried out.By rhTNFR-Fc antagonism TNF-α to the killing of target cell L929 cell strain come
Detect the biological activity of rhTNFR-Fc.
Sample activity (AU/ml)=[standard items ED50(ng/ml)/product to be tested ED50(ng/ml)] × standard items potency (AU/
Ml) × sample to be tested extension rate
It the results are shown in Table 2.
1, the L929 cell of logarithmic growth phase, with fully dispersed cell after 0.25% trypsase conventional digestion, every time
It surveys (1 piece of 96 orifice plate) living and takes about 2 × 106Supernatant is abandoned in cell, centrifugation, and culture solution B is added by the cell density tune of cell culture fluid
It is whole to 1.5 × 105It after/ml, is added in 96 well culture plates, the hole 0.1ml/, is placed in 37 DEG C, cultivated in 5% carbon dioxide incubator
Night (18~for 24 hours).
2, standard items are prepared: taking the TNFR:Fc working standard of a 10 μ g/ml, is added and is diluted with TNF-α (40U/ml)
10 μ g/ml actinomycin D solution be diluted to concentration be 500ng/ml.
3, according to the protein quantification of sample to be determined, it is diluted to 1mg/ml with sterile water for injection first, it is sterile in 1.5ml
It is gradually diluted to 500ng/ml with the actinomycin D solution of the diluted 10 μ g/ml of TNF-α (40U/ml) in centrifuge tube, is paid attention to every
Secondary extension rate is no more than 10 times.
4, the dilution of standard items and test sample
The dilution of continuous doubling dilution working standard or sample (contains same concentrations in all pipes after diluting
TNF-α and actinomyces D, but the concentration of TNFR:Fc is different.It is actual dilute when being added in the hole containing isometric culture solution in next step
Degree of releasing will be at this time 2 times).
5, the sample of the 100 various dilutions of μ l, 3 holes of each dilution are added in every hole.Negative control is added dilute with TNF-α
The actinomycin D solution of the 40U/ml released.It is added in positive control and is diluted with serum-free RPMI-1640/DMEM (1:1) culture solution
10 μ g/ml actinomycin D solution.
6,37 degree culture 18-24 hours in 5%CO2 incubator.
7, endpoint determination: 40.0 μ l violet staining liquid are added in every hole, dye ten minutes, with tap water rinse 3~4 times,
Until originally water colorless.96 orifice plates are patted dry as far as possible, make that destainer, every hole 100 is added to 96 orifice plates without residual moisture in plate
μ l is mixed, and absorption value is read at microplate reader 570nm.It the results are shown in Table 2.
Table 2, active contrast and experiment
| LOGEC50 | |
| TNFR-Fc | 3.812 |
| TNFR-linker-Fc | 3.518 |
It is calculated by table 2 it is found that under same protein concentration, the protein active of TNFR75-Fc ratio TNFR75-linker-Fc
Improve 8 percentage points.
Claims (2)
1. a kind of method for improving TNFR75-Fc track fusion amount, which comprises the steps of: construct one kind
Recombination TNFR75-Fc fusion containing base sequence described in SEQ ID NO:3；It constructs one kind and contains SEQ ID NO:3 institute
The recombinant vector for the base sequence stated, the carrier are pCI-gs-TNFR75-Fc, which is carrying out highly-expressing cell strain
In screening process, MSX concentration is finally increased to 500 μm；PCI-gs is transformed by pCI-neo, utilizes pCI-gs's
EcoR I single endonuclease digestion site, insertion are reflected through the correct rhTNFR-Fc genetic fragment of determined dna sequence result with Spe1 single endonuclease digestion
The both forward and reverse directions for determining Insert Fragment, obtain pCI-gs-TNFR75-Fc.
2. such as claim 1 the method, which is characterized in that during the SEQ ID NO:3 Fusion gene construction,
18 base complementrities that 18 bases and human IgG1 Fc genetic fragment 5 ' are held are introduced in 3 ' the end primers of TNFR75；
In the SEQ ID NO:3 sequence human soluble TNFR75 cDNA's the preparation method comprises the following steps:
1), PCR expands TNFR75 cDNA
B: 5 ' GTC ACA of A: 5 ' 3 ' primer of TC AAG CTT ATG GCT CCC GTC GCC GTC TGG of design primer
AGA TTT GGG CTC GTC GCC AGT GCT CCC TTC AGC 3 ',
Another set primer is designed, linker sequence, the sequence (Gly4Ser) 3, for a coding 15 will be introduced in primer B
The hydrophobic peptides of a amino acid, their mobility is preferable, does not influence the native three dimensional structure of TNFR and Fc, linker
DNA sequence be 5 '-GGT GGC GGT GGA AGC GGC GGT GGC GGA AGC GGC GGT GGC GGC AGC
- 3 '；
Design introduces the primer B of linker sequence；Linker-B: 5 ' GCT GCC GCC ACC GCC GCT TCC of primer
GCC ACC GCC GCT TCC ACC GCC ACC GTC ；
Using people in loop total serum IgE as template, respectively using primer A and B as template, with A and Linker-B
For template, RT-PCR reaction is carried out, 94 DEG C of denaturation 5min are started the cycle over, 94 DEG C of 30sec, 58 DEG C of 30sec, 72 DEG C of 1min,
Totally 32 circulations, last 72 DEG C of extensions 8min；It obtains coding sTNFR75 and compares the genetic fragment of sTNFR75-linker；
2), the identification of PCR product and gel recycling
Above-mentioned condition is subjected to PCR, is identified on agarose gel electrophoresis, finds target gene molecular weight in product
The band of 800bp recycles the segment；
The clone of Fc sections of genetic fragments of human IgG1's heavy chain constant region in the SEQ ID NO:3 sequence:
C: 5 ' GAG CCC AAA TCT TGT GAC GAG CCC AAA TCT TGT GAC AAA 3 ' of design primer, primer
D:
5'AGT GAA TTC TCA TTT ACC CGG AGA CAG GGA 3'；
Design primer linker-C:GGT GGC GGT GGA AGC GGC GGT GGC GGA AGC GGC GGT GGC GGC
AGC GAG ；
Using IgG1 Fc/T easy vector plasmid as template, respectively using C and D as primer, using Linker-C and D as primer,
RT-PCR reaction is carried out, 94 DEG C of denaturation 5min are started the cycle over, 94 DEG C of 20sec, 62 DEG C of 30sec, 72 DEG C of 2min, and totally 35
Circulation, last 72 DEG C of extensions 10min；Amplification obtains encoding human IgG1 Fc and compares the gene piece of linker-IgG1 Fc
Section.
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| CN108355141B (en) * | 2018-01-11 | 2019-06-18 | 广西壮族自治区人民医院 | A kind of drug and preparation method thereof of the treatment allergic disease based on TIM-4-Fc fusion protein |
| CN112494658B (en) * | 2020-12-04 | 2024-07-12 | 苏州药明生物技术有限公司 | Stable Fc fusion protein preparation |
| WO2023044774A1 (en) * | 2021-09-24 | 2023-03-30 | Sichuan Clover Biopharmaceuticals, Inc. | Tpo mimetic fusion proteins and methods of use submission of sequence listing as ascii text file |
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| 重组人肿瘤坏死因子受体-Fc融合蛋白质控方法和标准的研究;张翊 等;《药学学报》;20130331;165-168 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105200073B (en) | 2019-03-01 |
| CN102382850A (en) | 2012-03-21 |
| CN105200073A (en) | 2015-12-30 |
| CN105177032A (en) | 2015-12-23 |
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