CN102603879A - Recombinant soybean allergen, mutant and preparation methods and application of recombinant soybean allergen and mutant - Google Patents
Recombinant soybean allergen, mutant and preparation methods and application of recombinant soybean allergen and mutant Download PDFInfo
- Publication number
- CN102603879A CN102603879A CN2012100890786A CN201210089078A CN102603879A CN 102603879 A CN102603879 A CN 102603879A CN 2012100890786 A CN2012100890786 A CN 2012100890786A CN 201210089078 A CN201210089078 A CN 201210089078A CN 102603879 A CN102603879 A CN 102603879A
- Authority
- CN
- China
- Prior art keywords
- recombinant
- allergen
- reorganization
- original soybean
- mutant
- 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 recombinant soybean allergen, a mutant and preparation methods and application of the recombinant soybean allergen and the mutant. The amino acid sequence of the recombinant soybean allergen disclosed by the invention is shown as SEQ ID NO:1. According to the invention, by a genetic engineering technology, a coding gene of the recombinant soybean allergen is artificially synthesized after codon optimization is carried out; an antigenic epitope of the recombinant soybean allergen is analyzed by adopting bioinformatics software; and by a site-specific mutagenesis method, directional replacement is carried out for one or a plurality of sites with high antigenicity to reduce the allergen property of the recombinant soybean allergen so as to construct the recombinant soybean allergen mutant with low allergen property. The high-purity recombinant soybean allergen and a mutant protein thereof are obtained by carrying out protein expression under the condition of manual control. Compared with a natural soybean allergen, the combination capacity of the recombinant protein mutant with the specificity IgE is obviously reduced and the recombinant soybean allergen and the mutant can be safely applied to diagnosis and treatment of allergic diseases and judgment on the allergen property of other proteins.
Description
Technical field
The present invention relates to the genetically engineered field, be specifically related to a kind of reorganization original soybean sensitive and two mutants.
Background technology
Its protein-high of tatoin, high-energy, low-fiber characteristics and become the most frequently used in the world food and feedstuff raw material.Soybean sub product---dregs of beans is as a kind of outstanding plant feed protein source, and consumption generally can reach 25% ~ 30% in daily ration, is fine vegetable protein source.Soybean is one of main protein source of human and animal, but also is simultaneously one of 8 types of main sensitization foods of United Nations's regulation.Sunlover 10 has brought certain food safety problem for the responsive crowd of soybean in the widespread use of food-processing industry.Investigation finds that about 2% grownup and 6% ~ 8% children suffer from phagopyrism.Most food sensitinogens cause people's I type anaphylaxis; Understood untoward reaction at the soybean digestive process or after sucking powdered soybean; Mainly show as and have a stomach upset or allergic dermatitis; Cardinal symptom is that mouthful all erythema, lip swelling, oral cavity pain, glossopharyngeum are swollen, nausea and vomiting etc. but, threaten but generally do not constitute life.
At present, treatment anaphylactic disease main method has: 1) utilize antihistamine drug and the steroid medicine can relief of symptoms, but can not thoroughly eradicate and might bring spinoff.2) specific active immunotherapy is considered to the unique etiotropic treat-ment of anaphylactic disease, has obtained widespread consensus.And carry out specific active immunotherapy at present mainly is to adopt the sensibiligen vat liquor; Another problem is but outstanding unusually thus: because extract component is complicated; But also contain a large amount of heterogenetic antigens, and cause the stdn difficulty, had a strong impact on result of treatment and clinical application standard; Simultaneously, because the existence of allergenicity, even adopt simple stdn sensibiligen to carry out the also certain risk of immunotherapy.Adopt biological chemistry to separate and to obtain purer sensibiligen, but this method purifying process complicacy and yielding poorly will consume a large amount of manpower and financial resources, so its widespread use has received bigger restriction with purification technique.
Comparatively speaking; Utilize genetic engineering technique; Method through rite-directed mutagenesis obtains low-allergen property or does not have the two mutants of the reorganization original soybean sensitive of allergenicity, can reduce the risk the therapeutic process from the source to a certain extent, can keep the BA of original soybean sensitive simultaneously.In addition, the working condition of recombinant allergens is stable, and standardized program is simple, helps being widely used in clinical diagnosis and treatment.Therefore, reorganization reduction sensibiligen will be a kind of good alternative method, and vast potential for future development is arranged.
Summary of the invention
The objective of the invention is to the problem of above-mentioned existence and the deficiency of crude soya bean sensibiligen extracting solution; On the basis of clonal expression reorganization original soybean sensitive; Utilize the epitope of information biology software analysis original soybean sensitive; The high site of one or more antigenicities of suddenling change, thereby the original soybean sensitive two mutants of acquisition low-allergen property, expectation can be applied to the treatment of anaphylactic disease safely and efficiently.
Another object of the present invention provides a kind of gene of encode above-mentioned reorganization original soybean sensitive and two mutants thereof.
Another object of the present invention provides a kind of recombinant plasmid vector that contains said gene.
Another object of the present invention provides a kind of recombinant expressed host that said gene transforms of containing.
Another object of the present invention provides the preparation method of a kind of above-mentioned reorganization original soybean sensitive and two mutants thereof.
Another object of the present invention provides a kind of preparation method of original soybean sensitive as medicine or diagnostic reagent that recombinate.
For realizing above-mentioned purpose, the present invention adopts following technical scheme:
Reorganization original soybean sensitive of the present invention is the varient that naturally occurring original soybean sensitive deutero-non-natural exists, its nucleotide sequence after codon optimized, the reorganization soybean allergy protogene of synthetic total length.Adopt its epitope of information biology software analysis; Autotelic displacement is carried out in site to one or more antigenicity indexes are high; Obtain the gene of reorganization original soybean sensitive two mutants; Make it on the basis that keeps original BA and space structure, can reduce the allergenicity of reorganization original soybean sensitive effectively.Utilize escherichia expression system then, under manually operated condition, obtain highly purified reorganization original soybean sensitive and mutant protein thereof.Compare with the crude soya bean sensibiligen, the bonding properties of this recombinant protein two mutants and specific IgE obviously reduces, and can be applied to basic prevention of supersensitivity and treatment safely.
A kind of reorganization original soybean sensitive, its aminoacid sequence is shown in SEQ ID NO:1.
The two mutants of above-mentioned reorganization original soybean sensitive; It is deutero-two mutants on the basis of two mutants of reorganization original soybean sensitive; Wherein the B cell is or/and the conservative amino acid residues that at least one surface of t cell epitope exposes is replaced by absent variable another residue of the same position in any known homologous protein aminoacid sequence in the taxonomy order in naturally occurring sensibiligen source, and this two mutants has and the essentially identical alpha-carbon skeleton of the sensibiligen of natural appearance tertiary structure.
The recombinate preparation method of two mutants of original soybean sensitive of the present invention; It is deutero-two mutants on the basis of two mutants of reorganization original soybean sensitive; Wherein the B cell is or/and the conservative amino acid residues that at least one surface of t cell epitope exposes is replaced by absent variable another residue of the same position in any known homologous protein aminoacid sequence in the taxonomy order in naturally occurring sensibiligen source, and this two mutants has and the essentially identical alpha-carbon skeleton of the sensibiligen of natural appearance tertiary structure.
Further, said expressive host is an escherichia expression system, and this expression system name of this expression system is called
Escherichia coliJM109-GJR m4, in China's typical culture collection center preservation, deposit number is CCTCC M 2011156, and preservation date is on April 29th, 2011, and the preservation place is a China. Wuhan. Wuhan University; Promptly obtain the described reorganization original soybean sensitive of claim 1 behind this expressive host expression and purification.
The most detailed preparing method's step is following:
(1) from DB, obtain the nucleotide sequence of original soybean sensitive, according to the codon preference type of expressive host it is carried out codon optimizedly, optimize that GC content is 35 ~ 65% in the sequence of back, the original acid sequence is constant;
(2) nucleotide sequence after synthetic is optimized;
(3) behind double digestion, be connected with expression vector, be built into recombinant vectors;
(4) recombinant vectors is transformed in the expressive host, select positive conversion product;
(5) under 20 ~ 38 ℃, cultivate positive conversion product, use concentration is that the IPTG of 0.01 ~ 2.0mmol/L induces expression of recombinant proteins, inducing temperature is 4 ~ 38 ℃;
(6) the gained culture is behind the cracking host cell, and purifying obtains highly purified recombinant protein.
The preparation method of the two mutants of reorganization original soybean sensitive according to the invention comprises the steps:
(a) the dominant antigen epi-position of the aminoacid sequence of the said reorganization original soybean sensitive of employing information biology software prediction claim 1;
(b) adopt method of fixed points to change the high site of antigenicity, replace original amino-acid residue with the absent variable amino-acid residue of same position in any known homologous protein aminoacid sequence in the taxonomy order in naturally occurring sensibiligen source;
(c) gene fragment of acquisition original soybean sensitive two mutants after enzyme is cut, is connected with expression vector, is built into the mutant recombinant vectors;
(d), obtain highly purified recombination mutation albumen according to the method for abovementioned steps (4) ~ (6).
Reorganization original soybean sensitive according to the invention and two mutants thereof can be used to prepare medicine, and the medicine that makes is used to treat allergic disorder and makes original soybean sensitive and even other type patient hypersensitive are produced immunological tolerance; Perhaps be used for diagnosis of allergies property disease or judge the height of the allergen property of other food and medicine.
Compared with prior art, the present invention has following beneficial effect:
The present invention utilizes genetic engineering technique; The encoding sox of synthetic reorganization original soybean sensitive after codon optimized; And adopt its epitope of information biology software analysis, through the method for rite-directed mutagenesis, carry out the orientation displacement to the high site of one or more antigenicities; To reduce its allergen property, be built into the reorganization original soybean sensitive two mutants of low-allergen property.Be utilized in and carry out protein expression under the manually operated condition, obtain highly purified reorganization original soybean sensitive and mutant protein thereof.Compare with the crude soya bean sensibiligen, the binding ability of this recombinant protein two mutants and specific IgE obviously reduces, and can be applied to the Clinics and Practices of anaphylactic disease safely and even judge the height of other albumen allergenicity.
Description of drawings
Fig. 1 recombinate proteic expression of original soybean sensitive and purifying electrophorogram.Be respectively Marker among the figure from left to right; Induce thalline; The ultrasonication supernatant; Purifying protein.Marker is respectively from top to bottom: 97.4,66.2,45.0,31.0,21.0, and 14.4kD.
Embodiment
Come further to explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.
The cloning and expression of embodiment 1 reorganization original soybean sensitive
(1) confirming of reorganization original soybean sensitive nucleotide sequence: the nucleotide sequence (X60043) that from ncbi database, obtains crude soya bean sensibiligen Gly m4; Add affinity tag 6*HIS, STREPII or the S albumen that makes things convenient for purifying at the C of this sequence end; Add restriction enzyme sites such as Nde I, Eco RI, Xho I, Pst I at sequence of N end and C end respectively then, the target gene fragment Gly m4 of the original soybean sensitive that obtains recombinating.
(2) according to colibacillary codon preference; The nucleotide sequence of goal gene Gly m4 is carried out codon optimized, make that goal gene is more suitable in the host bacterium, expressing, optimizing back GC content is 35 ~ 65%; The original acid sequence is constant, shown in SEQ ID NO:1.
(3) the Gly m4 gene order after synthetic is optimized.
(4) behind double digestion, purpose fragment Gly m4 is cloned on the prokaryotic expression carrier pET, make up recombinant expression plasmid.
(5) recombinant expression plasmid pET-Gly m4 is transformed among the expressive host bacterium Rosetta.
(6) the expressive host bacterium Rosetta that selects to contain recombinant expression plasmid pET-Gly m4 in the LB substratum 30-37 ℃ cultivated 1-6 hour, adding concentration is 0.01 ~ 2.0 mmol/L IPTG abduction delivering 2-8 hour, inducing temperature is 4-38 ℃.After inducing, centrifugal collection thalline carries out the proteic expression amount of SDS-PAGE testing goal.
(7) sonioation method cracking thalline obtains target protein, and lysate is 15-100 mM Tris-Hcl, 50-300 mM NaCl, 0.05-1.0 mM EDTA.
(8) supernatant that obtains after the ultrasonication obtains highly purified target protein (see figure 1) through the affinity column of 6*HIS, STREPII or S albumen label.
Confirming of the Characterization of antigenic epitopes of embodiment 2 reorganization original soybean sensitives and mutational site
(1) the online software platform of utilization comprises SYFPEITHI, MHCPred, SYFPEITHI, BIMAS; The NetMHC II, MHC-THREAD, EpiPredict; HLA-DR4 binding, ProPred, RankPep; SVMHC, PREDEP, PREDICT etc. analyze the antigenic index of the aminoacid sequence of reorganization original soybean sensitive.The analysis of comprehensive each software, the result shows that the antigen value in 54-73,79-98,135-157 zone in the reorganization original soybean sensitive aminoacid sequence is higher.
(2) carry out amino-acid substitution to antigen value than higher one or more sites, with the reduction antigenicity.The improved aminoacid sequence of antigenicity is carried out the analysis of above-mentioned antigens property again, so repeatedly, filter out the significantly reduced mutational site of antigenicity.
The structure of embodiment 3 reorganization original soybean sensitive two mutants
(1) to the mutational site of confirming, according to the sudden change test kit method design two mutants primer is provided, Tm value general requirement is greater than 78 ℃, and primer length is more suitable between 25-45 base.
(2) according to the sudden change test kit requirement, with the reorganization original soybean sensitive recombinant expression plasmid be template, PCR suddenlys change.
(3) with behind the enzymic digestion PCR product that provides in the test kit, the transformed competence colibacillus cell, the picking positive colony, whether order-checking detects sudden change successful.
SEQUENCE LISTING
< 110>The First Affiliated Hospital of Kunming Medical School
< 120>a kind of reorganization original soybean sensitive and two mutants
<130> 2011
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 158
<212> PRT
< 213>artificial sequence
<400> 1
Met Gly Val Phe Thr Phe Glu Asp Glu Ile Asn Ser Pro Val Ala Pro
1 5 10 15
Ala Thr Leu Tyr Lys Ala Leu Val Thr Asp Ala Asp Asn Val Ile Pro
20 25 30
Lys Ala Leu Asp Ser Phe Lys Ser Val Glu Asn Val Glu Gly Asn Gly
35 40 45
Gly Pro Gly Thr Ile Lys Lys Ile Thr Phe Leu Glu Asp Gly Glu Thr
50 55 60
Lys Phe Val Leu His Lys Ile Glu Ser Ile Asp Glu Ala Asn Leu Gly
65 70 75 80
Tyr Ser Tyr Ser Val Val Gly Gly Ala Ala Leu Pro Asp Thr Ala Glu
85 90 95
Lys Ile Thr Phe Asp Ser Lys Leu Val Ala Gly Pro Asn Gly Gly Ser
100 105 110
Ala Gly Lys Leu Thr Val Lys Tyr Glu Thr Lys Gly Asp Ala Glu Pro
115 120 125
Asn Gln Asp Glu Leu Lys Thr Gly Lys Ala Lys Ala Asp Ala Leu Phe
130 135 140
Lys Ala Ile Glu Ala Tyr Leu Leu Ala His Pro Asp Tyr Asn
145 150 155
Claims (8)
- One kind the reorganization original soybean sensitive; It is characterized in that it being the two mutants that the deutero-non-natural exists on the basis of reorganization original soybean sensitive; Wherein the B cell is or/and the conservative amino acid residues that at least one surface of t cell epitope exposes is replaced by absent variable another residue of the same position in any known homologous protein aminoacid sequence in the taxonomy order in naturally occurring sensibiligen source, and this two mutants has and the essentially identical alpha-carbon skeleton of the sensibiligen of natural appearance tertiary structure.
- 2. the two mutants of the said reorganization original soybean sensitive of claim 1 is characterized in that its aminoacid sequence is shown in SEQ ID NO:1.
- 3. the preparation method of the said reorganization original soybean sensitive of claim 1; The gene of the said reorganization original soybean sensitive of claim 1 of it is characterized in that comprising the steps: encoding obtains through codon optimized according to the situation of corresponding expressive host; And with the acquisition recombinant vectors after; Transformed into escherichia coli, yeast or plant obtain recombinant host, obtain the described reorganization original soybean sensitive of claim 1 behind the protein expression.
- 4. according to the preparation method of the said reorganization original soybean sensitive of claim 3, it is characterized in that said expressive host is an escherichia expression system, this expression system name is called Escherichia coliJM109-GJR m4, in China's typical culture collection center preservation, deposit number is CCTCC M 2011156, and preservation date is on April 29th, 2011, and the preservation place is a China. Wuhan. Wuhan University; Promptly obtain the described reorganization original soybean sensitive of claim 1 behind this expressive host expression and purification.
- 5. according to the preparation method of the said reorganization original soybean sensitive of claim 3, it is characterized in that comprising the steps:(1) from DB, obtain the nucleotide sequence of original soybean sensitive, according to the codon preference of expressive host it is carried out codon optimizedly, optimize that GC content is 35 ~ 65% in the sequence of back, the original acid sequence is constant;(2) nucleotide sequence after synthetic is optimized;(3) behind double digestion, be connected with expression vector, be built into recombinant vectors;(4) recombinant vectors is transformed in the expressive host, select positive conversion product;(5) under 20 ~ 38 ℃, cultivate positive conversion product, use concentration is that the inductor of 0.01 ~ 2.0mmol/L is induced expression of recombinant proteins, inducing temperature is 4 ~ 38 ℃;(6) the gained culture is behind the cracking host cell, and purifying obtains highly purified recombinant protein.
- 6. the preparation method of the two mutants of the said reorganization original soybean sensitive of claim 2 is characterized in that comprising the steps:(a) the dominant antigen epi-position of the aminoacid sequence of the said reorganization original soybean sensitive of employing information biology software prediction claim 1;(b) adopt method of fixed points to change the high site of antigenicity, replace original amino-acid residue with the absent variable amino-acid residue of same position in any known homologous protein aminoacid sequence in the taxonomy order in naturally occurring sensibiligen source;(c) gene fragment of acquisition original soybean sensitive two mutants after enzyme is cut, is connected with expression vector, is built into the mutant recombinant vectors;(d), obtain highly purified recombination mutation albumen according to the method for the said step of claim 5 (4) ~ (6).
- 7. the said reorganization original soybean sensitive of the arbitrary claim of claim 1-6 or its two mutants be in the application of preparation in the medicine, and this medicine is used to treat allergic disorder and makes original soybean sensitive and even other type patient hypersensitive are produced immunological tolerance; Perhaps be used for diagnosis of allergies property disease or judge the height of the allergenicity of other food and medicine.
- 8. medicine described in the claim 7 or diagnostic reagent; It is characterized in that it comprises according to recombinant allergens any in the claim 1 ~ 6; Acceptable vehicle and/or carrier in optional and the medicinal or diagnostic reagent, and optional adjuvants and/or coupling agent combination.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100890786A CN102603879A (en) | 2012-03-30 | 2012-03-30 | Recombinant soybean allergen, mutant and preparation methods and application of recombinant soybean allergen and mutant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100890786A CN102603879A (en) | 2012-03-30 | 2012-03-30 | Recombinant soybean allergen, mutant and preparation methods and application of recombinant soybean allergen and mutant |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102603879A true CN102603879A (en) | 2012-07-25 |
Family
ID=46521700
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012100890786A Pending CN102603879A (en) | 2012-03-30 | 2012-03-30 | Recombinant soybean allergen, mutant and preparation methods and application of recombinant soybean allergen and mutant |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102603879A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112225815A (en) * | 2020-09-30 | 2021-01-15 | 四川携光生物技术有限公司 | Milk, wheat, peanut and soybean allergen fusion protein and construction method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293710A (en) * | 1998-03-16 | 2001-05-02 | 阿尔克-阿贝洛有限公司 | Mutant recombinant allergens |
-
2012
- 2012-03-30 CN CN2012100890786A patent/CN102603879A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293710A (en) * | 1998-03-16 | 2001-05-02 | 阿尔克-阿贝洛有限公司 | Mutant recombinant allergens |
Non-Patent Citations (2)
| Title |
|---|
| MITTAG,D.,ET AL: "NCBI Reference sequence:NP_001236038.1,stress-induced protein SAM22[Glycine max]", 《NCBI》, 28 November 2011 (2011-11-28) * |
| 方旭前等: "大豆过敏原与低过敏原种质创新", 《遗传HEREDITAS(BEIJING)》, vol. 28, no. 8, 31 December 2006 (2006-12-31), pages 1043 - 1050 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112225815A (en) * | 2020-09-30 | 2021-01-15 | 四川携光生物技术有限公司 | Milk, wheat, peanut and soybean allergen fusion protein and construction method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113621052B (en) | Recombinant I-type humanized collagen polypeptide and preparation method and application thereof | |
| Bredehorst et al. | What establishes a protein as an allergen? | |
| CN100577804C (en) | Novel mutant allergens | |
| CN104519896B (en) | For treating the nucleic acid of allergy | |
| CN104056251A (en) | Epitopes Related To Coeliac Disease | |
| CN101624422B (en) | Schistosoma japonicum recombinant multi-epitope antigens, method for expressing and purifying same and application thereof | |
| CN102584966A (en) | Recombinant peanut allergen and mutant and preparation method and application of recombinant peanut allergen and mutant | |
| EA028228B1 (en) | Endopeptidase composition hydrolyzing gluten oligopeptides, process for producing same and use | |
| CN103834623B (en) | People source urate oxidase with catalytic activity | |
| CN102295695B (en) | Recombinant human follicle stimulating hormone and preparation thereof | |
| JP7097434B2 (en) | Human FGF21 mutant, its production method, and its use | |
| Rahman et al. | Identification, characterization and epitope mapping of proteins encoded by putative allergenic napin genes from Brassica rapa | |
| CN106085934B (en) | Food-grade Nattokinase expresses bacterium | |
| CN102603879A (en) | Recombinant soybean allergen, mutant and preparation methods and application of recombinant soybean allergen and mutant | |
| CN102307894B (en) | A variant of mite allergen and uses thereof | |
| CN102617726A (en) | Recombinant horse allergen and mutant and preparation methods and applications thereof | |
| CN102690340A (en) | Recombined Brazil nut allergen and mutant and preparation method and applications thereof | |
| CN110585445A (en) | Application of mushroom serine protease inhibitor in preparing sobering-up and liver-protecting medicine and medicine for preventing and treating alcoholic liver injury | |
| CN105980562A (en) | Immunogenic compositions and vaccines derived from bacterial surface receptor proteins | |
| CN102617719A (en) | Recombinant rice allergen and mutant and preparation methods and applications thereof | |
| CN101580846A (en) | Human cytoglobin for preventing and curing cirrhosis and preparation method thereof | |
| CN102675439A (en) | Preparation method and application of recombination ryegrass pollen allergen and mutant thereof | |
| CN103059140B (en) | For treating the hypoallergenic chimeric protein belonging to common milfoil (Parietaria judaica) lipid transfer family of allergy | |
| CN103864939A (en) | mGM-CSF/beta hCG fusion protein, and preparation method and application thereof | |
| CA2843804C (en) | Hypoallergenic variants of mal d 1, the major allergen from malus domestica |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20120725 |