CN105669871A - Fusion protein of thymulin alpha1 - Google Patents
Fusion protein of thymulin alpha1 Download PDFInfo
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- CN105669871A CN105669871A CN201610243306.9A CN201610243306A CN105669871A CN 105669871 A CN105669871 A CN 105669871A CN 201610243306 A CN201610243306 A CN 201610243306A CN 105669871 A CN105669871 A CN 105669871A
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- fusion protein
- thymosin alpha
- constant region
- immunoglobulin
- thymulin
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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
- C07K14/575—Hormones
- C07K14/57581—Thymosin; Related peptides
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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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses fusion protein of thymulin alpha1. The fusion protein is formed by connecting the thymulin alpha1 or active fragments of the thymulin alpha1with a constant region of immune globulin through connecting peptide or directly. The fusion protein can be expressed in eukaryotic cells and can also be expressed in prokaryotic cells. According to the fusion protein of the thymulin alpha1, a pronucleus carrier containing a target gene is prepared through the recombination DAN technology and expressed, separated and purified in escherichia coli, and compared with archetype thymulin alpha1, the obtained fusion protein of the thymulin alpha1 is longer in in-vivo half-life period and more efficient in anti-tumor effect.
Description
Technical field
The invention belongs to biotech medicine product technical field, be specifically related to the fusion protein of a kind of Thymosin alpha 1.
Background technology
Thymosin alpha 1 is a kind of micromolecule active polypeptide with immunoregulation effect, Goldstein separates from thymopoietin the 5th component (TF5) in phase late 1970s and is purified into a kind of small molecule bioactive polypeptide, there is biological activity like Thymic, Thymosin alpha 1 is asked in name, it is made up of 28 aminoacid, Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-L ys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Val-Val-Glu-G lu-Ala-Glu-Asn-OH. Thymosin alpha 1 is now widely used to clinic, is mainly used in constitutional and secondary immunodeficiency disease, such as chronic hepatitis B, severe hepatitis, antiviral therapy AIDS etc., and is used as the adjuvant drug of various malignant tumor early stage chemotherapy, radiotherapy. But, there is the half-life and short excite or the shortcoming such as cause the immune resistant function of tumor locus low in the existing Thymosin alpha 1 used clinically.
Immunoglobulin IgG is one of most rich in protein in blood, and its half-life in blood was up to 21 days. Immunoglobulin is generally made up of four peptide chains, namely by light chain (the L chain of two identical molecular weights, about 25kd) connecting via disulfide bond of forming of heavy chain (H chain, about 50kd) that the molecular weight identical with two is bigger form four peptide chain molecules. The L chain of IgG has a variable region (LH) and constant region LH.
The H chain of IgG is respectively arranged with a variable region (VH) and the functional areas such as three constant regions (CH1, CH2 and CH3) and the hinge region between CH1 and CH2 are constituted. VL and VH is the position being combined with antigen, and CH2 has complement Clq binding site, the classical activated channel of energy complement activation; CH3 has the function in conjunction with mononuclear cell, macrophage, granulocyte, B cell and NK cellular Fc Receptor.
Immunoglobulin constant region fc is mainly made up of CH2 and CH3. When, after antigen with antibodies, Fc can combine with Fc receptor (FcR), activating complement system, at antibody dependent cellular phagocytosis, the cytotoxicity of antibody dependent cellular mediation, the aspects such as inflammatory reaction are stimulated to play important important function. There is bibliographical information by Fc and some important cytokines clinically, such as interleukin-2, blood coagulation factor VIII, or and soluble recepter, as Tumor Necrosis Factor Receptors extracellular region is combined into fusion protein, can extend the prototype factor or receptor circulating half-life and or increase original biological activity (PeppelK, CrawfordD, BeutlerB.Atumornecrosisfactor (TNF) receptor-IgGheavychainchimericproteinasabivalentantagoni stofTNFactivity.JExpMed.1991Dec1;174 (6): 1483-9.).
Summary of the invention
Present invention aim at improving the fusion protein of a kind of Thymosin alpha 1, by Thymosin alpha 1 or its active fragment, by connection peptides or be directly formed by connecting with constant region for immunoglobulin. This fusion protein both can also at procaryotic cell expression at eukaryotic cell. Present invention recombinant DNA technology is prepared for the prokaryotic vector containing genes of interest, and at expression in escherichia coli and separation purification, the fusion protein of obtained Thymosin alpha 1, compared with prototype Thymosin alpha 1, has longer Half-life in vivo and more efficient antitumous effect.
The concrete technical scheme of the present invention is as follows:
The fusion protein of a kind of Thymosin alpha 1, by Thymosin alpha 1 or its active fragment, by connection peptides or be directly formed by connecting with constant region for immunoglobulin.
Preferably, described constant region for immunoglobulin is the constant region of heavy chain immunoglobulin.
Preferably, described immunoglobulin is human IgG 4, it is preferred that comprise immunoglobulin hinge region, and constant region CH2 and constant region CH3.
Preferably, shown in the aminoacid sequence of described Thymosin alpha 1 or its active fragment such as SEQIDNO:1 or 2, or, the C terminal amino acid residue of aminoacid sequence shown in SEQIDNO:1 or 2 is replaced with any of 19 kinds of natural amino acids outside original acid residue or disappearance. 20 kinds of natural amino acids are glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, agedoite, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.
Preferably, the N end of described Thymosin alpha 1 or its active fragment is acetylizad or non-acetylizad.
Preferably, described constant region for immunoglobulin aminoacid sequence is such as shown in SEQIDNO:3.
The fusion protein of Thymosin alpha 1 of the present invention, it is preferable that aminoacid sequence is such as shown in SEQIDNO:4.
Another object of the present invention is to the application providing Thymosin alpha 1 of the present invention in preparation treatment constitutional and secondary immunodeficiency disease and antitumor drug.
Preferably, described immunodeficiency symptoms includes chronic hepatitis B, severe hepatitis, acquired immune deficiency syndrome (AIDS), and described tumor is melanoma.
The fusion protein of Thymosin alpha 1 of the present invention can by the following method prepare more:
(1) acquisition of gene
The method utilizing gene chemical synthesis obtains Thymosin alpha 1 or its active fragment, by connection peptides or its complementary series of nucleotide sequence directly and corresponding to constant region for immunoglobulin.
(2) gene of synthesis is connected with prokaryotic expression carrier, converts escherichia coli, carry out abduction delivering with lactose.
(3) expression product is easily separated purification.
The invention have the advantages that
Pharmacodynamics test proves that the fusion protein of the present invention has stronger antitumor action and longer Half-life in vivo compared with prototype Thymosin alpha 1.
Accompanying drawing explanation
Fig. 1 is that SDS-PAGE analyzes purification Thymosin alpha 1 fusion protein (wherein M: molecular weight marker of the present invention, 1: the abduction delivering restructuring Thymosin alpha 1 fusion protein of 1 hour, 2: the abduction delivering restructuring Thymosin alpha 1 fusion protein of 2 hours, 3: the abduction delivering restructuring Thymosin alpha 1 fusion protein of 4 hours, 4: the abduction delivering restructuring Thymosin alpha 1 fusion protein of 4 hours.
Fig. 2 suppresses melanoma effect in Thymosin alpha 1 fusion protein body of the present invention.
Detailed description of the invention
The structure of embodiment 1 Thymosin alpha 1 fusion protein expression vector
The method utilizing gene chemical synthesis obtains the complementary series SEQIDNO:5 sequence two ends of the nucleotide sequence corresponding to SEQIDNO:1 and SEQIDNO:3 and devises NcoI and HindIII restriction enzyme site; and the 5' end of NcoI and HindIII restriction enzyme site respectively devises three guanine protection bases; complementary series is carried out 94 DEG C of degeneration 5min; 54 DEG C of annealing 10min; then NcoI and HindIII enzyme action is carried out; it is cloned into prokaryotic expression carrier pET32a; convert to escherichia coli, polymerase chain method screening positive transformant. Polymerase chain method primer is:
P1:5 '-CCATGGCTAGCGATGCGGCCGTGGAT-3 ' SEQIDNO:6
P2:5 '-AAGCTTTTATTTACCCGGAGACAGGG-3 ' SEQIDNO:7
Polymerase chain method amplification system is:
Primer P1 (25 μm of ol/L) and primer P2 (25 μm of ol/L) each 10 μ l, 10 × PCR reaction buffer (Mg2+free) 5 μ l, MgCl2 (25mmol/L) 3 μ l, dNTPsMix (25mmol/L) 0.5 μ l, Taq DNA polymerase 0.5 μ l, complements to 50 μ l with ddH2O. The reaction condition of polymerase chain method is 94 DEG C of degeneration 1min, and 54 DEG C of annealing 2min, 72 DEG C extend 2min, carry out 30 circulations altogether.
Finally carrying out nucleotide sequencing analysis, the result of sequencing is consistent with SEQIDNO:5, illustrates that sequence is successfully loaded expression vector.
The induction of embodiment 2 recombiant protein with separate purification
By recombinant bacterium from the single colony inoculation plate streaking to the LB culture medium of 5ml, shake and educate overnight, it is seeded in the 500ml LB liquid medium of ammonia benzyl resistance with the inoculum concentration of 1% (V/V), 37 degree shake and educate 2.5h after, when OD600 is about 0.6, the lactose of addition 1% (V/V) 0.5mol/L shakes to educate 1,2,3,4 hours induces destination protein to express, and electrophoresis detection, sees Fig. 1. 4 degree 13000 revs/min, centrifugal 10 minutes, collect thalline, add the buffer (sodium phosphate of 20mmol/LpH7.4 of 20 times of thalline volumes, 0.5mol/LNaCl) after suspension thalline, ice bath carries out ultrasonication thalline, then 4 degree 13000 revs/min, centrifugal 10 minutes, draw supernatant and be splined on metal chelate chromatography post, first with the buffer (sodium phosphate of 20mmol/LpH7.4 of 10 times of column volumes, 0.5mol/LNaCl) washing, then with the elution buffer (sodium phosphate of 20mmol/LpH7.4 of 5 times of column volumes, 0.5mol/LNaCl, 0.1mol/LImidazole) eluting, and collect eluent, then loading is to SephadexG-25 gel column, eluting is carried out with 0.01mmol/LNH4HCO3 (pH8.6), collection penetrates liquid, carry out detecting in 280nm place with spectrophotometer and calculate protein concentration, last lyophilizing is also stored in 4 degree of refrigerators. result obtains the destination protein of the pure rank of electrophoresis, and molecular weight is consistent with theoretic.
Melanoma experiment in embodiment 3 animal body
Mus melanin tumour b16 F10 cell 1000rpm after the trypsinization of 0.05% is centrifuged 5 minutes, is resuspended in PBS, adjusts to every milliliter of 5000000 cells, and at C57BL/6 (6-8 week) about 500000 cells of mice side subcutaneous vaccination. When tumor average volume reaches 100mm34 groups are divided at random by mice, 8/group, except negative control PBS group and positive control Taxol group, one group of administration Thymosin alpha 1 (dosage is 0.25mg/kg/d), (dosage is 0.25mg/kg/d to one group of administration Thymosin alpha 1 fusion protein, with the molar relationship such as the dosage of Thymosin alpha 1 is), administering mode is that every day is inoculated tumour offside subcutaneous injection (except paclitaxel group, for injection in every 3 days once, being 10mg/kg) every time.Final therapeutic effect represents via tumor weight suppression ratio: (1-treatment group tumors tumor weight/negative control group tumor tumor weight) × 100%
Result is as in figure 2 it is shown, when treatment was to the 11st day, the tumor control rate of Thymosin alpha 1 fusion protein is 30.4%, and the tumor control rate of Thymosin alpha 1 is 53.4% (p < 0.05). Experimental result illustrates that the Thymosin alpha 1 fusion protein of present invention design can actually significantly inhibit the tumor growth in Mice Body, and the effect of neoplasm growth is better than prototype Thymosin alpha 1.
Internal pharmacokinetics determination experiment in embodiment 4 animal body
SD rat, cleaning grade, male and female half and half, fasting, freely drink water, give Thymosin alpha 1 fusion protein with the single dose tail vein of 2.632mg/kg, respectively upon administration 0.5, l, 3,5,10,24,36h, 48h, 60h, 72h rat eye socket takes blood, and 12000r/min is centrifuged 2min, takes supernatant. ELISA method detects. First with phosphate buffer, the monoclonal antibody of Thymosin alpha 1 being diluted to 1ug/mL, add in ELISA Plate 96 orifice plate, it is 100ul that every hole adds volume, and 4 degree are coated overnight. Next day, discard and be coated liquid, wash 3 times with cleaning mixture. Add confining liquid, every hole 300uL, 37 degree of incubation 4h, discard confining liquid, wash 3 times with cleaning mixture. Adding the blood serum sample of dilution, every hole 100uL (does blank well, negative control hole and Positive control wells) simultaneously, hatches 1h in 37 degree, is washed out 3 times. In each reacting hole, add goat anti rat IgG (with the confining liquid 1:8000 dilution) 100uL of HRP labelling, hatch 1 hour for 37 DEG C, wash 3 times. Then every hole adds tmb substrate solution 0.1ml, 37 DEG C 20 minutes. Adding stop buffer 2M sulphuric acid 0.05ml, to measure A450 in microplate reader, (with reference to wavelength 630nm), place measured each hole trap, and result is as shown in table 1. Data by analysis, found that the peak time at the blood medicine peak of Thymosin alpha 1 fusion protein of the present invention is about 36 hours, than long 22 times of the peak time (1.67 hours) at the blood medicine peak of Thymosin alpha 1 in bibliographical information.
Table 1 fusion protein concentration (ng/L) of the present invention
Claims (10)
1. the fusion protein of a Thymosin alpha 1, it is characterised in that by Thymosin alpha 1 or its active fragment, by connection peptides or be directly formed by connecting with constant region for immunoglobulin.
2. the fusion protein of Thymosin alpha 1 as claimed in claim 1, it is characterised in that described constant region for immunoglobulin is the constant region of heavy chain immunoglobulin.
3. the fusion protein of Thymosin alpha 1 as claimed in claim 2, is characterized in that described immunoglobulin is human IgG 4.
4. the fusion protein of Thymosin alpha 1 as claimed in claim 3, it is characterised in that described in be comprise immunoglobulin hinge region, constant region CH2 and constant region CH3.
5. the fusion protein of Thymosin alpha 1 as claimed in claim 1, it is characterized in that shown in the aminoacid sequence of described Thymosin alpha 1 or its active fragment such as SEQIDNO:1 or 2, or, the C terminal amino acid residue of aminoacid sequence shown in SEQIDNO:1 replaces with any of 19 kinds of natural amino acids outside original acid residue or disappearance.
6. the fusion protein of Thymosin alpha 1 as claimed in claim 5, it is characterised in that the N end of described Thymosin alpha 1 or its active fragment is acetylizad or non-acetylizad.
7. the fusion protein of Thymosin alpha 1 as claimed in claim 1, it is characterised in that described constant region for immunoglobulin aminoacid sequence is such as shown in SEQIDNO:3.
8. the fusion protein of Thymosin alpha 1 as claimed in claim 1, it is characterised in that the fusion protein aminoacid sequence of Thymosin alpha 1 is such as shown in SEQIDNO:4.
9. the fusion protein of the Thymosin alpha 1 as described in any one of claim 1-8 application in preparation treatment constitutional and secondary immunodeficiency disease and antitumor drug.
10. the fusion protein of Thymosin alpha 1 as claimed in claim 9 application in preparing antitumor drug, it is characterized in that described immunodeficiency symptoms includes chronic hepatitis B, chronic hepatitis A, severe hepatitis, serious symptom hepatitis A and acquired immune deficiency syndrome (AIDS), described tumor includes melanoma, pulmonary carcinoma, breast carcinoma, gastric cancer and colon cancer.
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| CN201610243306.9A CN105669871A (en) | 2016-04-19 | 2016-04-19 | Fusion protein of thymulin alpha1 |
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| CN201610243306.9A CN105669871A (en) | 2016-04-19 | 2016-04-19 | Fusion protein of thymulin alpha1 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107098978A (en) * | 2017-05-05 | 2017-08-29 | 中国药科大学 | A kind of antitumor and Immune-enhancing effect double effects fusion protein |
Citations (4)
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| CN101534865A (en) * | 2005-10-19 | 2009-09-16 | Ibc药品公司 | Methods and compositions for generating bioactive assemblies of increased complexity and uses |
| CN101967196A (en) * | 2010-11-10 | 2011-02-09 | 夏志南 | Interferon fusion protein, preparation thereof and application thereof |
| CN104168914A (en) * | 2011-09-23 | 2014-11-26 | 昂考梅德药品有限公司 | VEGF/DLL4 binding agents and uses thereof |
| CN105111315A (en) * | 2015-09-29 | 2015-12-02 | 海南医学院 | MIP (macrophage inflammatory protein) 3alpha-Fc fusion protein and application thereof |
-
2016
- 2016-04-19 CN CN201610243306.9A patent/CN105669871A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101534865A (en) * | 2005-10-19 | 2009-09-16 | Ibc药品公司 | Methods and compositions for generating bioactive assemblies of increased complexity and uses |
| CN101967196A (en) * | 2010-11-10 | 2011-02-09 | 夏志南 | Interferon fusion protein, preparation thereof and application thereof |
| CN104168914A (en) * | 2011-09-23 | 2014-11-26 | 昂考梅德药品有限公司 | VEGF/DLL4 binding agents and uses thereof |
| CN105111315A (en) * | 2015-09-29 | 2015-12-02 | 海南医学院 | MIP (macrophage inflammatory protein) 3alpha-Fc fusion protein and application thereof |
Non-Patent Citations (1)
| Title |
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| 张鹏 等: "胸腺肽α1的研究进展", 《东南大学学报(医学版)》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107098978A (en) * | 2017-05-05 | 2017-08-29 | 中国药科大学 | A kind of antitumor and Immune-enhancing effect double effects fusion protein |
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Application publication date: 20160615 |