CN106520820A - Method for preparing pro-brain natriuretic peptide epitope by virtue of Bacillus brevis (B.brevis) - Google Patents
Method for preparing pro-brain natriuretic peptide epitope by virtue of Bacillus brevis (B.brevis) Download PDFInfo
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- CN106520820A CN106520820A CN201610916266.XA CN201610916266A CN106520820A CN 106520820 A CN106520820 A CN 106520820A CN 201610916266 A CN201610916266 A CN 201610916266A CN 106520820 A CN106520820 A CN 106520820A
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- 101800000407 Brain natriuretic peptide 32 Proteins 0.000 title claims abstract description 28
- 102400000667 Brain natriuretic peptide 32 Human genes 0.000 title claims abstract description 28
- 101800002247 Brain natriuretic peptide 45 Proteins 0.000 title claims abstract description 28
- 241000193764 Brevibacillus brevis Species 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 20
- HPNRHPKXQZSDFX-OAQDCNSJSA-N nesiritide Chemical compound C([C@H]1C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)CNC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CO)C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 HPNRHPKXQZSDFX-OAQDCNSJSA-N 0.000 title claims abstract description 12
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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Abstract
The invention discloses a method for preparing a pro-brain natriuretic peptide epitope by virtue of Bacillus brevis (B.brevis). According to the method, the nucleotide sequence of N-terminal pro-brain natriuretic peptide linear epitope (NT-BNP<12-21>) capable of being bound with a pro-brain natriuretic peptide antibody is replaced by the nucleotide sequence of a coding region FG of human fibronectin 3 (Fn3) to form a fusion gene of Fn3 and NT-BNP<12-21> linear epitope, the fusion gene is inserted into the downstream of a signal peptide P2 of shuttle plasmid pNCMO2 to construct recombinant plasmid pNCMO2-Fn3-NT-BNP<12-21>, and the recombinant plasmid is enabled to express the secretory fusion protein formed through fusion of the skeleton protein Fn3 and NT-BNP<12-21> linear epitope with high efficiency in B.brevis. The method disclosed by the invention is capable of simply and rapidly preparing the secretory fusion protein having the antigenicity equivalent to that of pro-brain natriuretic peptide with high efficiency and low cost.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of using high efficient expression secreted protein expression system preparation people source
Fibronectin (Fn3) shows the method for antigen plasma pro-brain natriuretic peptide levels N-terminal (NT-BNP) linear epitope.
Background technology
In ELISA detection kit preparation process, need corresponding antigen as standard items.Wherein, it is protein-based
Antigen occupies very big ratio.How proteinaceous antigen is efficiently produced, be a key problem in technology of such product.The present invention is adopted
Employment source fibronectin (Fn3) display protein matter class antigen linear epitope, trial set up one using bacillus brevis height
The general-purpose platform of effect production secretary protein class linear epitope.
NT-BNP is released to when cardiac muscle is upset or loses in the blood plasma of people, and the long half time in blood plasma is dense
Degree is high, more stable in vitro.Therefore, NT-BNP is widely used in examining for angiocardiopathy at present as standard detection antigen
In disconnected treatment and prognosis application.NT-BNP includes chemical synthesis, Escherichia coli as the source of standard detection antigen
(Escherichia coli, E.coli) restructuring, people's cell restructuring are separated with patients with heart failure blood plasma.Due to other several sources
It is relatively costly, or less stable, current main source is that Escherichia coli restructuring is obtained.But Recombinant protein expression melts
Hop protein, the protein expression of upstream need IPTG to induce, and the protein purification in downstream needs a series of step such as smudge cells, than
It is more complicated.
Bacillus brevis (Bacillus brevis, B.brevis) expression system is being capable of high efficient expression secreting type and excellent
The expression system of dystrophin matter, is particularly suited for producing secretary protein.The system has the following advantages：(1) it is not bright
Aobvious codon-bias, while expression product is also not easily formed inclusion body, albumen is processed and direct across after cell membrane
There is no aggregation in being discharged into culture medium, reclaim and purifying protein is relatively simple；(2) hardly to exocytosis protease,
Beneficial to the stability of destination protein；(3) for the secretary protein of eukaryotic origin, typically there is S -- S structure, according to the study its
The factor that can promote disulfide formation in albumen is there is in zymotic fluid, can promote the folding of foreign protein.
The present invention shows antigen plasma pro-brain natriuretic peptide levels linear list using bacillus brevis expression people source fibronectin (Fn3)
Position, can be directly secreted into the foreign protein of expression in nutrient solution by the approach, so as to greatly simplify downstream foreign protein
Isolate and purify process.Be capable of achieving using the shown antigen of bacillus brevis secreting, expressing fibronectin one or more
Epi-position, produces the antigen protein substitute with the high stable equivalent with antigen protein.
The content of the invention
The present invention provides a kind of using secreted protein expression system bacillus brevis Prepare restructuring people source Fn3-N- akrencephalon
Pro-BNP epitope (NT-BNP12-21) method, reduce production cost, realize that quick, large-scale separation is recombinated for people
N- akrencephalon pro-BNP epitopes.
The technical scheme is that：By the epitope nucleotides sequence column permutation people that can be combined with plasma pro-brain natriuretic peptide levels antibody
The coding FG region nucleotide sequences of fibronectin Fn3, then by above Fusion gene construction to shuttle vector pNCMO2On, profit
With bacillus brevis expression system efficiently expressing recombinant human source Fn3-N- akrencephalon pro-BNP epitope Fn3-BNP12-21.Specifically
Step is as follows：
1. people's fibronectin Fn3 shows NT-BNP12-21Linear epitope fusion protein expression vector builds：
(1) by NT-BNP12-21Linear epitope design introduces coding BNP linear epitopes in the FG areas of Fn3 in FG loop areas
Short peptide sequence, constitute people fibronectin Fn3 and NT-BNP12-21Linear epitope fusion Fn3-BNP12-21, synthesis
Fn3 shows NT-BNP12-21The genetic fragment of linear epitope, the separation for introducing 6 histidines of coding in 5 ' end of fusion are pure
Change label, be easy to isolate and purify；
(2) by above-mentioned fusion Fn3-BNP12-21Shuttle vector pNCMO is building up to by Nco I and I restriction enzyme sites of Xho2
P2 strong promoters downstream, formed fusion expression vector pNCMO2-Fn3-BNP12-21；
2. people's fibronectin Fn3 shows NT-BNP12-21The expression of linear epitope fusion protein：
(1) by fusion expression vector pNCMO2-Fn3-BNP12-21Convert into Escherichia coli, in amicillin resistance
Screening positive clone on LB flat boards, final concentration of 100 μ g/ml of the ampicillin in LB culture mediums, LB culture medium prescriptions are every
Contain 10 grams of tryptone, yeast extract 5g, sodium chloride 10g and 15g agar powder in rising culture medium；
(2) positive transformant identified is converted into bacillus brevis, is screened on the MT flat boards of neomycin resistance
Positive colony, in the final concentration of 10 μ g/ml of MT culture mediums, MT culture medium prescriptions are containing glucose in every liter of culture medium to neomycin
20g, multivalence peptone 10g, meat leaching powder 5g, yeast extract 20g, FeSO4·7H2O 10mg、MnSO4·4H2O 10mg、ZnSO4·
7H2O1mg, pH=7.2-7.4；
(3) positive transformant identified is seeded in MT the or 2SY fluid nutrient mediums containing neomycin resistance, it is new mould
The final concentration of 50 μ g/ml of plain liquid medium within, incubated overnight collect supernatant nutrient solution, using SDS-PAGE electrophoresis and
Western Blot method testing goal protein bands, 2SY culture medium prescriptions are 20g containing glucose, soya peptone in every liter of culture medium
40g, yeast extract 5g, CaCl2·2H2O 0.15g, pH=7.2-7.4；
3. fusion protein is isolated and purified：
Supernatant nutrient solution being collected, the fusion protein of His-Tag labels being carried with ni-sepharose purification, the albumen is recombination human source
Fn3 merges N- akrencephalon pro-BNP epitope BNP12-21Albumen.
The invention has the beneficial effects as follows：The expression vector that the present invention is adopted is for pNCMO2, the carrier be in B.brevis and
Shuttle plasmid between E.coli.In E.coli after construction expression plasmid, proceeding to B.brevis carries out protein expression.Host Strains
The P2 promoters of cell wall protein are used as pNCMO2Expression promoter, as P2 promoters do not work in E.coli,
Be conducive to the clone of genes of interest；But it is then exponentially other strong promoter in B.brevis, can efficiently expresses purpose
Albumen.
The present invention shows antigen plasma pro-brain natriuretic peptide levels linear list using bacillus brevis expression people source fibronectin (Fn3)
Position, can be directly secreted into the foreign protein of expression in nutrient solution by the approach, so as to greatly simplify downstream foreign protein
Isolate and purify process, so as to it is quick, simple, it is efficient preparation people restructuring N- akrencephalon pro-BNP epitopes.
The present invention is applicable not only to the preparation of secreting type people fibronectin-plasma pro-brain natriuretic peptide levels epitope, is also suitable
In the preparation of other recombinant proteins.Therefore, the present invention prepares people source Fn3-BNP extensive12-21Recombinant protein and other weights
There is high value of practical in the production of group polypeptide.
Description of the drawings
Fn3-BNP of the accompanying drawing 1 for the present invention12-21Gene structure figure.
PNCMO of the accompanying drawing 2 for the present invention2-Fn3-BNP12-21Fusion expression vector collection of illustrative plates.
Fn3-BNP of the accompanying drawing 3 for the present invention12-21The SDS-PAGE that recombinant protein is expressed in different culture media,
Wherein：Band 1,2,3 is positive transformant HB116-pNCMO2-Fn3-BNP12-21Bacterial strain cultivates 24h in MT culture mediums, centrifugation
Gained Supernatant samples, 4,5,6 is positive transformant HB116-pNCMO2-Fn3-BNP12-21Bacterial strain is cultivated in 2SY culture mediums
24h, centrifugation gained Supernatant samples, M is standard protein.
Fn3-BNP of the accompanying drawing 4 for the present invention12-21Recombinant protein Western Blot detect figure result figure.Wherein, band 1
For the Western Blot detection figure results in embodiment one, band 2 is Western Blot detection figure results in embodiment two.
Fn3-BNP of the accompanying drawing 5 for the present invention12-21Recombinant protein SDS-PAGE Jing after ni-sepharose purification, wherein, band
1st, 2,3 it is respectively the Fn3-BNP after the ni-sepharose purification obtained by embodiment one to three12-21Fusion protein, M are standard protein.
Restructuring Fn3-BNP of the accompanying drawing 6 for the present invention12-21Albumen and the anti-BNP in mouse source12-21The monoclonal of amino acid residue resists
Body 13G12 binding abilities are analyzed.
Specific embodiment
Below in conjunction with the accompanying drawings and specific embodiment the present invention is further illustrated, but have no effect on the present invention guarantor
Shield scope.
Embodiment one
1. people's fibronectin Fn3 shows NT-BNP12-21Linear epitope fusion protein expression vector builds：
(1) (EMBL logins number for pattern gene to adopt people's fibronectin type III domain protein (Fn3)
AJ320527), by NT-BNP12-21Linear epitope design introduces coding BNP epitopes in the FG areas of Fn3 in FG loop areas
Short peptide sequence (CTGGAAACCTCGGGCCTGCAGGAACAACGT encodes the 12-21 amino acid residue LETSGLQEQR of BNP),
Commission Nanjing Genscript Biotechnology Co., Ltd. synthesizes Fn3-BNP according to sequence table [1]12-21Gene, it is last in fusion 5 '
That holds introducing 6 histidines of coding isolates and purifies label, is easy to isolate and purify；The fusion structure of formation is shown in accompanying drawing 1；
(2) by above-mentioned fusion Fn3-BNP12-21Shuttle vector pNCMO is building up to by Nco I and I restriction enzyme sites of Xho2
P2 strong promoters downstream, formed fusion expression vector pNCMO2-Fn3-BNP12-21, its nucleotide sequence such as sequence table [2] institute
Show, Vector map is shown in accompanying drawing 2；
2. people's fibronectin Fn3 shows NT-BNP12-21The expression of linear epitope fusion protein：
(1) by fusion expression vector pNCMO2-Fn3-BNP12-21Felt according to precious bioengineering (Dalian) Co., Ltd JM109
It is transformed in JM109 coli strains by state cell specification, is trained in the LB containing ampicillin (100 μ g/ml) resistance
On foster base flat board, 37 DEG C of culture 24h.LB culture medium prescriptions：In every liter of culture medium containing 10 grams of tryptone, yeast extract 5g,
Sodium chloride 10g and agar powder 15g；
(2) picking positive colony is inoculated in the LB fluid nutrient mediums containing ampicillin (100 μ g/ml) resistance, 37 DEG C,
200rpm overnight incubations, extract plasmid, after Nco I and I double digestions of Xho are accredited as the positive, by recombinant plasmid pNCMO2-
Fn3-BNP12-21Illustrate according to the NTP methods in precious bioengineering (Dalian) Co., Ltd bacillus brevis expression system HB200, turn
Change into bacillus brevis expression system HB116, in ovobiocin resistance (10 μ g/ml) MT culture mediums, 30 DEG C of overnight incubations,
Plasmid is extracted, Nco I was learnt from else's experience and I double digestions of Xho is accredited as the transformant HB116-pNCMO of the positive2-Fn3-BNP12- 21 carry out
The expression of fusion protein.MT culture medium prescriptions：In every liter of culture medium, 20g containing glucose, multivalence peptone 10g, meat leaching powder 5g, yeast are carried
Take thing 20g, FeSO4·7H2O 10mg、MnSO4·4H2O 10mg、ZnSO4·7H2O 1mg, pH=7.2-7.4；
(3) by the positive transformant HB116-pNCMO for identifying2-Fn3-BNP12- 21 inoculations are to 5ml ovobiocins
In the MT culture mediums of resistance (50 μ g/ml), under the conditions of 200rpm, 30 DEG C, concussion and cultivate 24h.By bacterium solution centrifugal force it is
12000g, is centrifuged 10min, collects supernatant, adds SDS-PAGE sample-loading buffers, after thermal cracking, carries out SDS-PAGE electrophoresis,
As a result see accompanying drawing 3；
(4) go to the supernatant obtained in step 2 (3) wet on pvdf membrane, after confining liquid closing, add one anti-(1
: 2500 dilution, anti-BNP monoclonal antibodies, purchased from HyTest companies) be incubated at room temperature, two resist for horseradish enzyme mark goat-anti it is little
After mouse IgG (1: 4000 dilution) incubation cleaning, the luminous detections of Western-Blot are carried out, accompanying drawing 4 is as a result seen；
3. fusion protein is isolated and purified：
The supernatant that step 2 (3) is obtained is obtained into purpose fusion protein F n3-BNP after ni-sepharose purification12-21, pass through
SDS-PAGE electrophoresis carries out purity of protein detection, and SDS-PAGE is shown in accompanying drawing 5, determines immune serum using ELISA method
Potency.Fusion protein is coated with 96 hole elisa Plates Jing after stepwise dilution, after confining liquid closing, adds the production of HyTest companies
The anti-NT-proBNP in mouse source12-21Monoclonal antibody 13G12 (1 of amino acid residue:2500 dilutions), make empty with seralbumin
It is white to compare.Two resist for HRPO mark goat anti-mouse igg antibody (1: 4000 dilution), and substrate is TMB, and develop the color 10min
Afterwards with the sulfuric acid terminating reaction of 2M.Determine the OD values at wavelength 450nm.As shown in Figure 6, as a result fusion protein F n3-BNP12-21
With good BNP anti-with mouse source12-21The monoclonal antibody 13G12 binding ability of amino acid residue.
Embodiment two
1. people's fibronectin Fn3 shows NT-BNP12-21Linear epitope fusion protein expression vector builds：
(1) (EMBL logins number for pattern gene to adopt people's fibronectin type III domain protein (Fn3)
AJ320527), by NT-BNP12-21Linear epitope design introduces coding BNP epitopes in the FG areas of Fn3 in FG loop areas
Short peptide sequence (CTGGAAACCTCGGGCCTGCAGGAACAACGT encodes the 12-21 amino acid residue LETSGLQEQR of BNP),
Commission Nanjing Genscript Biotechnology Co., Ltd. synthesizes Fn3-BNP according to sequence table [1]12-21Gene, it is last in fusion 5 '
That holds introducing 6 histidines of coding isolates and purifies label, is easy to isolate and purify；The fusion structure of formation is shown in accompanying drawing 1；
(2) by above-mentioned fusion Fn3-BNP12-21Shuttle vector pNCMO is building up to by Nco I and I restriction enzyme sites of Xho2
P2 strong promoters downstream, formed fusion expression vector pNCMO2-Fn3-BNP12-21, its nucleotide sequence such as sequence table [2] institute
Show, Vector map is shown in accompanying drawing 2；
2. people's fibronectin Fn3 shows NT-BNP12-21The expression of linear epitope fusion protein：
(1) by fusion expression vector pNCMO2-Fn3-BNP12-21Felt according to precious bioengineering (Dalian) Co., Ltd JM109
It is transformed in JM109 coli strains by state cell specification, is trained in the LB containing ampicillin (100 μ g/ml) resistance
On foster base flat board, 37 DEG C of culture 24h.LB culture medium prescriptions：In every liter of culture medium containing 10 grams of tryptone, yeast extract 5g,
Sodium chloride 10g and agar powder 15g；
(2) picking positive colony is inoculated in the LB fluid nutrient mediums containing ampicillin (100 μ g/ml) resistance, 37 DEG C,
200rpm overnight incubations, extract plasmid, after Nco I and I double digestions of Xho are accredited as the positive, by recombinant plasmid pNCMO2-
Fn3-BNP12-21Illustrate according to the NTP methods in precious bioengineering (Dalian) Co., Ltd bacillus brevis expression system HB200, turn
Change into bacillus brevis expression system HB116, in ovobiocin resistance (10 μ g/ml) MT culture mediums, 30 DEG C of overnight incubations,
Plasmid is extracted, Nco I was learnt from else's experience and I double digestions of Xho is accredited as the transformant HB116-pNCMO of the positive2-Fn3-BNP12-21Carry out
The expression of fusion protein.MT culture medium prescriptions：In every liter of culture medium, 20g containing glucose, multivalence peptone 10g, meat leaching powder 5g, yeast are carried
Take thing 20g, FeSO4·7H2O 10mg、MnSO4·4H2O 10mg、ZnSO4·7H2O 1mg, pH=7.2-7.4；
(3) by the positive transformant HB116-pNCMO for identifying2-Fn3-BNP12-21Inoculation is anti-to 5ml ovobiocins
In the 2SY culture mediums of property (50 μ g/ml), under the conditions of 200rpm, 30 DEG C, concussion and cultivate 24h.By bacterium solution centrifugal force it is
12000g, is centrifuged 10min, collects supernatant, adds SDS-PAGE sample-loading buffers, after thermal cracking, carries out SDS-PAGE electrophoresis,
As a result see accompanying drawing 3.2SY culture medium prescriptions are as follows：20g containing glucose in every liter of culture medium, soya peptone 40g, yeast extract 5g,
CaCl2·2H2O 0.15g, pH=7.2-7.4；
(4) go to the supernatant obtained in step 2 (3) wet on pvdf membrane, after confining liquid closing, add one anti-(1
: 3000 dilution, anti-BNP monoclonal antibodies, purchased from HyTest companies) be incubated at room temperature, two resist for horseradish enzyme mark goat-anti it is little
After mouse IgG (1: 4000 dilution) incubation cleaning, the luminous detections of Western-Blot are carried out, accompanying drawing 4 is as a result seen；
3. fusion protein is isolated and purified：
The supernatant that step 2 (3) is obtained is obtained into purpose fusion protein F n3-BNP after ni-sepharose purification12-21, pass through
SDS-PAGE electrophoresis carries out purity of protein detection, and SDS-PAGE is shown in accompanying drawing 5, determines immune serum using ELISA method
Potency.Fusion protein is coated with 96 hole elisa Plates Jing after stepwise dilution, after confining liquid closing, adds the production of HyTest companies
The anti-NT-proBNP in mouse source12-21Monoclonal antibody 13G12 (1 of amino acid residue:3000 dilutions), make empty with seralbumin
It is white to compare.Two resist for HRPO mark goat anti-mouse igg antibody (1: 4000 dilution), and substrate is TMB, and develop the color 10min
Afterwards with the sulfuric acid terminating reaction of 2M.Determine the OD values at wavelength 450nm.As shown in Figure 6, as a result fusion protein F n3-BNP12-21
With good BNP anti-with mouse source12-21The monoclonal antibody 13G12 binding ability of amino acid residue.
Embodiment three
1. people's fibronectin Fn3 shows NT-BNP12-21Linear epitope fusion protein expression vector builds：
(1) (EMBL logins number for pattern gene to adopt people's fibronectin type III domain protein (Fn3)
AJ320527), by NT-BNP12-21Linear epitope design introduces coding BNP epitopes in the FG areas of Fn3 in FG loop areas
Short peptide sequence (CTGGAAACCTCGGGCCTGCAGGAACAACGT encodes the 12-21 amino acid residue LETSGLQEQR of BNP),
Commission Nanjing Genscript Biotechnology Co., Ltd. synthesizes Fn3-BNP according to sequence table [1]12-21Gene, it is last in fusion 5 '
That holds introducing 6 histidines of coding isolates and purifies label, is easy to isolate and purify；The fusion structure of formation is shown in accompanying drawing 1；
(2) by above-mentioned fusion Fn3-BNP12-21Shuttle vector pNCMO is building up to by Nco I and I restriction enzyme sites of Xho2
P2 strong promoters downstream, formed fusion expression vector pNCMO2-Fn3-BNP12-21, its nucleotide sequence such as sequence table [2] institute
Show, Vector map is shown in accompanying drawing 2；
2. people's fibronectin Fn3 shows NT-BNP12-21The expression of linear epitope fusion protein：
(1) by fusion expression vector pNCMO2-Fn3-BNP12-21Felt according to precious bioengineering (Dalian) Co., Ltd JM109
It is transformed in JM109 coli strains by state cell specification, is trained in the LB containing ampicillin (100 μ g/ml) resistance
On foster base flat board, 37 DEG C of culture 24h.LB culture medium prescriptions：In every liter of culture medium containing 10 grams of tryptone, yeast extract 5g,
Sodium chloride 10g and agar powder 15g；
(2) picking positive colony is inoculated in the LB fluid nutrient mediums containing ampicillin (100 μ g/ml) resistance, 37 DEG C,
200rpm overnight incubations, extract plasmid, after Nco I and I double digestions of Xho are accredited as the positive, by recombinant plasmid pNCMO2-
Fn3-BNP12-21Illustrate according to the NTP methods in precious bioengineering (Dalian) Co., Ltd bacillus brevis expression system HB200, turn
Change into bacillus brevis expression system HB116, in ovobiocin resistance (10 μ g/ml) MT culture mediums, 30 DEG C of overnight incubations,
Plasmid is extracted, Nco I was learnt from else's experience and I double digestions of Xho is accredited as the transformant HB116-pNCMO of the positive2-Fn3-BNP12-21Carry out
The expression of fusion protein.MT culture medium prescriptions：In every liter of culture medium, 20g containing glucose, multivalence peptone 10g, meat leaching powder 5g, yeast are carried
Take thing 20g, FeSO4·7H2O 10mg、MnSO4·4H2O 10mg、ZnSO4·7H2O 1mg, pH=7.2-7.4；
(3) by the positive transformant HB116-pNCMO for identifying2-Fn3-BNP12-21Inoculation is anti-to 5ml ovobiocins
In the 2SY culture mediums of property (50 μ g/ml), under the conditions of 200rpm, 30 DEG C, concussion and cultivate 48h.By bacterium solution centrifugal force it is
10000g, is centrifuged 10min, collects supernatant, adds SDS-PAGE sample-loading buffers, after thermal cracking, carries out SDS-PAGE electrophoresis,
As a result see accompanying drawing 3.2SY culture medium prescriptions are as follows：20g containing glucose in every liter of culture medium, soya peptone 40g, yeast extract 5g,
CaCl2·2H2O 0.15g, pH=7.2-7.4；
(4) go to the supernatant obtained in step 2 (3) wet on pvdf membrane, after confining liquid closing, add one anti-(1
: 2500 dilution, anti-BNP monoclonal antibodies, purchased from HyTest companies) be incubated at room temperature, two resist for horseradish enzyme mark goat-anti it is little
After mouse IgG (1: 5000 dilution) incubation cleaning, the luminous detections of Western-Blot are carried out, accompanying drawing 4 is as a result seen；
3. fusion protein is isolated and purified：
The supernatant that step 2 (3) is obtained is obtained into purpose fusion protein F n3-BNP after ni-sepharose purification12-21, pass through
SDS-PAGE electrophoresis carries out purity of protein detection, and SDS-PAGE is shown in accompanying drawing 5, determines immune serum using ELISA method
Potency.Fusion protein is coated with 96 hole elisa Plates Jing after stepwise dilution, after confining liquid closing, adds the production of HyTest companies
The anti-NT-proBNP in mouse source12-21Monoclonal antibody 13G12 (1 of amino acid residue:2500 dilutions), make empty with seralbumin
It is white to compare.Two resist for HRPO mark goat anti-mouse igg antibody (1: 5000 dilution), and substrate is TMB, and develop the color 10min
Afterwards with the sulfuric acid terminating reaction of 2M.Determine the OD values at wavelength 450nm.As shown in Figure 6, as a result fusion protein F n3-BNP12-21
With good BNP anti-with mouse source12-21The monoclonal antibody 13G12 binding ability of amino acid residue.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any those familiar with the art in the technical scope of present disclosure, according to the ordinary technical knowledge of this area
And universal method, technology according to the present invention scheme and its inventive concept equivalent or change in addition, all should cover at this
Within bright protection domain.
<110>University Of Dalian
<120>A kind of method that utilization bacillus brevis prepares plasma pro-brain natriuretic peptide levels epitope
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 336
<212> DNA
<213>It is artificial synthesized
<400> 1
gctcaggttt ctgatgttcc gcgtgacctg gaagttgttg ctgcgacccc gactagcctg 60
ctgatcagct gggatgctcc tgcagttacc gtgcgttatt accgtatcac gtacggtgaa 120
accggtggta actccccggt tcaggaattc actgtacctg gttccaagtc tactgctacc 180
atcagcggcc tgaaaccggg tgtcgactat accatcactg tatacgctgt tactggcgaa 240
acctcgggcc tgcaggaaca atccccaatc tcgattaact accgtaccgg tggcagcggt 300
ggtcatcatc atcatcatca cggtaaaaaa ggtaaa 336
<210> 2
<211> 5530
<212> DNA
<213>It is artificial synthesized
<400> 2
tgatccgaca taatggacag gtgaataacg aaccacgaaa aaaactttaa atttttttcg 60
aaggcgccgc aacttttgat tcgctcaggc gtttaatagg atgtaattgt gagcggataa 120
caattattct gcatggcttt cctgcgaaag gaggtgacac gcgcttgcag gattcgggct 180
ttaaaaagaa agatagatta acaacaaata ttccccaaga acaatttgtt tatactagag 240
gaggagaaca caaggtcatg aaaaaaagaa gggtcgttaa cagtgtattg cttctgctac 300
tgctagctag tgcactcgca cttactgttg ctcccatggc tttcgctgct caggtttctg 360
atgttccgcg tgacctggaa gttgttgctg cgaccccgac tagcctgctg atcagctggg 420
atgctcctgc agttaccgtg cgttattacc gtatcacgta cggtgaaacc ggtggtaact 480
ccccggttca ggaattcact gtacctggtt ccaagtctac tgctaccatc agcggcctga 540
aaccgggtgt cgactatacc atcactgtat acgctgttac tggcgaaacc tcgggcctgc 600
aggaacaatc cccaatctcg attaactacc gtaccggtgg cagcggtggt catcatcatc 660
atcatcacgg taaaaaaggt aaatagtaag cttggtggtg gtggttcact cgagcggcat 720
tatagtgcgg aggctttttc gcatgcaggt agggaacaat tacattgtct ttgattgtaa 780
aaatgctgtt gacaggacac taaatggtgt cgtattctca aagtaacacc atttggtgtc 840
caattgcaag tcatttggta accttaattg gatcagacaa ggtaaaggat aaaacagcac 900
aattccaaga aaaacacgat ttagaaccta aaaagaacga atttgaacta actcataacc 960
gagaggtaaa aaaagaacga agtcgagatc agggaatgag tttataaaat aaaaaaagca 1020
cctgaaaagg tgtctttttt tgatggtttt gaacttgttc tttcttatct tgatacatat 1080
agaaataacg tcatttttat tttagttgct gaaaggtgcg ttgaagtgtt ggtatgtatg 1140
tgttttaaag tattgaaaac ccttaaaatt ggttgcacag aaaaacccca tctgttaaag 1200
ttataagtga ctaaacaaat aactaaatag atgggggttt cttttaatat tatgtgtcct 1260
aatagtagca tttattcaga tgaaaaatca agggttttag tggacaagac aaaaagtgga 1320
aaagtgagac cttggagaga aaagaaaatc gctaatgttg attactttga acttctgcat 1380
attcttgaat ttaaaaaggc tgaaagagta aaagattgtg ctgaaatatt agagtataaa 1440
caaaatcgtg aaacaggcga aagaaagttg tatcgagtgt ggttttgtaa atccaggctt 1500
tgtccaatgt gcaactggag gagagcaatg aaacatggca ttcagtcaca aaaggttgtt 1560
gctgaagtta ttaaacaaaa gccaacagtt cgttggttgt ttctcacatt aacagttaaa 1620
aatgtttatg atggcgaaga attaaataag agtttgtcag atatggctca aggatttcgc 1680
cgaatgatgc aatataaaaa aattaataaa aatcttgttg gttttatgcg tgcaacggaa 1740
gtgacaataa ataataaaga taattcttat aatcagcaca tgcatgtatt ggtatgtgtg 1800
gaaccaactt attttaagaa tacagaaaac tacgtgaatc aaaaacaatg gattcaattt 1860
tggaaaaagg caatgaaatt agactatgat ccaaatgtaa aagttcaaat gattcgaccg 1920
aaaaataaat ataaatcgga tatacaatcg gcaattgacg aaactgcaaa atatcctgta 1980
aaggatacgg attttatgac cgatgatgaa gaaaagaatt tgaaacgttt gtctgatttg 2040
gaggaaggtt tacaccgtaa aaggttaatc tcctatggtg gtttgttaaa agaaatacat 2100
aaaaaattaa accttgatga cacagaagaa ggcgatttga ttcatacaga tgatgacgaa 2160
aaagccgatg aagatggatt ttctattatt gcaatgtgga attgggaacg gaaaaattat 2220
tttattaaag agtagttcaa caaacgggcc agtttgttga agattagatg ctataattgt 2280
tattaaaagg attgaaggat gcttaggaag acgagttatt aatagctgaa taagaacggt 2340
gctctccaaa tattcttatt tagaaaagca aatctaaaat tatctgaaaa gggaatgaga 2400
atagtgaatg gaccaataat aatgactaga gaagaaagaa tgaagattgt ccatgaaatt 2460
aaggaacgaa tattggataa atatggggat gatgttaagg ctattggtgt ttatggctct 2520
cttggtcgtc agactgatgg gccctattcg gatattgaga tgatgtgtgt catgtcaaca 2580
gaggaagcag agttcagcca tgaatggaca accggtgagt ggaaggtgga agtgaatttt 2640
gatagcgaag agattctact agattatgca tctcaggtgg aatcagattg gccgcttaca 2700
catggtcaat ttttctctat tttgccgatt tatgattcag gtggatactt agagaaagtg 2760
tatcaaactg ctaaatcggt agaagcccaa acgttccacg atgcgatttg tgcccttatc 2820
gtagaagagc tgtttgaata tgcaggcaaa tggcgtaata ttcgtgtgca aggaccgaca 2880
acatttctac catccttgac tgtacaggta gcaatggcag gtgccatgtt gattggtctg 2940
catcatcgca tctgttatac gacgagcgct tcggtcttaa ctgaagcagt taagcaatca 3000
gatcttcctt caggttatga ccatctgtgc cagttcgtaa tgtctggtca actttccgac 3060
tctgagaaac ttctggaatc gctagagaat ttctggaatg ggattcagga gtggacagaa 3120
cgacacggat atatagtgga tgtgtcaaaa cgcataccat tttgaacgat gacctctaat 3180
aattgttaat catgttggtt acgtatttat taacttctcc tagtattagt aattatcatg 3240
gctgtcatgg cgcattaacg gaataaaggg tgtgcttaaa tcgggcctgc atgacgaaag 3300
ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg 3360
tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 3420
cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 3480
aaaaggaaga gtatgagtat tcaacatttc cgtgtccccc ttattccctt ttttgcggca 3540
ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 3600
cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 3660
agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 3720
gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 3780
cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 3840
gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 3900
ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 3960
gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 4020
gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 4080
cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 4140
ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 4200
gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 4260
gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 4320
gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 4380
ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 4440
gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 4500
gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 4560
caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 4620
ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 4680
tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 4740
ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 4800
tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 4860
cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 4920
gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 4980
ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 5040
gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 5100
agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcca 5160
tgcaggctga ataaaagata cgagagacct ctcttgtatc ttttttattt tgagtggttt 5220
tgtccgttac actagaaaac cgaaagacaa taaaaatttt attcttgctg agtctggctt 5280
tcggtaagct agacaaaacg gacaaaataa aaattggcaa gggtttaaag gtggagattt 5340
tttgagtgat cttctcaaaa aatactacct gtcccttgct gatttttaaa cgagcacgag 5400
agcaaaaccc ccctttgctg aggtggcaga gggcaggttt ttttgtttct tttttctcgt 5460
aaaaaaaaga aaggtcttaa aggttttatg gttttggtcg gcactgccga cagcctcgca 5520
gagcacacac 5530
Claims (1)
1. a kind of method that utilization bacillus brevis prepares plasma pro-brain natriuretic peptide levels epitope, it is characterised in that comprise the following steps：
1), people's fibronectin Fn3 shows NT-BNP12-21Linear epitope fusion protein expression vector builds：
(1) by NT-BNP12-21Linear epitope design introduces the short of coding BNP linear epitopes in the FG areas of Fn3 in FG loop areas
Peptide sequence, constitutes people fibronectin Fn3 and NT-BNP12-21Linear epitope fusion Fn3-BNP12-21, synthesize Fn3 exhibitions
Show NT-BNP12-21The genetic fragment of linear epitope, introduces 6 histidines of coding in fusion 5 ' end and isolates and purifies mark
Sign, be easy to isolate and purify；
(2) by above-mentioned fusion Fn3-BNP12-21Shuttle vector pNCMO is building up to by Nco I and I restriction enzyme sites of Xho2P2
Strong promoter downstream, forms fusion expression vector pNCMO2-Fn3-BNP12-21；
2), people's fibronectin Fn3 shows NT-BNP12-21The expression of linear epitope fusion protein：
(1) by fusion expression vector pNCMO2-Fn3-BNP12-21Convert into Escherichia coli, put down in the LB of amicillin resistance
Screening positive clone on plate, final concentration of 100 μ g/ml of the ampicillin in LB culture mediums, LB culture medium prescriptions are per liter of training
Contain 10 grams of tryptone, yeast extract 5g, sodium chloride 10g and 15g agar powder in foster base；
(2) positive transformant identified is converted into bacillus brevis, screens positive on the MT flat boards of neomycin resistance
Clone, in the final concentration of 10 μ g/ml of MT culture mediums, MT culture medium prescriptions are 20g containing glucose in every liter of culture medium, many to neomycin
Valency peptone 10g, meat leaching powder 5g, yeast extract 20g, FeSO4·7H2O 10mg、MnSO4·4H2O 10mg、ZnSO4·
7H2O1mg, pH=7.2-7.4；
(3) positive transformant identified is seeded in containing in neomycin MT or 2SY fluid nutrient medium, neomycin is trained in liquid
The final concentration of 50 μ g/ml of foster base, incubated overnight collect supernatant nutrient solution, using SDS-PAGE electrophoresis and Western Blot methods
Testing goal protein band, 2SY culture medium prescriptions are 20g containing glucose, soya peptone 40g, yeast extract in every liter of culture medium
5g、CaCl2·2H2O 0.15g, pH=7.2-7.4；
3), fusion protein is isolated and purified：
Supernatant nutrient solution is collected, with fusion protein of the ni-sepharose purification with His-Tag labels, the albumen as recombination human source Fn3
Fusion N- akrencephalon pro-BNP epitope BNP12-21Albumen.
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|---|---|---|---|
| CN201610916266.XA CN106520820B (en) | 2016-10-20 | 2016-10-20 | A method of plasma pro-brain natriuretic peptide levels epitope is prepared using bacillus brevis |
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| CN108179152A (en) * | 2017-12-29 | 2018-06-19 | 大连大学 | A kind of method that antigen substitute is prepared based on the dual anti-former epitope of III type structure domain views of people's fibronectin |
| CN115044601A (en) * | 2022-05-23 | 2022-09-13 | 大连大学 | Electrotransformation method for transferring pNCMO2 plasmid into brevibacillus brevis HB200 |
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| CN108179152A (en) * | 2017-12-29 | 2018-06-19 | 大连大学 | A kind of method that antigen substitute is prepared based on the dual anti-former epitope of III type structure domain views of people's fibronectin |
| CN115044601A (en) * | 2022-05-23 | 2022-09-13 | 大连大学 | Electrotransformation method for transferring pNCMO2 plasmid into brevibacillus brevis HB200 |
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