CN101429519A - Process for producing recombinant insulin-like growth factor-1(IGF-1) amalgamation protein - Google Patents
Process for producing recombinant insulin-like growth factor-1(IGF-1) amalgamation protein Download PDFInfo
- Publication number
- CN101429519A CN101429519A CN 200810072372 CN200810072372A CN101429519A CN 101429519 A CN101429519 A CN 101429519A CN 200810072372 CN200810072372 CN 200810072372 CN 200810072372 A CN200810072372 A CN 200810072372A CN 101429519 A CN101429519 A CN 101429519A
- Authority
- CN
- China
- Prior art keywords
- igf
- fusion rotein
- recombinant human
- growth factor
- genetic engineering
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention discloses a method for preparing recombined human insulin growth factor-1(IGF-1) fused protein, which belongs to the field of biological medicine field in biological technique. The invention adopts the genetic engineering fungus fermentation method for production: a. designing and synthesizing recombining human IGF-1 fused protein gene; b. constructing an expression vector of the recombined human IGF-1 fused protein; c. utilizing the expression vector to convert the host to construct the genetic engineering fungus; and d. utilizing the genetic engineering fungus to ferment the recombined human IGF-1 fused protein. In the method, according to the characteristics that the first amino acid of the natural IGF-1 is glycine(GLy, G), a leading peptide is added before the natural IGF-1, and a hydroxylamine specific cracking part(Asn-Gly peptide bond) is designed between the leading peptide and the natural IGF-1, thereby making the expression products stable and lowering purification cost. In the method, the fused protein in serial expression is used, wherein the N end is thioredoxin, and the C end is IGF-1, so that the expression products are more stable, and the separation method is simple and cheap. The method has wide application prospect.
Description
Technical field
The present invention relates to the biomedicine field in the biotechnology, especially the DNA recombinant technology promptly makes up Trx and people IGF-1 fusion rotein, and by its method of gene engineering colibacillus fermentative preparation.
Background technology
Rillderktercht in 1976 from human serum, separate obtain insulin-like growth factor-i (insulin-like growth factor-1, IGF-1), new era of beginning IGF-1 research.IGF-I has effects such as promoting to grow, to promote material generation, extremely payes attention in recent years, is treating aspects such as insulin resistance diabetes, nerve injury, cretinism, osteoporosis, katabolism disease, has broad application prospects.At present, external IGF-1 treatment diabetes, the industrialization of amyotrophy lateral sclerosis disease, but China does not have launch (product that same names is arranged, but production method is not open) as yet abroad.Adopting gene engineering method at present is to produce unique effective ways of IGF-I.
The expression study of domestic report hlGF-1 in intestinal bacteria is many, selects the prokaryotic expression carrier pRSET B plasmid that contains phage promoter for use as the Wu Lanxiao of No.1 Military Medical Univ..The copy number height of plasmid in intestinal bacteria expressed strong.Can be when they select preceding Nde 1 of ATG and HindIII restriction enzyme site clone foreign gene with the excision of leading peptide encoding sequence, recombinant plasmid transformed expressive host bacterium BL21 has realized the non-fusion expression of goal gene after IPTG induces.But the first amino acid of expression product is methionine(Met), and natural hIGF-1 does not contain methionine(Met).Other author's expression product contains methionine(Met) equally.The product that contains methionine(Met) must be removed methionine(Met), will increase the cost of product greatly like this.And the short expression product that merges (only several amino acid) type of bibliographical information adult form or N end is arranged is extremely unsettled, is difficult in the general intestinal bacteria and stablizes high expression level.
The pET serial carrier is the prokaryotic expression system that is most widely used at present.The pET serial carrier is to utilize intestinal bacteria T7 phage re-reading system to express.The T7 RNA polymerase of T7 expression system optionally activates transcribing of T7 phage promoter.It is a kind of highly active RNA polymerase, fast about 5 times of the speed of its synthetic mRNA than e. coli rna polysaccharase, and the gene transcription of escherichia coli host own is less competitive than the T7 phage and transcribes system.PET32 is the fusion protein expression vector in the pET serial carrier.It contains a highly soluble polypeptide-Trx, and (thioredoxin Trx), has the formation effect of catalysis disulfide linkage.In addition, (6xHis tag) is very convenient to the purifying of target protein for 6 * Histidine fragment that this carrier contains because 6 * Histidine fragment can with the combination of Ni-NTA agarose, with the direct upper prop of sample, wash-out just can be purified into the fusion rotein that has His Tag.。
Fusion rotein can carry out the selectivity cutting with proteolytic ferment or chemical reagent and obtain the little peptide of purpose.Because zymin costs an arm and a leg, the cost height, this method is not suitable for scale operation.
Summary of the invention:
In order to overcome the deficiencies in the prior art, the objective of the invention is to provide a kind of product stable, separation method is simple, and cost is low, be applicable to the Trx and the people IGF-1 fusion rotein of large-scale production, and by its method of gene engineering colibacillus fermentative preparation.
The present invention solves technical scheme that its technical problem adopts: the preparation method of a kind of recombinant human insulin-like growth factor-1 (IGF-1) fusion rotein is characterized in that: adopt genetic engineering fungus fermentation method production: a. recombinant human IGF-1 fusion rotein genes involved design and synthetic; B. make up recombinant human IGF-1 fusion protein expression vector; C. transform the host with described expression vector and make up genetic engineering bacterium; D. with described genetic engineering bacterium fermentation recombinant human IGF-1 fusion rotein.
The constructional feature of described genes involved is that two albumen of composition fusion rotein are respectively Trx and IGF-1, has introduced one two peptide bond Asn-Gly between two albumen; Trx can promote the disulfide linkage of IGF-1 to form, and makes albumen with soluble form with may increasing that activity form occurs arranged; Two peptide bonds can rupture under the effect of azanol as the specificity cracking section of azanol, discharge IGF-1, in addition, contain 6 Histidine series, thereby make things convenient for purifying process greatly.
The technical process of described gene fermentation recombinant human IGF-1 fusion rotein is as follows: select single bacterium colony genetic engineering bacterium, carry out amplification culture, abduction delivering is collected bacterium liquid and is carried out cytoclasis, isolates inclusion body, destroys inclusion body, discharges fusion rotein, crosses NI
2+-metal chelating column obtains fusion rotein, and fusion rotein is cracking in hydroxylamine solution, discharges recombinant human IGF-1, separation and purification IGF-1, and desalination again, freeze drying example, quality inspection gets final sample.
Described carrier is commercial pET32a (+), and substitute.
Described host cell is DH5 α and substitute thereof.
Be to adopt the cracking condition of optimizing in the described fusion rotein, azanol concentration is 2-3M, and the temperature of reaction is 40-45 ℃, adopts LiOH control pH8.5-9.5, and the reaction times is 5-10 hours.
1. according to the primary structure of people IGF-1, designed the fusion rotein of a tandem expression, the N end is Trx, and the C end is IGF-1, and middle dipeptides for design voluntarily is used for hydroxylamine cleavage;
2. according to the dna sequence dna of the natural IGF-1 of people, designed two PCR primers, added EcoRI and HindIII recognition sequence respectively, two peptide sequences and protectiveness base, by EcoRI and site-directed pET32a (+) expressive plasmid of being cloned into of HindIII, obtain expression vector;
3. the positive colony fermentation condition optimization obtains the product of high expression level;
4. adopt Ni
2+-metal-chelating column chromatography method obtains fusion rotein;
5. fusion rotein discharges the IGF-1 monomer through hydroxylamine cleavage;
6. adopt Ni
2+-metal-chelating column chromatography is removed uncracked fusion rotein and cracked leading peptide, obtains the IGF-1 monomer.
The invention has the beneficial effects as follows: because the present invention is glycine (Gly according to first amino acid of natural IGF-I, G) characteristics, add leading peptide in natural IGF-I front, and the purifying cost is lowered at the special cracking position (Asn-Gly peptide bond) of an azanol of design between leading peptide and the IGF-1.Leading peptide is a Trx, can promote the formation of disulfide linkage, and makes albumen with soluble form with may increasing that activity form occurs arranged.In addition, leading peptide contains 6x Histidine series, therefore, not only can efficiently express, and can conveniently adopt Ni-metal affinity chromatography post with the leading peptide place to go, obtains natural complete IGF-I.
The present invention adopts the fusion rotein of tandem expression, and the N end is Trx, and the C end makes expression product more stable for IGF-1, and separation method is simple, and is inexpensive.Therefore, the present invention is with a wide range of applications.
Description of drawings
Fig. 1 pET32a (+) expression vector synoptic diagram.
The multiple clone site and the expressed sequence of Fig. 2 pET32a (+) expression vector.
RxTag represents the thioredoxin label among the figure, is made up of 109 amino acid; HisTag represent 6 histidine-tagged.
Fig. 3 PCR primer 1.AAC GGT coding Asn-Gly wherein, GAA TTC is the EcorI restriction enzyme site.
Fig. 4 PCR primer 2.5 amino acid series in back that comprise IGF-1, termination codon (TAG), HindIII restriction enzyme site (AAGCTT).
Fig. 5 pET32-IGF design of graphics.
The IGF-1DNA that Fig. 6 PCR obtains is at 1.5% agarose gel electrophoretogram.
Fig. 7 fusion rotein abduction delivering result's SDS-PAGE collection of illustrative plates.
Among the figure from left to right each swimming lane be respectively: IPTG induces 1 hour, 2 hours, 4 hours, 6 hours, the blank bacterium and the molecular weight marker of untransfected plasmid.
Fig. 8 inclusion body total protein is through NI
2+The fusion rotein SDS-PAGE collection of illustrative plates that obtains behind the metal chelating column.
Among the figure from left to right each swimming lane be respectively: the phosphoric acid buffer wash-out result who contains 400mM (elutriant dilution 5 times), 400mM, 10mM, 20,50,100mM imidazoles.
Collection of illustrative plates after the cracking of Fig. 9 fusion rotein.
Among the figure from left to right each swimming lane be respectively: molecular weight marker, crack protein not, crack protein.
The MALDI-TOF-MS collection of illustrative plates of the target protein IGF-1 of purifying after the cracking of Figure 10 fusion rotein.
Embodiment
1. the clone of antigen-4 fusion protein gene
1.1 the design of tandem expression fusion rotein
It is Gly that its N of natural IGF-1 holds first amino acid, is not methionine(Met).That is to say that synthetic IGF-1 precursor just is secreted into the extracellular after must removing first methionine(Met) in human body cell matter.Therefore, if with the IGF-1 gene single expression that contains the gene codon ATG, separation and purification inconvenience is on the one hand removed also unusual trouble of methionine(Met) on the other hand.In the enforcement according to azanol can specificity the characteristics of cracking Asn-Gly peptide bond, and first amino acid of natural IGF-1 just in time is Asn, and there are not the characteristics of Asn-Gly two peptide sequences on the whole protein chain of IGF-1, when design, between leading peptide and IGF-1, design an Asn-Gly sequence, promote to express and make things convenient for purifying.
1.2 the gene primer of fusion rotein design
According to the multiple clone site sequence of pET32a (+), on two primers of 5 ' end and 3 ' end, add EcoRI and HindIII restriction endonuclease sites respectively.
1.3 the IGF-1 gene is synthetic
Adopt PCR method amplifying target genes, amplification program is as follows: 95 ℃ of pre-sex change, 5min; 95 ℃ of sex change, 1min; 64 ℃ of renaturation, 1min; Extend 72 ℃, 1min, totally 35 circulations, reacting the extension time for the last time is 10min.
Get an amount of PCR product and carry out 1.5-2% agarose gel electrophoresis, take pictures behind ethidium bromide staining, other PCR product adopts glue to reclaim test kit behind electrophoresis dying and extracts PCR product in the glue.
1.4 goal gene inserts expression vector pET32a (+)
Goal gene (PCR product) carries out agarose electrophoresis after with EcoRI and HindIII double digestion, reclaims dna fragmentation.PET32a (+) plasmid carries out enzyme with these two enzymes equally and cuts the back purifying.Target DNA fragment and carrier after enzyme is cut mixed be connected.
After the ligation reaction solution directly is added to competent DH5 α intestinal bacteria, is inoculated into then and carries out resistance screening on the agarose plate that contains penbritin.6 bacterium colonies of picking, amplification back preparation plasmid in a small amount, and be template with this plasmid, adopt above-mentioned PCR primer to increase.Amplified production carries out 1.5-2% agarose gel electrophoresis, estimates the base number of product behind ethidium bromide staining.Base number is the positive clone of 230bp left and right sides person.The plasmid of positive colony is directly carried out the dna sequence dna order-checking.The correct bacterial classification that checks order is kept at (20 ℃) in the glycerine pipe.
2. the abduction delivering of genetic engineering bacterium
2.1. microbial culture and inducing: get in the 2ul glycerine pipe bacterium liquid and join 3ml and contain in the LB nutrient solution of penbritin, 37 ℃, 250 rev/mins, the shaking table overnight incubation.Get an amount of bacterium liquid next day, join 100 times of LB culture medium culturing 2h that contain penbritin, add IPTG and induce 5h.
2.2 cracking of bacterium: collect bacterium liquid, the centrifugal 10min of 4000rpm, resuspended with PBS, ultrasonic 180 times with the cracking bacterium.Lysate removes supernatant with the centrifugal 10min of 12000rpm, with 1 * PBS washing precipitation 3 times, and the centrifugal 1min of 12000rpm, resuspended with buffer B, incubated at room 2h (composition of buffer B: 100MmNaH2PO4,10Mm Tris-Cl, 8M urea, pH8.0).With the centrifugal 30min of 12000rpm, carefully pipette supernatant (bacterial lysate) then.
3. the separation of fusion rotein
(fusion rotein of sample is less than 15mg on every ml resin) places in the chromatography column with an amount of Ni-NTA resin, opening the post lower end cap allows liquid drain off naturally, after adding the phosphoric acid buffer balance bacterial lysate is added, behind the 30min with 30ml contain 0,10,20,50 respectively, the phosphoric acid buffer of 100mM imidazoles carries out stepwise elution, flow velocity is 2ml/min, and elutriant is not collected.Carry out wash-out with the damping fluid that contains the 400mM imidazoles at last, flow velocity is 1ml/min.Collect elutriant, lyophilize ,-20 ℃ of preservations.
4. hydroxylamine cleavage reaction
Purified, cryodesiccated fusion rotein adds hydroxylamine cleavage liquid (contain the 6M guanidine, the 2M azanol is adjusted to pH9 with 5M LiOH solution), 45 ℃ of water bath heat preservations 4-6 hours.After reaction finishes the reaction solution dialysis is gone up NTA-Ni+ agarose chromatography post after 20 hours once more,, collect elutriant, lyophilize ,-20 ℃ of preservations with the phosphoric acid buffer wash-out that does not contain imidazoles.
5. substance assistant laser desorpted ionized/flight time mass spectrum systems approach is identified target protein
It is substance assistant laser desorpted ionized/the flight time mass spectrum system that (MALDI-TOF-MS) is U.S. Sai Fuji company (CIPHERGEN) product.The molecular weight of split product was for being respectively 7640.0D and 7640.8D during 2 substance assistant laser desorpted ionized/flight time mass spectrum method of masurement (MALDI-TOF MS) were analyzed.The maximum error of measuring of MALDI-TOF-MS instrument is 0.5%, because the theoretical molecular of IGF-1 is 7649D, therefore theoretical value is 7610.7～7687.2 in limit of error.Tested proteic molecular weight can be thought IGF-1 in this scope.
SEQUENCE?LISTING
＜110〉Medical University Of Fujian
＜120〉preparation method of recombinant human insulin-like growth factor-1 (IGF-1) fusion rotein
<160>2
<170>Patentln?version?3.1
<210>1
<211>210
<212>DNA
＜213〉people
<400>1
<210>2
<211>70
<212>PRT
＜213〉people
<400>2
Claims (6)
1, the preparation method of a kind of recombinant human insulin-like growth factor-1 (IGF-1) fusion rotein is characterized in that adopting gene bacterium fermentative Production: a. recombinant human IGF-1-1 antigen-4 fusion protein gene design and synthetic; B. make up recombinant human IGF-1-1 fusion protein expression vector; C. transform the host with expression vector and make up genetic engineering bacterium; D. with genetic engineering bacterium fermentation recombinant human IGF-1 fusion rotein.
2, the preparation method of a kind of recombinant human insulin-like growth factor-1 according to claim 1 (IGF-1) fusion rotein, it is characterized in that: the constructional feature of described gene is that two albumen of composition fusion rotein are respectively Trx and IGF-1, has introduced one two peptide bond Asn-Gly between two albumen; The purifying process that contains 6 Histidine series.
3, the technical process of gene fermentation recombinant human IGF-1-1 fusion rotein according to claim 1 is as follows: select single bacterium colony genetic engineering bacterium, carry out amplification culture, abduction delivering, collect bacterium liquid and carry out cytoclasis, isolate inclusion body, destroy inclusion body, discharge fusion rotein, cross NI
2+-metallized metal chelate column obtains fusion rotein, and fusion rotein is cracking in hydroxylamine solution, discharges recombinant human IGF-1, separation and purification IGF-1, and desalination again, freeze drying example, quality inspection gets final sample.
4, the preparation method of a kind of recombinant human insulin-like growth factor-1 according to claim 1 (IGF-1) fusion rotein, it is characterized in that: described carrier is commercial pET32a (+), and substitute.
5, the preparation method of a kind of recombinant human insulin-like growth factor-1 according to claim 1 (IGF-1) fusion rotein, it is characterized in that: described host cell is DH5 α and substitute thereof.
6, the preparation method of a kind of recombinant human insulin-like growth factor-1 according to claim 1 (IGF-1) fusion rotein, it is characterized in that: be to adopt the cracking condition of optimizing in the described fusion rotein, azanol concentration is 2-3M, the temperature of reaction is 40-45 ℃, adopt LiOH control pH8.5-9.5, the reaction times is 5-10 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810072372 CN101429519A (en) | 2008-12-16 | 2008-12-16 | Process for producing recombinant insulin-like growth factor-1(IGF-1) amalgamation protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810072372 CN101429519A (en) | 2008-12-16 | 2008-12-16 | Process for producing recombinant insulin-like growth factor-1(IGF-1) amalgamation protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101429519A true CN101429519A (en) | 2009-05-13 |
Family
ID=40645157
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200810072372 Pending CN101429519A (en) | 2008-12-16 | 2008-12-16 | Process for producing recombinant insulin-like growth factor-1(IGF-1) amalgamation protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101429519A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776259A (en) * | 2011-05-13 | 2012-11-14 | 吴江近岸蛋白质科技有限公司 | Preparation method of long insulin-like growth factor 1 mutant |
| CN101993495B (en) * | 2009-08-12 | 2013-07-24 | 上海近岸科技有限公司 | Protein mixture and preparation method thereof |
| CN108998463A (en) * | 2018-08-24 | 2018-12-14 | 苏州红冠庄国药股份有限公司 | A kind of preparation process of source of people insulin growth factor-1 (IGF-1) compound |
| CN110205336A (en) * | 2019-05-22 | 2019-09-06 | 华南理工大学 | A kind of recombinant expression method of Brevinin-2GUb polypeptide and application |
| CN113376380A (en) * | 2021-05-31 | 2021-09-10 | 华南农业大学 | ELISA kit for detecting dog IL-6 and application thereof |
| CN114292800A (en) * | 2021-12-27 | 2022-04-08 | 普健生物（武汉）科技有限公司 | Recombinant cell for recombinant expression of IGF-1 gene and recombinant expression method |
| CN114644715A (en) * | 2022-03-04 | 2022-06-21 | 苏州红冠庄国药股份有限公司 | Preparation method of fusion protein of IGFBP-3 and IGF-1 and compound |
-
2008
- 2008-12-16 CN CN 200810072372 patent/CN101429519A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993495B (en) * | 2009-08-12 | 2013-07-24 | 上海近岸科技有限公司 | Protein mixture and preparation method thereof |
| CN102776259A (en) * | 2011-05-13 | 2012-11-14 | 吴江近岸蛋白质科技有限公司 | Preparation method of long insulin-like growth factor 1 mutant |
| CN102776259B (en) * | 2011-05-13 | 2016-04-20 | 吴江近岸蛋白质科技有限公司 | The preparation method of long type-1 insulin like growth factor mutant |
| CN108998463A (en) * | 2018-08-24 | 2018-12-14 | 苏州红冠庄国药股份有限公司 | A kind of preparation process of source of people insulin growth factor-1 (IGF-1) compound |
| CN110205336A (en) * | 2019-05-22 | 2019-09-06 | 华南理工大学 | A kind of recombinant expression method of Brevinin-2GUb polypeptide and application |
| CN113376380A (en) * | 2021-05-31 | 2021-09-10 | 华南农业大学 | ELISA kit for detecting dog IL-6 and application thereof |
| CN114292800A (en) * | 2021-12-27 | 2022-04-08 | 普健生物（武汉）科技有限公司 | Recombinant cell for recombinant expression of IGF-1 gene and recombinant expression method |
| CN114644715A (en) * | 2022-03-04 | 2022-06-21 | 苏州红冠庄国药股份有限公司 | Preparation method of fusion protein of IGFBP-3 and IGF-1 and compound |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101429519A (en) | Process for producing recombinant insulin-like growth factor-1(IGF-1) amalgamation protein | |
| CN107245494A (en) | Solution expression with high efficiency and purification process of the A β 42 in Escherichia coli | |
| CN106995811A (en) | A kind of algin catenase, its preparation method and application | |
| CN115960209B (en) | Recombinant humanized collagen and application thereof | |
| CN112725319A (en) | Alginate lyase FaAly7 with polyG substrate specificity and application thereof | |
| CN112980865A (en) | Construction method of recombinant human-like collagen engineering bacteria | |
| CN102465134B (en) | Method for preparing recombinant anthropogenic Cu/Zn superoxide dismutase | |
| CN115975004A (en) | Recombinant human fibronectin, preparation method and application thereof | |
| CN113801240B (en) | D-psicose-3-epimerase activity aggregate and preparation method and application thereof | |
| CN112322599B (en) | Transaminase UPTA, preparation method and application | |
| CN104603280A (en) | Method for the production of polypeptides | |
| CN1746306B (en) | Recombinant human interferon alpha 4 coded cDNA sequence, production and use thereof | |
| CN108277210A (en) | Mould ketenes hydrolase ZEN214 and encoding gene and application | |
| CN101921799B (en) | Preparation method of horse serum amyloid protein A1 and expression vector and genetic engineering bacteria thereof | |
| CN114276413A (en) | Preparation method of artificial antibacterial peptide G3 | |
| CN101948837A (en) | Method for producing human insulin growth factor-1 in recombinant Escherichia coli | |
| CN101857871B (en) | Method for purifying recombinant VP1 antigen of enterovirus type 71 viruses | |
| CN102277327B (en) | Colon bacillus for over-expressing RimL and application on preparing N-extrasin alpha acetylate | |
| CN112625117A (en) | Non-denaturing purification method and application of soluble recombinant teriparatide | |
| CN110205336A (en) | A kind of recombinant expression method of Brevinin-2GUb polypeptide and application | |
| CN103911338A (en) | Construction of engineering strain capable of highly expressing IGF-1 based on procaryotic codon preference | |
| CN100523172C (en) | Gene engineering bacteria of high efficiency expression of human alpha 1-thymulin and its construction method and use | |
| CN109022471A (en) | Produce the escherichia expression system of oxalate oxidase, the production method and its application of oxalate oxidase | |
| CN112941058B (en) | Recombinant clostridium histolyticum type II collagenase as well as preparation method and application thereof | |
| CN111471667B (en) | Chitosanase Csn-PT and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20090513 |