CN102617719A - Recombinant rice allergen and mutant and preparation methods and applications thereof - Google Patents
Recombinant rice allergen and mutant and preparation methods and applications thereof Download PDFInfo
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- CN102617719A CN102617719A CN2012100896119A CN201210089611A CN102617719A CN 102617719 A CN102617719 A CN 102617719A CN 2012100896119 A CN2012100896119 A CN 2012100896119A CN 201210089611 A CN201210089611 A CN 201210089611A CN 102617719 A CN102617719 A CN 102617719A
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Abstract
The invention discloses a recombinant rice allergen and a mutant and preparation methods and applications thereof. The recombinant rice allergen is a non-natural variant derived from a natural allergen, and an amino acid sequence of the recombinant rice allergen is shown as SEQIDNO:1. The preparation method of the recombinant rice allergen comprises the following steps of: performing codon optimization and artificially synthesizing an encoding gene of a recombinant rice allergen by using a gene engineering technology; analyzing an epitope by adopting bioinformatics software; and performing oriented displacement specific to one or more sites with high antigenicity through a site-specific mutagenesis method to lower the allergenicity and construct a recombinant rice allergen with low allergenicity. Protein expression is performed under a manual control condition, so that a high-purity recombinant rice allergen and a mutant protein thereof are obtained. Compared with a natural rice allergen, a recombinant protein mutant has remarkably-lowered binding ability with a specific IgE (immunoglobulin E) and can be safely applied to diagnosis and treatment of allergic diseases and even judgment of the allergenicity levels of other proteins.
Description
Technical field
The present invention relates to the genetically engineered field, be specifically related to a kind of reorganization rice sensibiligen and two mutants.
Background technology
Rice contains abundant vitamin substances particularly vitamin B group and vitamin E and mineral substance, low sugar, is an outstanding glucide source.Most people in the world, the people that particularly live in south, Asia and east with rice as staple food.In recent years, allergic diseases obtains the attention of various circles of society, and rice allergy more and more receives publicity.In Japan, Thailand etc. is that the country of staple food also finds the irritated case of rice with the rice, can fall ill per capita to growing up from the infant.Rice clinical condition hypersensitive is divided into part and systemic reaction.Local reaction is wheal, itch, flush, blush companion papule, bubble etc.; Systemic reaction comprises urticaria, asthma, rhinitis, vomiting, angioedema and baby's enterocolitis syndromes etc., has had a strong impact on patient's Health and Living quality.
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 also has certain risk even adopt simple stdn sensibiligen to carry out 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 rice sensibiligen of allergenicity, can reduce the risk the therapeutic process from the source to a certain extent, can keep the BA of rice sensibiligen 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 natural rice sensibiligen extracting solution; On the basis of clonal expression reorganization rice sensibiligen; Utilize the epitope of information biology software analysis rice sensibiligen; The high site of one or more antigenicities of suddenling change, thereby the rice allergen 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 rice sensibiligen 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 rice sensibiligen and two mutants thereof.
Another object of the present invention provides a kind of preparation method of rice sensibiligen as medicine or diagnostic reagent that recombinate.
For realizing above-mentioned purpose, the present invention adopts following technical scheme:
Reorganization rice sensibiligen of the present invention is the varient that naturally occurring rice sensibiligen deutero-non-natural exists, its nucleotide sequence after codon optimized, the reorganization rice sensibiligen gene 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 rice allergen mutants; Make it on the basis that keeps original BA and space structure, can reduce the allergenicity of reorganization rice sensibiligen effectively.Utilize escherichia expression system then, under manually operated condition, obtain highly purified reorganization rice sensibiligen and mutant protein thereof.Compare with the natural rice 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 rice sensibiligen, its aminoacid sequence is shown in SEQ ID NO:1.
The two mutants of above-mentioned reorganization rice sensibiligen; It is deutero-two mutants on the basis of the two mutants of reorganization rice sensibiligen; 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 rice sensibiligen of the present invention; It is deutero-two mutants on the basis of the two mutants of reorganization rice sensibiligen; 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; This expression system name is called Escherichia coli JM109-RA17, and in China's typical culture collection center preservation, deposit number is CCTCC NO:M 2011149; Preservation date is on April 29th, 2011, and the preservation place is a China. Wuhan. and Wuhan University; Promptly obtain the described reorganization rice of claim 1 sensibiligen behind this expressive host expression and purification.
The most detailed preparing method's step is following:
(1) from DB, obtain the nucleotide sequence of rice sensibiligen, 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 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 rice sensibiligen according to the invention comprises the steps:
(a) the dominant antigen epi-position of the aminoacid sequence of the said reorganization rice of employing information biology software prediction claim 1 sensibiligen;
(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 rice allergen 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 rice sensibiligen 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 rice sensibiligen 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 rice sensibiligen 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 rice allergen mutants of low-allergen property.Be utilized in and carry out protein expression under the manually operated condition, obtain highly purified reorganization rice sensibiligen and mutant protein thereof.Compare with the natural rice 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
The recombinate abduction delivering electrophorogram of rice allergen protein of Fig. 1.Be respectively Marker among the figure from left to right; Do not induce thalline; Induce thalline.Marker is respectively from top to bottom: 97.4,66.2,45.0,31.0, and 21.0,14.4 kD.
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 rice sensibiligen
(1) confirming of reorganization rice sensibiligen nucleotide sequence: nucleotide sequence (the NCBI accession number: NM_001065720) of from ncbi database, obtaining natural rice sensibiligen RAG1; 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, EcoR I, Xho I, Pst I at sequence of N end and C end respectively then, the target gene fragment RA17 of the rice sensibiligen that obtains recombinating.
(2) according to colibacillary codon preference; The nucleotide sequence of goal gene RAG1 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 RAG1 gene order after synthetic is optimized.
(4) behind double digestion, purpose fragment RAG1 is cloned on the prokaryotic expression carrier pET, make up recombinant expression plasmid.
(5) recombinant expression plasmid pET-RAG1 is transformed among the expressive host bacterium Rosetta.
(6) the expressive host bacterium Rosetta that selects to contain recombinant expression plasmid pET-RAG1 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 (see figure 1) of SDS-PAGE testing goal.
(7) sonioation method cracking thalline obtains the inclusion body of target protein, and lysate is 15-100 mM Tris-Hcl, 50-300 mM NaCl, 0.05-1.0 mM EDTA.
(8) carry out protein renaturation with dialysis method after, at 4 ℃ of centrifugal 15-30 min down, collect supernatant with the rotating speed of 8000-30000rpm.
(9) supernatant that obtains after the renaturation obtains highly purified target protein through the affinity column of 6*HIS, STREPII or S albumen label.
Confirming of the Characterization of antigenic epitopes of embodiment 2 reorganization rice sensibiligens 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 rice sensibiligen.The analysis of comprehensive each software, the result shows that the antigen value in 1-23,35-62,123-150 zone in the reorganization rice sensibiligen 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 rice allergen 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 requirement of sudden change test kit, be template with the recombinant expression plasmid of reorganization rice sensibiligen, 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 rice sensibiligen and two mutants
<130> 2011
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 163
<212> PRT
< 213>artificial sequence
<400> 1
Met Ala Ser Asn Lys Val Val Phe Ser Val Leu Leu Leu Val Val Leu
1 5 10 15
Ser Val Leu Ala Ala Ala Met Ala Thr Met Ala Asp His His Gln Val
20 25 30
Tyr Ser Pro Gly Glu Gln Cys Arg Pro Gly Ile Ser Tyr Pro Thr Tyr
35 40 45
Ser Leu Pro Gln Cys Arg Thr Leu Val Arg Arg Gln Cys Val Gly Arg
50 55 60
Gly Ala Ala Ser Ala Ala Asp Glu Gln Val Trp Gln Asp Cys Cys Arg
65 70 75 80
Gln Leu Ala Ala Val Asp Asp Gly Trp Cys Arg Cys Gly Ala Leu Asp
85 90 95
His Met Leu Ser Gly Ile Tyr Arg Glu Leu Gly Ala Thr Glu Ala Gly
100 105 110
His Pro Met Ala Glu Val Phe Pro Gly Cys Arg Arg Gly Asp Leu Glu
115 120 125
Arg Ala Ala Ala Ser Leu Pro Ala Phe Cys Asn Val Asp Ile Pro Asn
130 135 140
Gly Pro Gly Gly Val Cys Tyr Trp Leu Gly Tyr Pro Arg Thr Pro Arg
145 150 155 160
Thr Gly His
Claims (8)
1. reorganization rice sensibiligen; It is characterized in that it being the two mutants that the deutero-non-natural exists on the basis of reorganization rice sensibiligen; 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 described a kind of rice sensibiligen of recombinating of claim 1 is characterized in that its aminoacid sequence is shown in SEQ ID NO:1.
3. the preparation method of the said a kind of rice sensibiligen of recombinating of claim 1; The gene of the said reorganization rice of claim 1 sensibiligen 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 rice of claim 1 sensibiligen behind the protein expression.
4. according to the preparation method of the said a kind of rice sensibiligen of recombinating of claim 3; It is characterized in that said expressive host is an escherichia expression system; This expression system name is called Escherichia coli JM109-RA17, and in China's typical culture collection center preservation, deposit number is CCTCC NO:M 2011149; Preservation date is on April 29th, 2011, and the preservation place is a China. Wuhan. and Wuhan University; Promptly obtain the described reorganization rice of claim 1 sensibiligen behind this expressive host expression and purification.
5. the preparation method of a kind of rice sensibiligen of recombinating according to claim 3 is characterized in that comprising the steps:
(1) from DB, obtain the nucleotide sequence of rice sensibiligen, 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 described a kind of rice sensibiligen of recombinating of claim 2 is characterized in that comprising the steps:
(a) the dominant antigen epi-position of the aminoacid sequence of the said reorganization rice of employing information biology software prediction claim 1 sensibiligen;
(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 rice allergen 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. claim 1-6 described reorganization rice sensibiligen or its two mutants be in the application of preparation in the medicine, and this medicine is used to treat allergic disorder and makes rice sensibiligen 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.
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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 |
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| 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 (3)
| Title |
|---|
| TANAKA,T等: "NP_001059184.1", 《NCBI REFERENCE SEQUENCE》 * |
| 任晓霞: "苦荞过敏原TBa的定点突变及免疫活性研究", 《农业科技辑》 * |
| 王文高等: "低过敏大米研究进展", 《粮食与油脂》 * |
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Application publication date: 20120801 |