CN105441464A - System for codon optimization and pichia pastoris expression of genes of cellobiohydrolase II - Google Patents
System for codon optimization and pichia pastoris expression of genes of cellobiohydrolase II Download PDFInfo
- Publication number
- CN105441464A CN105441464A CN201510801789.5A CN201510801789A CN105441464A CN 105441464 A CN105441464 A CN 105441464A CN 201510801789 A CN201510801789 A CN 201510801789A CN 105441464 A CN105441464 A CN 105441464A
- Authority
- CN
- China
- Prior art keywords
- beta
- hydrolase
- glucancellobio
- gene
- expression vector
- 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
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a system for codon optimization and pichia pastoris expression of genes of cellobiohydrolase II (CBH II) derived from trichoderma reesei. According to the system, a Gene Designer (DNA 2.0, Menlo Park, CA, USA) is used for carrying out codon directivity optimization on CBH II (NCBI Reference Sequence: XM_006962518.1) to construct a pPIC9K-cbh2 expression vector, and Pichia pastoris GS115 is taken as a host to obtain a transformant by virtue of an electro-transformation method. Detections show that the genes of CBH II subjected to codon optimization can be stably and efficiently expressed in pichia pastoris, the enzyme activity of a recombinant strain CMC is 0.14U/mL, and the protein content of a fermented supernatant is 0.15mg/mL.
Description
Technical field
The present invention relates to genetically engineered field, be specifically related to the structure of Trichodermareesei 1,4-BETA-D-glucancellobio-hydrolase II (CBHII) gene codon optimization and pPIC9K-cbh2 expression vector.
Background technology
Cellulase is the general name of a class polycomponent enzyme, by being difficult to for the benefit of bioavailable glucose of bioavailable cellulose hydrolysis, can therefore be considered to one of zymin had a high potential.Cellulase belongs to glycoside hydrolase, the cellulase system of degraded cellulose can at least comprise three class components: endo-type-beta-glucanase (endo-1,4-β-D-glucanase, EC3.2.1.4), circumscribed-type-beta-glucanase (exo-1,4-β-D-glucanase, EC3.2.1.91) and beta-glucosidase (β-1,4-D-glucosidase, EC3.2.1.21).And Mierocrystalline cellulose main body is made up of Glucopyranose, and connect with β-Isosorbide-5-Nitrae glycosidic link, form macromole linear polymer, monomer is cellobiose molecule, is difficult to noncrystalline domain two portions that the crystallizing field of degrading and short texture, microorganism easily degrade forms primarily of Stability Analysis of Structures, microorganism.
Enzyme component in cellulase system is not act solely on Mierocrystalline cellulose thus cellulolytic, they realize hydrolysis function by mutually acting synergistically, wherein endo-type-beta-glucanase acts on noncrystalline domain, hydrolysis β-1,4 glycosidic links, can block linear fibre element polymer and generate a large amount of Mierocrystalline cellulose small molecules; Circumscribed-type-beta-glucan enzymic hydrolysis Isosorbide-5-Nitrae-β-D-glycosidic link, acts on linear fibre element polymer end, generates cellobiose molecule; Cellobiose hydrolysis is then become glucose by beta-glucosidase.Mutually acted synergistically by cellulase prozyme component, by biopolymer-Mierocrystalline cellulose the abundantest on the earth, the glucose that microorganism can utilize easily can be converted into, produce biofuel, in order to alleviate current energy shortage problem by fermentation.
Cellulase wide material sources, the biology producing cellulase comprises insect, mollusk, protozoon, bacterium and fungi.But these self-produced enzymes innately have certain proportioning defect, such as: in the cellulase of Trichodermareesei CBHII and beta-glucosidase underproduce, and in aspergillus niger the secretion peak phase of each enzyme different, these factors are to the restricted effect of cellulose hydrolysis.In addition, in different ligno-cellulosic materials, there were significant differences for the ratio of Mierocrystalline cellulose, hemicellulose and xylogen, causes same class commercial fibres element zymin or self-produced enzyme can not play best hydrolysis result.So, optimize customization cellulase, make each component reach optimum proportioning, optimum hydrolysis effect could be obtained when minimum enzyme dosage.Therefore, in order to optimize customization cellulase, obtain cellulase list enzyme component very crucial.
In the last few years, heterogenous expression cellulose enzyme gene obtained single enzyme component is study hotspot.Pichia spp (Pichiapastoris) can realize posttranslational modification effect, as glycosylation, disulfide formation, albumen can correctly be folded, and obtain activated target protein, in addition, pichia spp does not produce endogenous lignocellulolyticenzymes component, and extracellular protein composition uncomplicated, recombinant yeast pichia pastoris bacterium fermented supernatant fluid even directly can use as single zymin without purifying, therefore, cellulose components CBHII heterogenous expression is realized using pichia spp as Host Strains to obtain CBHII component.
Summary of the invention
The object of the invention is the Trichodermareesei CBHII gene providing a kind of optimization, and the sequence GC content after optimization is reduced to 41.5% by 55.4%, and builds pPIC9K-cbh2 expression vector, to obtain the expression transformant of stability and high efficiency.
CBHII gene codon provided by the invention optimize and pichia yeast expression system concrete steps as follows:
1, codon bias optimization:
The present invention realizes codon optimized to CBHII, its nucleotide sequence is as shown in SEQIDNO.1.
SEQIDNO.1：
Codon optimized front CBHII aminoacid sequence is as shown in SEQIDNO.2.
SEQIDNO.2：
The present invention utilizes GeneDesigner (DNA2.0, MenloPark, CA, USA) to realize optimizing the CBHII nucleotide sequence codon bias shown in SEQIDNO.1, and after optimizing, nucleotide sequence is as shown in SEQIDNO.3.
SEQIDNO.3
After codon is optimized, CBHII aminoacid sequence is identical with SEQIDNO.2.
2, expression vector establishment:
Using plasmid pPIC9K as carrier, the CBHII gene after optimizing is inserted promotor AOX1 downstream, 5 ' end restriction enzyme site is EcoRI, and 3 ' end restriction enzyme site is NotI, builds pPIC9K-cbh2 expression vector.
3, recombinant bacterial strain is obtained:
Using SacI as pPIC9K-cbh2 expression vector linearisation sites, realize expression vector linearizing, proceed to pichia spp by electricity.
4, induction condition of enzyme production is optimized:
Using BMMY as culture medium, optimize potassium phosphate buffer pH value in inductive dose and substratum, realize CBHII stability and high efficiency and produce enzyme.
Beneficial effect of the present invention:
1, present invention achieves the optimization of Trichodermareesei 1,4-BETA-D-glucancellobio-hydrolase II (CBHII) gene codon skewed popularity, deflection object is Pichia yeast, reduce GC content in optimizing process, be reduced to 41.5% by 55.4%, improve recombinant yeast pichia pastoris bacterium expression efficiency.
2, the present invention is in Trichodermareesei 1,4-BETA-D-glucancellobio-hydrolase II (CBHII) gene codon skewed popularity optimizing process, utilize RNA secondary structure folding software RNAstructure to realize secondary structure to the nucleotide sequence after optimization to fold, and adjust base sequence, initiator codon end base is made to be open loop structure, be beneficial to rrna to combine, improve recombinant yeast pichia pastoris bacterium expression efficiency.
3, the present invention constructs the Expression vector pPIC9K-cbh2 containing alcohol oxygen type promotor AOX1, using methyl alcohol as inductor, controls methyl alcohol addition, increases substantially restructuring target protein expression amount.
Accompanying drawing explanation
Fig. 1: Trichodermareesei 1,4-BETA-D-glucancellobio-hydrolase II (CBHII) codon bias optimizes front and back nucleotide alignments figure.
Fig. 2: Trichodermareesei 1,4-BETA-D-glucancellobio-hydrolase II (CBHII) codon bias optimizes front and back Amino acid sequences alignment figure.
Fig. 3: Expression vector pPIC9K-cbh2 plasmid map.
Fig. 4: whether nucleic acid electrophoresis detects Expression vector pPIC9K-cbh2 and successfully construct, wherein:
No. 1 swimming lane is gained band after utilizing restriction enzyme EcoRI, NotI double digestion;
No. 2 swimming lanes are gained bands after utilizing restriction enzyme SacI single endonuclease digestion.
Fig. 5: utilize 250mL triangular flask, liquid amount is 30mL, and methyl alcohol addition is 0.5%, in 220rpm, cultivates CMC Enzyme activity assay result after 120h at 30 DEG C.
Fig. 6: utilize 500mL triangular flask, liquid amount is 50mL, and methyl alcohol addition is 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, and in 220rpm, after cultivating 192h at 30 DEG C, CMC enzyme is lived and biomass detected result.
Fig. 7: utilize 500mL triangular flask, liquid amount is 50mL, regulates potassium phosphate buffer pH in BMMY substratum to be 5.0,5.5,6.0,6.5,7.0,7.5, and in 220rpm, after cultivating 192h at 30 DEG C, CMC enzyme is lived and biomass detected result.
Fig. 8: utilize 500mL triangular flask, liquid amount is 50mL, in 220rpm, cultivates at 30 DEG C, and every 24h sampling, carries out CMC Enzyme activity assay comparing result to nutrient solution after nutrient solution before optimum culture condition and optimum culture condition.
Embodiment
Below by example, the present invention will be described in detail.
Embodiment 1: Trichodermareesei 1,4-BETA-D-glucancellobio-hydrolase II (CBHII) gene codon optimization and expression vector establishment.
1, the present invention utilizes GeneDesigner (DNA2.0, MenloPark, CA, USA) CBHII (NCBIReferenceSequence:XM_006962518.1) the nucleotide sequence codon bias realized as shown in SEQIDNO.1 is optimized, and after optimizing, nucleotide sequence is as shown in SEQIDNO.3.Consistent with original amino acid by the aminoacid sequence after codon optimized, as shown in SEQIDNO.2.Using PichiapastorisGS115 as host, contrasted by nucleotide sequence after codon bias optimization and original nucleotide sequences and see Fig. 1, contrasted by aminoacid sequence after codon bias optimization and original amino acid and see Fig. 2.
2, with 5 ' end restriction enzyme site for EcoRI, 3 ' end restriction enzyme site be NotI as double enzyme site, insert pPIC9K plasmid vector AOX1 promotor downstream, construction of expression vector pPIC9K-cbh2, Fig. 3 is shown in by expression vector collection of illustrative plates.And by double digestion, expression vector is verified, to determine that Expression vector pPIC9K-cbh2 successfully constructs, enzyme is cut rear nucleic acid electrophoresis figure and is seen Fig. 4.
Embodiment 2: linearizing expression vector also carries out electricity conversion and screening superior strain.
1, using SacI as pPIC9K-cbh2 expression vector linearisation sites, realize expression vector linearizing, nucleic acid electrophoresis is verified the plasmid after linearizing, sees Fig. 4.PichiapastorisGS115 is proceeded to by electricity, obtain recombinant bacterial strain, utilize bacterium colony PCR, by amplified band clearly bacterium colony obtain superior strain by shake flask fermentation, sample when 120h and utilize DNS method to measure fermented supernatant fluid CMC enzyme and live, see Fig. 5, wherein, 1 unit enzyme unit definition alive is under 50 DEG C of water bath condition, can transform the enzyme amount of 1 μm of ol substrate in 1 minute.
Embodiment 3: optimize induction condition of enzyme production.
1, using BMMY as culture medium, using methyl alcohol as inductor, carry out product enzyme with methyl alcohol addition 0.5%, 1.0%, 1.5%, 2.0%, 2.5% respectively, utilize in 192h place DNS method to measure fermented supernatant fluid CMC enzyme and live, and measuring its cell concentration, optimum result is shown in Fig. 6.
2, by regulating potassium phosphate buffer pH in BMMY substratum 5.0,5.5,6.0,6.5,7.0,7.5 to carry out the optimization of products enzyme, utilize in 192h place DNS method to measure the work of fermented supernatant fluid CMC enzyme, and measure its cell concentration, optimum result is shown in Fig. 7.After optimization, Fig. 8 is shown in the contrast alive of front CMC enzyme with optimization.
Claims (7)
1. a codon optimized and pichia yeast expression system for 1,4-BETA-D-glucancellobio-hydrolase II gene, is characterized in that codon optimized as follows with the step of expression system construction:
1) codon bias optimization: the present invention utilizes software GeneDesigner to realize the CBHII nucleotide sequence codon bias optimization shown in SEQIDNO.1, and after optimizing, nucleotide sequence is as shown in SEQIDNO.3.
2) expression vector establishment: using plasmid pPIC9K as carrier, inserts promotor AOX1 downstream by the CBHII gene after optimizing, and 5 ' end restriction enzyme site is EcoRI, and 3 ' end restriction enzyme site is NotI, builds pPIC9K-cbh2 expression vector.
3) recombinant bacterial strain is obtained: using SacI as pPIC9K-cbh2 expression vector linearisation sites, realize expression vector linearizing, proceed to pichia spp by electricity.
4) optimize induction condition of enzyme production: using BMMY as culture medium, optimize potassium phosphate buffer pH value in inductor addition and substratum, realize CBHII stability and high efficiency and produce enzyme.
2. the codon optimized and pichia yeast expression system of a kind of 1,4-BETA-D-glucancellobio-hydrolase II gene according to claim 1, its step 1) in the CBHII of indication be by the Trichodermareesei 1,4-BETA-D-glucancellobio-hydrolase II gene such as shown in SEQIDNO.1 or SEQIDNO.3 coded by nucleotide sequence.
3. the codon optimized and pichia yeast expression system of a kind of 1,4-BETA-D-glucancellobio-hydrolase II gene according to claim 1, its step 1) the middle 1,4-BETA-D-glucancellobio-hydrolase II gene codon skewed popularity optimization described, after optimizing, CBHII nucleotide sequence is as shown in SEQIDNO.3.
4. the codon optimized and pichia yeast expression system of a kind of 1,4-BETA-D-glucancellobio-hydrolase II gene according to claim 1, its step 1) the middle 1,4-BETA-D-glucancellobio-hydrolase II gene described, its aminoacid sequence is as shown in SEQIDNO.2.
5. the codon optimized and pichia yeast expression system of a kind of 1,4-BETA-D-glucancellobio-hydrolase II gene according to claim 1, its step 2) the middle expression vector described is pPIC9K-cbh2 expression vector.
6. the codon optimized and pichia yeast expression system of a kind of 1,4-BETA-D-glucancellobio-hydrolase II gene according to claim 1, its step 3) the middle recombinant bacterial strain described, refer to the recombinant yeast pichia pastoris bacterium containing optimizing rear cbh2 gene.
7. the codon optimized and pichia yeast expression system of a kind of 1,4-BETA-D-glucancellobio-hydrolase II gene according to claim 1, its step 4) the middle recombinant pichia yeast strain described can stability and high efficiency product 1,4-BETA-D-glucancellobio-hydrolase II albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510801789.5A CN105441464A (en) | 2015-11-19 | 2015-11-19 | System for codon optimization and pichia pastoris expression of genes of cellobiohydrolase II |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510801789.5A CN105441464A (en) | 2015-11-19 | 2015-11-19 | System for codon optimization and pichia pastoris expression of genes of cellobiohydrolase II |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105441464A true CN105441464A (en) | 2016-03-30 |
Family
ID=55552095
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510801789.5A Pending CN105441464A (en) | 2015-11-19 | 2015-11-19 | System for codon optimization and pichia pastoris expression of genes of cellobiohydrolase II |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105441464A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434735A (en) * | 2016-10-12 | 2017-02-22 | 江南大学 | Method of increasing yield of lignocellulose substrate hydrolase |
| CN109609522A (en) * | 2018-11-27 | 2019-04-12 | 湖南美可达生物资源股份有限公司 | Macleaya cordata dihydrobenzo phenanthridines oxidase gene optimization and its application |
| CN109609521A (en) * | 2018-11-27 | 2019-04-12 | 湖南美可达生物资源股份有限公司 | Macleaya cordata protopine -6- hydroxylation enzyme gene optimization and its application |
| CN109797160A (en) * | 2018-09-26 | 2019-05-24 | 天津科技大学 | The construction method of heterogenous expression exoglucanase Cel6A, Cel7A in a kind of Pichia pastoris |
| CN110684704A (en) * | 2019-10-11 | 2020-01-14 | 天津科技大学 | Gene engineering strain of synechocystis PCC6803 for producing cellulase and construction method thereof |
| CN111850027A (en) * | 2020-06-12 | 2020-10-30 | 天津科技大学 | Pichia pastoris engineering strain for heterologous expression of cellulase gene CBH II and application |
| CN114517203A (en) * | 2022-03-18 | 2022-05-20 | 湖南美可达生物资源股份有限公司 | Macleaya cordata berberine bridge enzyme gene optimization sequence and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101624601A (en) * | 2009-07-30 | 2010-01-13 | 中国热带农业科学院热带生物技术研究所 | Method for regulating and expressing exoglucanase by pGAP of pichia pastoris |
| CN102719459A (en) * | 2012-05-02 | 2012-10-10 | 中国热带农业科学院热带生物技术研究所 | Production method of endo-1,4-beta-D-glucanase, exo-1,4-beta-D-glucanase and beta-glucosidase |
-
2015
- 2015-11-19 CN CN201510801789.5A patent/CN105441464A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101624601A (en) * | 2009-07-30 | 2010-01-13 | 中国热带农业科学院热带生物技术研究所 | Method for regulating and expressing exoglucanase by pGAP of pichia pastoris |
| CN102719459A (en) * | 2012-05-02 | 2012-10-10 | 中国热带农业科学院热带生物技术研究所 | Production method of endo-1,4-beta-D-glucanase, exo-1,4-beta-D-glucanase and beta-glucosidase |
Non-Patent Citations (4)
| Title |
|---|
| NASSAPAT BOONVITTHYA等: "Comparison of the heterologous expression of Trichoderma reesei endoglucanase Ⅱ and cellobiohydrolase Ⅱ in the yeasts Pichia pastoris and Yarrowia lipolytica", 《MOLECULAR BIOTECHNOLOGY》 * |
| 付玲: "毕赤酵母表达载体pPIC9K的优化", 《中国优秀硕士学位论文数据库基础科学辑》 * |
| 李国庆等: "黑曲霉外切葡聚糖酶基因密码子的优化及表达", 《南京林业大学学报》 * |
| 林国滟等: "（中性纤维素酶外切葡聚糖CBH II的异源表达", 《微生物学杂志》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434735A (en) * | 2016-10-12 | 2017-02-22 | 江南大学 | Method of increasing yield of lignocellulose substrate hydrolase |
| CN109797160A (en) * | 2018-09-26 | 2019-05-24 | 天津科技大学 | The construction method of heterogenous expression exoglucanase Cel6A, Cel7A in a kind of Pichia pastoris |
| CN109609522A (en) * | 2018-11-27 | 2019-04-12 | 湖南美可达生物资源股份有限公司 | Macleaya cordata dihydrobenzo phenanthridines oxidase gene optimization and its application |
| CN109609521A (en) * | 2018-11-27 | 2019-04-12 | 湖南美可达生物资源股份有限公司 | Macleaya cordata protopine -6- hydroxylation enzyme gene optimization and its application |
| CN109609522B (en) * | 2018-11-27 | 2020-10-09 | 湖南美可达生物资源股份有限公司 | Macleaya cordata dihydrobenzophenanthridine oxidase gene optimization sequence and application thereof |
| CN109609521B (en) * | 2018-11-27 | 2020-12-29 | 湖南美可达生物资源股份有限公司 | Optimized sequence of macleaya cordata protopine-6-hydroxylase gene and application thereof |
| CN110684704A (en) * | 2019-10-11 | 2020-01-14 | 天津科技大学 | Gene engineering strain of synechocystis PCC6803 for producing cellulase and construction method thereof |
| CN111850027A (en) * | 2020-06-12 | 2020-10-30 | 天津科技大学 | Pichia pastoris engineering strain for heterologous expression of cellulase gene CBH II and application |
| CN114517203A (en) * | 2022-03-18 | 2022-05-20 | 湖南美可达生物资源股份有限公司 | Macleaya cordata berberine bridge enzyme gene optimization sequence and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105441464A (en) | System for codon optimization and pichia pastoris expression of genes of cellobiohydrolase II | |
| Zhang et al. | Development of the cellulolytic fungus Trichoderma reesei strain with enhanced β-glucosidase and filter paper activity using strong artifical cellobiohydrolase 1 promoter | |
| Lee et al. | Co-fermentation using recombinant Saccharomyces cerevisiae yeast strains hyper-secreting different cellulases for the production of cellulosic bioethanol | |
| Li et al. | Effect of pH on cellulase production and morphology of Trichoderma reesei and the application in cellulosic material hydrolysis | |
| Chen et al. | Isolation and characterization of a β-glucosidase from Penicillium decumbens and improving hydrolysis of corncob residue by using it as cellulase supplementation | |
| Singhania et al. | An integrative process for bio-ethanol production employing SSF produced cellulase without extraction | |
| CN101827939B (en) | Enzymatic hydrolysis of lignocellulosic feedstocks using accessory enzymes | |
| Srivastava et al. | Application of cellulases in biofuels industries: an overview | |
| CN105420269A (en) | Construction method for co-expression hemoglobin VHb and cellulase protein in pichia pastoris | |
| EP2076594A1 (en) | Process for enzymatic hydrolysis of pretreated lignocellulosic feedstocks | |
| CN105802854B (en) | Cellulase high-yield strain and application thereof | |
| CN102686736A (en) | Methods for improving the efficiency of simultaneous saccharification and fermentation reactions | |
| Qi et al. | Coordinated β-glucosidase activity with the cellulosome is effective for enhanced lignocellulose saccharification | |
| Jain et al. | Functional expression of a thermostable endoglucanase from Thermoascus aurantiacus RCKK in Pichia pastoris X-33 and its characterization | |
| Sreeja-Raju et al. | Penicillium janthinellum NCIM1366 shows improved biomass hydrolysis and a larger number of CAZymes with higher induction levels over Trichoderma reesei RUT-C30 | |
| Valadares et al. | The secretome of two representative lignocellulose-decay basidiomycetes growing on sugarcane bagasse solid-state cultures | |
| Shinkawa et al. | Feasibility study of on-site solid-state enzyme production by Aspergillus oryzae | |
| Liu et al. | Cocktail production of an endo-β-xylanase and a β-glucosidase from Trichoderma reesei QM 9414 in Escherichia coli | |
| Todaka et al. | Heterologous expression and characterization of an endoglucanase from a symbiotic protist of the lower termite, Reticulitermes speratus | |
| Pirota et al. | Saccharification of biomass using whole solid‐state fermentation medium to avoid additional separation steps | |
| Liu et al. | Co-production of a whole cellulase system in Escherichia coli | |
| Rani et al. | Expression of novel glucose tolerant β-glucosidase on cell surface by Rhodotorula glutinis isolate | |
| Cavka et al. | Biorefining of wood: combined production of ethanol and xylanase from waste fiber sludge | |
| da Silva Delabona et al. | The impact of putative methyltransferase overexpression on the Trichoderma harzianum cellulolytic system for biomass conversion | |
| Cai et al. | Salt-tolerant mechanism of marine Aspergillus niger cellulase cocktail and improvement of its activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20160330 |
|
| WD01 | Invention patent application deemed withdrawn after publication |