CN106520820B - A method of plasma pro-brain natriuretic peptide levels epitope is prepared using bacillus brevis - Google Patents
A method of plasma pro-brain natriuretic peptide levels epitope is prepared using bacillus brevis Download PDFInfo
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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
- C12N15/63—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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- 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
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- 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
A kind of method for preparing plasma pro-brain natriuretic peptide levels epitope using bacillus brevis (Bacillus brevis, B.brevis) of disclosure of the invention.This method will be with the plasma pro-brain natriuretic peptide levels N-terminal linear epitope (NT-BNP in conjunction with plasma pro-brain natriuretic peptide levels antibody12‑21) nucleotides sequence column permutation people fibronectin (fibronectin 3, Fn3) coding the area FG nucleotide sequence, constitute Fn3 and NT-BNP12‑21Linear epitope fusion is inserted into shuttle plasmid pNCMO2P2 signal peptide downstream, construct pNCMO2‑Fn3‑NT‑BNP12‑21Recombinant plasmid, so that recombinant plasmid skelemin Fn3 of energy high efficient expression secreting type in B.brevis merges NT-BNP12‑21The fusion protein of linear epitope.The present invention can prepare simple, quickly, efficiently at low cost secreting type have and the comparable fusion protein of plasma pro-brain natriuretic peptide levels antigenicity.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of utilization high efficient expression secreted protein expression system preparation source of people
The method of fibronectin (Fn3) displaying antigen plasma pro-brain natriuretic peptide levels N-terminal (NT-BNP) linear epitope.
Background technique
In ELISA detection kit preparation process, need using corresponding antigen as standard items.Wherein, protein-based
Antigen occupies very big ratio.How proteinaceous antigen is efficiently produced, is a key problem in technology of such product.The present invention adopts
With source of people fibronectin (Fn3) display protein matter class antigen linear epitope, trial is established one high using bacillus brevis
The general-purpose platform of effect production secretary protein class linear epitope.
NT-BNP is in the blood plasma for being released to people when cardiac muscle is stimulated or loses, 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 cardiovascular disease as standard detection antigen at present
In disconnected treatment and prognosis application.NT-BNP includes chemical synthesis, Escherichia coli as the source of standard detection antigen
(Escherichia coli, E.coli) recombination, people's cell recombination and the separation of patients with heart failure blood plasma etc..Due to other several sources
Higher cost or stability are poor, and main source is that Escherichia coli recombination obtains at present.But Recombinant protein expression melts
The protein expression of hop protein, upstream needs IPTG to induce, and the protein purification in downstream needs a series of step such as smudge cells, than
It is more complex.
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 suitable for production secretary protein.The system has the advantage that (1) is not bright
Aobvious codon-bias, while expression product is also not easily formed inclusion body, after albumen crosses over cell membrane, is processed and direct
It is discharged into culture medium without assembling, recycling and purifying protein are relatively simple；(2) hardly to exocytosis protease,
Conducive to the stability of destination protein；(3) for the secretary protein of eukaryotic origin, generally there is S -- S structure, according to the study its
There is the factors that can promote disulfide bond formation in albumen in fermentation liquid, can promote the folding of foreign protein.
The present invention shows antigen plasma pro-brain natriuretic peptide levels linear list using bacillus brevis expression source of people fibronectin (Fn3)
The foreign protein of expression, can be directly secreted into culture solution, to greatly simplify downstream foreign protein by position by the approach
Isolate and purify process.The one or more using the shown antigen of bacillus brevis secreting, expressing fibronectin can be achieved
Epitope, to produce the antigen protein substitute having with the equivalent high stable of antigen protein.
Summary of the invention
The present invention provides a kind of utilization secreted protein expression system bacillus brevis preparation and reorganization source of people Fn3-N- akrencephalon
Pro-BNP epitope (NT-BNP12-21) method, reduce production cost, realize quickly, large-scale separation preparation people recombination
N- akrencephalon pro-BNP epitope.
The technical solution of the present invention is as follows: will be with the epitope nucleotides sequence column permutation people in conjunction with plasma pro-brain natriuretic peptide levels antibody
The coding FG region nucleotide sequence of fibronectin Fn3, then by the above Fusion gene construction to shuttle vector pNCMO2On, benefit
With bacillus brevis expression system efficiently expressing recombinant human source Fn3-N- akrencephalon pro-BNP epitope Fn3-BNP12-21.Specifically
Steps are as follows:
1. people's fibronectin Fn3 shows NT-BNP12-21The building of linear epitope fusion protein expression vector:
(1) by NT-BNP12-21Linear epitope design introduces coding BNP linear epitope in the area FG of Fn3, in the area FG loop
Short peptide sequence, constitute people's fibronectin Fn3 and NT-BNP12-21Linear epitope fusion Fn3-BNP12-21, synthesis
Fn3 shows NT-BNP12-21The genetic fragment of linear epitope, it is pure in the separation that 5 ' end of fusion introduces 6 histidines of coding
Change label, convenient for isolating and purifying；
(2) by above-mentioned fusion Fn3-BNP12-21Shuttle vector pNCMO is building up to by Nco I and I restriction enzyme site of Xho2
P2 strong promoter 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-BNP12-21Conversion is into Escherichia coli, in amicillin resistance
Screening positive clone on LB plate, ampicillin are every in final concentration of 100 μ g/ml, the LB culture medium prescription of LB culture medium
It rises and contains 10 grams of tryptone, yeast extract 5g, sodium chloride 10g and 15g agar powder in culture medium；
(2) positive transformant identified is converted into bacillus brevis, is screened on the MT plate of neomycin resistance
Positive colony, neomycin are to contain glucose in every liter of culture medium in final concentration of 10 μ g/ml, the MT culture medium prescription of MT culture medium
20g, multivalence peptone 10g, meat soak powder 5g, yeast extract 20g, FeSO4·7H2O 10mg、MnSO4·4H2O 10mg、ZnSO4·
7H2O 1mg, pH=7.2-7.4；
(3) positive transformant identified is seeded in MT the or 2SY fluid nutrient medium containing neomycin resistance, it is new mould
The plain final concentration of 50 μ g/ml of liquid medium within, is incubated overnight, and collects supernatant culture solution, using SDS-PAGE electrophoresis and
Western Blot method testing goal protein band, 2SY culture medium prescription 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 isolates and purifies:
Supernatant culture solution is collected, the fusion protein of His-Tag label is had with ni-sepharose purification, which is recombination human source
Fn3 merges N- akrencephalon pro-BNP epitope BNP12-21Albumen.
The beneficial effects of the present invention are: the expression vector that the present invention uses is pNCMO2, the carrier be in B.brevis and
Shuttle plasmid between E.coli.After constructing expression plasmid in E.coli, it is transferred to B.brevis and carries out protein expression.Host
The P2 promoter of bacterium cell wall protein is as pNCMO2Expression promoter, since P2 promoter does not act as in E.coli
With being conducive to the clone of target gene；But it is then exponentially other strong promoter in B.brevis, can efficiently expresses mesh
Albumen.
The present invention shows antigen plasma pro-brain natriuretic peptide levels linear list using bacillus brevis expression source of people fibronectin (Fn3)
The foreign protein of expression, can be directly secreted into culture solution, to greatly simplify downstream foreign protein by position by the approach
Isolate and purify process, thus quickly, simply, efficiently prepare people recombinate N- akrencephalon pro-BNP epitope.
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 is in large scale preparation source of people Fn3-BNP12-21Recombinant protein and other heavy
There is high value of practical in the production of group polypeptide.
Detailed description of the invention
Attached drawing 1 is Fn3-BNP of the invention12-21Gene structure figure.
Attached drawing 2 is pNCMO of the invention2-Fn3-BNP12-21Fusion expression vector map.
Attached drawing 3 is Fn3-BNP of the invention12-21The SDS-PAGE electrophoresis that recombinant protein is expressed in different medium,
Wherein: band 1,2,3 is positive transformant HB116-pNCMO2-Fn3-BNP12-21Bacterial strain is cultivated for 24 hours in MT culture medium, centrifugation
Gained Supernatant samples, 6,7,8 be positive transformant HB116-pNCMO2-Fn3-BNP12-21Bacterial strain is cultivated in 2SY culture medium
For 24 hours, centrifugation gained Supernatant samples, M is standard protein.
Attached drawing 4 is Fn3-BNP of the invention12-21Recombinant protein Western Blot detects figure result figure.Wherein, band 1
For the Western Blot detection figure in embodiment one as a result, band 2 is Western Blot detection figure result in embodiment two.
Attached drawing 5 is Fn3-BNP of the invention12-21Recombinant protein SDS-PAGE electrophoresis after ni-sepharose purification, wherein band
1,2,3 be respectively the Fn3-BNP after the resulting ni-sepharose purification of embodiment one to three12-21Fusion protein, M are standard protein.
Attached drawing 6 is recombination Fn3-BNP of the invention12-21Albumen and the anti-BNP of source of mouse12-21The monoclonal of amino acid residue is anti-
The analysis of body 13G12 binding ability.
Specific embodiment
With reference to the accompanying drawing and specific embodiment the present invention is further illustrated, but have no effect on guarantor of the invention
Protect range.
Embodiment one
1. people's fibronectin Fn3 shows NT-BNP12-21The building of linear epitope fusion protein expression vector:
(1) using people's fibronectin type III domain protein (Fn3) for mode gene, (EMBL logins number
AJ320527), by NT-BNP12-21Linear epitope design introduces coding BNP epitope in the area FG of Fn3, in the area FG loop
Short peptide sequence (CTGGAAACCTCGGGCCTGCAGGAACAACGT encodes the 12-21 amino acid residue LETSGLQEQR of BNP),
Nanjing Genscript Biotechnology Co., Ltd. is entrusted to synthesize Fn3-BNP according to SEQ ID NO:112-21Gene, in fusion 5 '
Introducing 6 histidines of coding in end isolate and purify label, convenient for isolating and purifying；The fusion structure of formation is shown in attached drawing 1；
(2) by above-mentioned fusion Fn3-BNP12-21Shuttle vector pNCMO is building up to by Nco I and I restriction enzyme site of Xho2
P2 strong promoter downstream, formed fusion expression vector pNCMO2-Fn3-BNP12-21, nucleotide sequence such as SEQ ID NO:2
Shown, Vector map is shown in attached drawing 2；
2. people's fibronectin Fn3 shows NT-BNP12-21The expression of linear epitope fusion protein:
(1) by fusion expression vector pNCMO2-Fn3-BNP12-21According to precious bioengineering (Dalian) Co., Ltd JM109
Competent cell specification is transformed into JM109 coli strain, in the LB for containing ampicillin (100 μ g/ml) resistance
On culture medium flat plate, 37 DEG C of cultures are for 24 hours.LB culture medium prescription: contain 10 grams of tryptone, yeast extract in every liter of culture medium
5g, sodium chloride 10g and agar powder 15g；
(2) picking positive colony is inoculated in the LB liquid medium containing ampicillin (100 μ g/ml) resistance, 37 DEG C,
200rpm overnight incubation extracts plasmid, after Nco I and I double digestion of Xho are accredited as the positive, by recombinant plasmid pNCMO2-
Fn3-BNP12-21Illustrate according to the NTP method in precious bioengineering (Dalian) Co., Ltd bacillus brevis expression system HB200,
Conversion is into bacillus brevis expression system HB116, and in ovobiocin resistance (10 μ g/ml) MT culture medium, 30 DEG C were cultivated
Night extracts plasmid, takes and is accredited as positive transformant HB116-pNCMO by Nco I and I double digestion of Xho2-Fn3-BNP12-21
Carry out the expression of fusion protein.MT culture medium prescription: 20g containing glucose, multivalence peptone 10g, meat soak powder 5g, ferment in every liter of culture medium
Female extract 20g, FeSO4·7H2O 10mg、MnSO4·4H2O 10mg、 ZnSO4·7H2O 1mg, pH=7.2-7.4；
(3) the positive transformant HB116-pNCMO that will be identified2-Fn3-BNP12-21Strain inoculated is anti-to 5ml ovobiocin
In the MT culture medium of property (50 μ g/ml), under the conditions of 200rpm, 30 DEG C, shake culture is for 24 hours.It is by bacterium solution centrifugal force
12000g is centrifuged 10min, collects supernatant, and SDS-PAGE sample-loading buffer is added, and after thermal cracking, carries out SDS-PAGE electricity
Swimming, the results are shown in attached figure 3；
(4) it goes to the supernatant obtained in step 2 (3) is wet on pvdf membrane, after confining liquid is closed, primary antibody is added
(1: 2500 dilution, anti-BNP monoclonal antibody are purchased from HyTest company) is incubated at room temperature, and secondary antibody is that horseradish enzyme marks goat-anti
After mouse IgG (1: 4000 dilution) is incubated for cleaning, the luminous detection of Western-Blot is carried out, the results are shown in attached figure 4；
3. fusion protein isolates and purifies:
The supernatant that step 2 (3) obtain 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 electrophoresis is shown in attached drawing 5, measures immune serum using ELISA method
Potency.Fusion protein is coated with 96 hole elisa Plates after diluting step by step, and after confining liquid is closed, the production of HyTest company is added
The anti-NT-proBNP of source of mouse12-21The monoclonal antibody 13G12 (1:2500 dilution) of amino acid residue, is made with seralbumin
Blank control.Secondary antibody is that horseradish peroxidase marks goat anti-mouse igg antibody (1: 4000 dilution), substrate TMB, colour developing
Reaction is terminated with the sulfuric acid of 2M after 10min.Measure the OD value at wavelength 450nm.As a result as shown in Fig. 6, fusion protein F n3-
BNP12-21With the good and anti-BNP of source of mouse12-21The monoclonal antibody 13G12 binding ability of amino acid residue.
Embodiment two
1. people's fibronectin Fn3 shows NT-BNP12-21The building of linear epitope fusion protein expression vector:
(1) using people's fibronectin type III domain protein (Fn3) for mode gene, (EMBL logins number
AJ320527), by NT-BNP12-21Linear epitope design introduces coding BNP epitope in the area FG of Fn3, in the area FG loop
Short peptide sequence (CTGGAAACCTCGGGCCTGCAGGAACAACGT encodes the 12-21 amino acid residue LETSGLQEQR of BNP),
Nanjing Genscript Biotechnology Co., Ltd. is entrusted to synthesize Fn3-BNP according to SEQ ID NO:112-21Gene, in fusion 5 '
Introducing 6 histidines of coding in end isolate and purify label, convenient for isolating and purifying；The fusion structure of formation is shown in attached drawing 1；
(2) by above-mentioned fusion Fn3-BNP12-21Shuttle vector pNCMO is building up to by Nco I and I restriction enzyme site of Xho2
P2 strong promoter downstream, formed fusion expression vector pNCMO2-Fn3-BNP12-21, nucleotide sequence such as SEQ ID NO:2
Shown, Vector map is shown in attached drawing 2；
2. people's fibronectin Fn3 shows NT-BNP12-21The expression of linear epitope fusion protein:
(1) by fusion expression vector pNCMO2-Fn3-BNP12-21According to precious bioengineering (Dalian) Co., Ltd JM109
Competent cell specification is transformed into JM109 coli strain, in the LB for containing ampicillin (100 μ g/ml) resistance
On culture medium flat plate, 37 DEG C of cultures are for 24 hours.LB culture medium prescription: contain 10 grams of tryptone, yeast extract in every liter of culture medium
5g, sodium chloride 10g and agar powder 15g；
(2) picking positive colony is inoculated in the LB liquid medium containing ampicillin (100 μ g/ml) resistance, 37 DEG C,
200rpm overnight incubation extracts plasmid, after Nco I and I double digestion of Xho are accredited as the positive, by recombinant plasmid pNCMO2-
Fn3-BNP12-21Illustrate according to the NTP method in precious bioengineering (Dalian) Co., Ltd bacillus brevis expression system HB200,
Conversion is into bacillus brevis expression system HB116, and in ovobiocin resistance (10 μ g/ml) MT culture medium, 30 DEG C were cultivated
Night extracts plasmid, takes and is accredited as positive transformant HB116-pNCMO by Nco I and I double digestion of Xho2-Fn3-BNP12-21
Carry out the expression of fusion protein.MT culture medium prescription: 20g containing glucose, multivalence peptone 10g, meat soak powder 5g, ferment in every liter of culture medium
Female extract 20g, FeSO4·7H2O 10mg、MnSO4·4H2O 10mg、 ZnSO4·7H2O 1mg, pH=7.2-7.4；
(3) the positive transformant HB116-pNCMO that will be identified2-Fn3-BNP12-21Strain inoculated is anti-to 5ml ovobiocin
In the 2SY culture medium of property (50 μ g/ml), under the conditions of 200rpm, 30 DEG C, shake culture is for 24 hours.It is by bacterium solution centrifugal force
12000g is centrifuged 10min, collects supernatant, and SDS-PAGE sample-loading buffer is added, and after thermal cracking, carries out SDS-PAGE electricity
Swimming, the results are shown in attached figure 3.2SY culture medium prescription is as follows: 20g containing glucose, soya peptone 40g, yeast extract in every liter of culture medium
5g、CaCl2·2H2O 0.15g, pH=7.2-7.4；
(4) it goes to the supernatant obtained in step 2 (3) is wet on pvdf membrane, after confining liquid is closed, primary antibody is added
(1: 3000 dilution, anti-BNP monoclonal antibody are purchased from HyTest company) is incubated at room temperature, and secondary antibody is that horseradish enzyme marks goat-anti
After mouse IgG (1: 4000 dilution) is incubated for cleaning, the luminous detection of Western-Blot is carried out, the results are shown in attached figure 4；
3. fusion protein isolates and purifies:
The supernatant that step 2 (3) obtain 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 electrophoresis is shown in attached drawing 5, measures immune serum using ELISA method
Potency.Fusion protein is coated with 96 hole elisa Plates after diluting step by step, and after confining liquid is closed, the production of HyTest company is added
The anti-NT-proBNP of source of mouse12-21The monoclonal antibody 13G12 (1:3000 dilution) of amino acid residue, is made with seralbumin
Blank control.Secondary antibody is that horseradish peroxidase marks goat anti-mouse igg antibody (1: 4000 dilution), substrate TMB, colour developing
Reaction is terminated with the sulfuric acid of 2M after 10min.Measure the OD value at wavelength 450nm.As a result as shown in Fig. 6, fusion protein F n3-
BNP12-21With the good and anti-BNP of source of mouse12-21The monoclonal antibody 13G12 binding ability of amino acid residue.
Embodiment three
1. people's fibronectin Fn3 shows NT-BNP12-21The building of linear epitope fusion protein expression vector:
(1) using people's fibronectin type III domain protein (Fn3) for mode gene, (EMBL logins number
AJ320527), by NT-BNP12-21Linear epitope design introduces coding BNP epitope in the area FG of Fn3, in the area FG loop
Short peptide sequence (CTGGAAACCTCGGGCCTGCAGGAACAACGT encodes the 12-21 amino acid residue LETSGLQEQR of BNP),
Nanjing Genscript Biotechnology Co., Ltd. is entrusted to synthesize Fn3-BNP according to SEQ ID NO:112-21Gene, in fusion 5 '
Introducing 6 histidines of coding in end isolate and purify label, convenient for isolating and purifying；The fusion structure of formation is shown in attached drawing 1；
(2) by above-mentioned fusion Fn3-BNP12-21Shuttle vector pNCMO is building up to by Nco I and I restriction enzyme site of Xho2
P2 strong promoter downstream, formed fusion expression vector pNCMO2-Fn3-BNP12-21, nucleotide sequence such as SEQ ID NO:2
Shown, Vector map is shown in attached drawing 2；
2. people's fibronectin Fn3 shows NT-BNP12-21The expression of linear epitope fusion protein:
(1) by fusion expression vector pNCMO2-Fn3-BNP12-21According to precious bioengineering (Dalian) Co., Ltd JM109
Competent cell specification is transformed into JM109 coli strain, in the LB for containing ampicillin (100 μ g/ml) resistance
On culture medium flat plate, 37 DEG C of cultures are for 24 hours.LB culture medium prescription: contain 10 grams of tryptone, yeast extract in every liter of culture medium
5g, sodium chloride 10g and agar powder 15g；
(2) picking positive colony is inoculated in the LB liquid medium containing ampicillin (100 μ g/ml) resistance, 37 DEG C,
200rpm overnight incubation extracts plasmid, after Nco I and I double digestion of Xho are accredited as the positive, by recombinant plasmid pNCMO2-
Fn3-BNP12-21Illustrate according to the NTP method in precious bioengineering (Dalian) Co., Ltd bacillus brevis expression system HB200,
Conversion is into bacillus brevis expression system HB116, and in ovobiocin resistance (10 μ g/ml) MT culture medium, 30 DEG C were cultivated
Night extracts plasmid, takes and is accredited as positive transformant HB116-pNCMO by Nco I and I double digestion of Xho2-Fn3-BNP12-21
Carry out the expression of fusion protein.MT culture medium prescription: 20g containing glucose, multivalence peptone 10g, meat soak powder 5g, ferment in every liter of culture medium
Female extract 20g, FeSO4·7H2O 10mg、MnSO4·4H2O 10mg、 ZnSO4·7H2O 1mg, pH=7.2-7.4；
(3) the positive transformant HB116-pNCMO that will be identified2-Fn3-BNP12-21Strain inoculated is anti-to 5ml ovobiocin
In the 2SY culture medium of property (50 μ g/ml), under the conditions of 200rpm, 30 DEG C, shake culture 48h.It is by bacterium solution centrifugal force
10000g is centrifuged 10min, collects supernatant, and SDS-PAGE sample-loading buffer is added, and after thermal cracking, carries out SDS-PAGE electricity
Swimming, the results are shown in attached figure 3.2SY culture medium prescription is as follows: 20g containing glucose, soya peptone 40g, yeast extract in every liter of culture medium
5g、CaCl2·2H2O 0.15g, pH=7.2-7.4；
(4) it goes to the supernatant obtained in step 2 (3) is wet on pvdf membrane, after confining liquid is closed, primary antibody (1 is added
: 2500 dilutions, anti-BNP monoclonal antibody are purchased from HyTest company) it is incubated at room temperature, secondary antibody is that horseradish enzyme marks goat-anti small
After mouse IgG (1: 5000 dilution) is incubated for cleaning, the luminous detection of Western-Blot is carried out；
3. fusion protein isolates and purifies:
The supernatant that step 2 (3) obtain 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 electrophoresis is shown in attached drawing 5, measures immune serum using ELISA method
Potency.Fusion protein is coated with 96 hole elisa Plates after diluting step by step, and after confining liquid is closed, it is raw that HyTest company is added
The anti-NT-proBNP of the source of mouse of production12-21The monoclonal antibody 13G12 (1:2500 dilution) of amino acid residue, is made with seralbumin
Blank control.Secondary antibody is that horseradish peroxidase marks goat anti-mouse igg antibody (1: 5000 dilution), substrate TMB, colour developing
Reaction is terminated with the sulfuric acid of 2M after 10min.Measure the OD value at wavelength 450nm.As a result as shown in Fig. 6, fusion protein F n3-
BNP12-21With the good and anti-BNP of source of mouse12-21The monoclonal antibody 13G12 binding ability of amino acid residue.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art within the technical scope of the present disclosure, according to the ordinary technical knowledge of this field
And universal method, it is subject to equivalent substitution or change according to the technical scheme of the invention and its inventive conception, should all covers in this hair
Within bright protection scope.
<110>University Of Dalian
<120>a kind of method for preparing plasma pro-brain natriuretic peptide levels epitope using bacillus brevis
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 336
<212> DNA
<213>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>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 for preparing plasma pro-brain natriuretic peptide levels epitope using bacillus brevis, which comprises the following steps:
1), people's fibronectin Fn3 shows NT-BNP12-21The building of linear epitope fusion protein expression vector:
(1) by NT-BNP12-21Linear epitope design introduces the short of coding BNP linear epitope in the area FG of Fn3, in the area FG loop
Peptide sequence constitutes people's fibronectin Fn3 and NT-BNP according to SEQ ID NO:112-21Linear epitope fusion Fn3-
BNP12-21, synthesis Fn3 displaying NT-BNP12-21The genetic fragment of linear epitope introduces 6 groups of coding in 5 ' end of fusion
Propylhomoserin isolates and purifies label, convenient for isolating and purifying；
(2) introducing obtained in step (1) is isolated and purified to the fusion Fn3-BNP of label12-21Pass through Nco I and Xho I
Restriction enzyme site is building up to shuttle vector pNCMO2P2 strong promoter 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-BNP12-21Conversion is flat in the LB of amicillin resistance into Escherichia coli
Screening positive clone on plate, ampicillin are every liter of training in final concentration of 100 μ g/ml, the LB culture medium prescription of LB culture medium
It supports and contains 10 grams of tryptone, yeast extract 5g, sodium chloride 10g and 15g agar powder in base；
(2) positive transformant identified is converted into bacillus brevis, is sieved on the MT culture medium flat plate of neomycin resistance
Positive colony is selected, neomycin is to contain glucose in every liter of culture medium in final concentration of 10 μ g/ml, the MT culture medium prescription of MT culture medium
20g, multivalence peptone 10g, meat soak powder 5g, yeast extract 20g, FeSO4·7H2O 10mg、MnSO4·4H2O 10mg、ZnSO4·
7H2O 1mg, pH=7.2-7.4；
(3) positive transformant identified is seeded in MT the or 2SY fluid nutrient medium containing neomycin, neomycin is in liquid
The final concentration of 50 μ g/ml of culture medium, is incubated overnight, and collects supernatant culture solution, utilizes SDS-PAGE electrophoresis and Western Blot
Method testing goal protein band, 2SY culture medium prescription is 20g containing glucose in every liter of culture medium, soya peptone 40g, yeast extract
Object 5g, CaCl2·2H2O 0.15g, pH=7.2-7.4；
3), fusion protein isolates and purifies:
Supernatant culture solution is collected, the fusion protein of His-Tag label is had with ni-sepharose purification, which is recombination human source Fn3
Merge N- akrencephalon pro-BNP epitope BNP12-21Albumen.
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| CN102952193A (en) * | 2011-12-29 | 2013-03-06 | 北京五康新兴科技有限公司 | Antigenic epitope displaying method based on single domain antibody |
| CN105154462A (en) * | 2015-08-25 | 2015-12-16 | 大连大学 | Method for building N-glycosylation receptor protein models in Escherichia coli by aid of skeleton proteins Fn3 (fibronectin type III domain) |
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| CN105154461A (en) * | 2015-08-25 | 2015-12-16 | 大连大学 | Method for building N-glycosylation efficiency detection receptor protein models in Escherichia coli by aid of skeleton proteins Fn3 (fibronectin type III domain) |
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