CN102690340A - Recombined Brazil nut allergen and mutant and preparation method and applications thereof - Google Patents
Recombined Brazil nut allergen and mutant and preparation method and applications thereof Download PDFInfo
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- CN102690340A CN102690340A CN2012100893873A CN201210089387A CN102690340A CN 102690340 A CN102690340 A CN 102690340A CN 2012100893873 A CN2012100893873 A CN 2012100893873A CN 201210089387 A CN201210089387 A CN 201210089387A CN 102690340 A CN102690340 A CN 102690340A
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Abstract
The invention discloses a recombined Brazil nut allergen and mutant and a preparation method and applications thereof. The recombined Brazil nut allergen is a non-natural variant derived from a natural allergen, and the amino acid sequence is shown as SEQIDNO:1. By means of the genetic engineering technologies, coding genes of the recombined Brazil nut allergen is artificially synthesized after codons are optimized, antigenic epitopes of the recombined Brazil nut allergen are analyzed by bioinformatics software, and directional displacement is performed to one or multiple locus with high antigenicity by directed mutation to lower the allergenicity of the locus so as to build a recombined Brazil nut allergen mutant with low allergenicity. A high-purity recombined Brazil nut allergen and mutant protein of the same are obtained by performing protein expression under manual control. Compared with natural allergens, combining capacity of the recombined protein mutant with a specific IgE is lowered obviously, and the recombined Brazil nut allergen and mutant can be safely applicable to diagnosis and treatment of allergic diseases and even judging of the level of allergenicity of other proteins.
Description
Technical field
The present invention relates to the genetically engineered field, be specifically related to a kind of reorganization Bere1 and two mutants.
Background technology
Brazil's nut (Brazil nut) belongs to Wang Rui section Brazil nut and belongs to (Family Lecythidaceae), tropical evergreen fruit trees, megaphanerophyte.Brazil's nut kernel sulfur-containing amino acid, fat, Serlabo, vitamin substances content are very abundant.In recent years; The Absatzvolumen of Brazil's nut constantly rises, and has the huge market share in Europe and North America, and annual turnover is above 3,300,000,000 dollars; Also constantly increase in Chinese market; But it is a sensibiligen, compares other sensibiligens such as egg and milk, and the anaphylaxis that nut causes usually is permanent.Brazil's nut clinical symptom hypersensitive is serious, even if eat the Bere1 food of trace, also can cause the intensive anaphylaxis, in public health and food safety field, receives much concern.On the other hand, because the ubiquity of cross allergy and complicacy exist, there is serious cross reaction in the irritated allergy with other types of Brazilian nut, has also increased its irritated probability that takes place.
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 Bere1 of allergenicity; Can reduce the risk the therapeutic process from the source to a certain extent, can keep the BA of Bere1 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 Bere1 extracting solution; On the basis of clonal expression reorganization Bere1; Utilize the epitope of information biology software analysis Bere1; The high site of one or more antigenicities of suddenling change, thereby the Bere1 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 Bere1 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 Bere1 and two mutants thereof.
Another object of the present invention provides a kind of preparation method of Bere1 as medicine or diagnostic reagent that recombinate.
For realizing above-mentioned purpose, the present invention adopts following technical scheme:
Reorganization Bere1 of the present invention is the varient that naturally occurring Bere1 deutero-non-natural exists, its nucleotide sequence after codon optimized, the reorganization Bere1 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 Bere1 two mutants; Make it on the basis that keeps original BA and space structure, can reduce the allergenicity of reorganization Bere1 effectively.Utilize escherichia expression system then, under manually operated condition, obtain highly purified reorganization Bere1 and mutant protein thereof.Compare with natural Bere1, 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 Bere1, its aminoacid sequence is shown in SEQ ID NO:1.
The two mutants of above-mentioned reorganization Bere1; It is deutero-two mutants on the basis of two mutants of reorganization Bere1; 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 Bere1 of the present invention; It is deutero-two mutants on the basis of two mutants of reorganization Bere1; 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-B2SQ3, and in China's typical culture collection center preservation, deposit number is CCTCC M 2011152; Preservation date is on April 29th, 2011, and the preservation place is a China. Wuhan. and Wuhan University; Promptly obtain the described reorganization Bere1 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 Bere1, 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 Bere1 according to the invention comprises the steps:
(a) the dominant antigen epi-position of the aminoacid sequence of the said reorganization Bere1 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 Bere1 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 Bere1 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 Bere1 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 Bere1 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 Bere1 two mutants of low-allergen property.Be utilized in and carry out protein expression under the manually operated condition, obtain highly purified reorganization Bere1 and mutant protein thereof.Compare with natural Bere1, 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 is reorganization Bere1 induction expression of protein electrophorogram.Be respectively Marker among the figure from left to right, do not induce bacterium, 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 Bere1
(1) confirming of reorganization Bere1 nucleotide sequence: nucleotide sequence (the GenBank accession number: M17146.1) of from ncbi database, obtaining natural Bere1; 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 Ber e1 of the Bere1 that obtains recombinating.
(2) according to colibacillary codon preference; The nucleotide sequence of goal gene Ber e1 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 Ber e1 gene order after synthetic is optimized.
(4) behind double digestion, purpose fragment Ber e1 is cloned on the prokaryotic expression carrier pET, make up recombinant expression plasmid.
(5) recombinant expression plasmid pET-Ber e1 is transformed among the expressive host bacterium Rosetta.
(6) the expressive host bacterium Rosetta that selects to contain recombinant expression plasmid pET-Ber e1 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 Bere1s 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 Bere1.The analysis of comprehensive each software, the result shows that the antigen value in 1-15,18-42,70-95,98-116 zone in the reorganization Bere1 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 Bere1 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 Bere1 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 Bere1 and two mutants
<130> 2011
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 146
<212> PRT
< 213>artificial sequence
<400> 1
Met Ala Lys Ile Ser Val Ala Ala Ala Ala Leu Leu Val Leu Met Ala
1 5 10 15
Leu Gly His Ala Thr Ala Phe Arg Ala Thr Val Thr Thr Thr Val Val
20 25 30
Glu Glu Glu Asn Gln Glu Glu Cys Arg Glu Gln Met Gln Arg Gln Gln
35 40 45
Met Leu Ser His Cys Arg Met Tyr Met Arg Gln Gln Met Glu Glu Ser
50 55 60
Pro Tyr Gln Thr Met Pro Arg Arg Gly Met Glu Pro His Met Ser Glu
65 70 75 80
Cys Cys Glu Gln Leu Glu Gly Met Asp Glu Ser Cys Arg Cys Glu Gly
85 90 95
Leu Arg Met Met Met Met Arg Met Gln Gln Glu Glu Met Gln Pro Arg
100 105 110
Gly Glu Gln Met Arg Arg Met Met Arg Leu Ala Glu Asn Ile Pro Ser
115 120 125
Arg Cys Asn Leu Ser Pro Met Arg Cys Pro Met Gly Gly Ser Ile Ala
130 135 140
Gly Phe
145
Claims (8)
- One kind the reorganization Bere1; It is characterized in that it being the two mutants that the deutero-non-natural exists on the basis of reorganization Bere1; 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 Bere1 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 Bere1 of recombinating of claim 1; The gene of the said reorganization Bere1 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 Bere1 of claim 1 behind the protein expression.
- 4. according to the preparation method of the said a kind of Bere1 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-B2SQ3, and in China's typical culture collection center preservation, deposit number is CCTCC M 2011152; Preservation date is on April 29th, 2011, and the preservation place is a China. Wuhan. and Wuhan University; Promptly obtain the described reorganization Bere1 of claim 1 behind this expressive host expression and purification.
- 5. the preparation method of a kind of Bere1 of recombinating according to claim 3 is characterized in that comprising the steps:(1) from DB, obtain the nucleotide sequence of Bere1, 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 Bere1 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 Bere1 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 Bere1 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 described reorganization Bere1 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 Bere1 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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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115611970A (en) * | 2021-07-15 | 2023-01-17 | 青岛蔚蓝生物集团有限公司 | Brazilian sweet mutant and application thereof |
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| CN1869071A (en) * | 2006-04-13 | 2006-11-29 | 广州医学院第二附属医院 | Recombination immune therapeutic protein and its expression method and application |
| CN1332029C (en) * | 1998-03-16 | 2007-08-15 | 阿尔克-阿贝洛有限公司 | Mutant recombinant allergens |
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- 2012-03-30 CN CN2012100893873A patent/CN102690340A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1332029C (en) * | 1998-03-16 | 2007-08-15 | 阿尔克-阿贝洛有限公司 | Mutant recombinant allergens |
| CN1869071A (en) * | 2006-04-13 | 2006-11-29 | 广州医学院第二附属医院 | Recombination immune therapeutic protein and its expression method and application |
Non-Patent Citations (2)
| Title |
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| GANDER,E.S., ET AL.: "2S albumin[Bertholletia excelsa]，GenBank序列号：CAA38362.1", 《GENBANK数据库》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115611970A (en) * | 2021-07-15 | 2023-01-17 | 青岛蔚蓝生物集团有限公司 | Brazilian sweet mutant and application thereof |
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Application publication date: 20120926 |