CN101280017A - Fused protein of human brain natriuretic peptide diad [(BNP)2] and human serum albumin (HAS), and preparation thereof - Google Patents
Fused protein of human brain natriuretic peptide diad [(BNP)2] and human serum albumin (HAS), and preparation thereof Download PDFInfo
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- CN101280017A CN101280017A CNA2008100249860A CN200810024986A CN101280017A CN 101280017 A CN101280017 A CN 101280017A CN A2008100249860 A CNA2008100249860 A CN A2008100249860A CN 200810024986 A CN200810024986 A CN 200810024986A CN 101280017 A CN101280017 A CN 101280017A
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Abstract
The invention discloses a fused protein from human brain natriuretic peptide(BNP)2 and human serum albumin (HSA) and the preparation method thereof, belonging to the technical field of long-effetive recombinant fused protein drugs. The invention introduces OE-PCR technology to splice two BNP cDNAs(called (BNP)2 for short) and HAS cDNA, without adding any connecting peptide therebetween, obtaining fused gene(BNP)2-HSA cDNA which is then integrated with the chromosome of the host and expresses in the expression system of the host. The fused protein of the invention comprises a first region with at least 85% of the sequence congenetic with human brain natriuretic peptide and a second region with at least 85%of the sequence congenetic with human serum albumin. Under the premise of not being changed in characteristics, the fused protein is capable of the substitution, deletion or addition of specific amino acid residues. The fused protein maintains the physiological characteristics and improves the solubility of human brain natriuretic peptide and prolongs the half life of human brain natriuretic peptide within human body; therefore the fused protein is of good application prospect in pharmacy.
Description
Technical field
The human brain natriuretic peptide diad [(BNP)
2] with the preparation method and the product of the fusion rotein of human serum albumin (HSA), belong to long-acting fusion rotein technical field of pharmaceuticals.
Background technology
Brain natriuretic peptide (Brain Natriuretic Peptide, BNP) be the sharp sodium peptide family member who found in the pig brain in 1988 by Sudoh etc., it is mainly synthetic by the ventricle secretion, and precursor contains 108 amino acid, discharges after the processing to contain 32 amino acid whose ripe BNP molecules.The main characteristic of BNP is the effect of antagonism renin-angiotensin-aldosterone system, and promptly by suppressing the secretion of feritin and aldosterone, increase glomerular filtration rate(GFR and inhibition kidney medulla collecting tubule sodium heavily absorb and promotes row's sodium, diuresis.Also can be by the effect of the direct nervous plain II of lax vascular smooth muscle and antagonizing vessel vasodilation, the inhibition smooth muscle cell proliferation influences blood vessel to be reinvented.Vein gives BNP can reduce heart failure patient pulmonary capillary wedge pressure (PCWP), right atrium pressure, systemic vascular resistance (SVR), and can alleviate water-sodium retention, improves hemodynamic state rapidly.August calendar year 2001 drugs approved by FDA the upright peptides (nesiritide) of recombinant human brain natriuretic peptide (rhBNP) medicine-Nai two be used for the clinical treatment acute heart failure.In October, 2005, China rhBNP (trade(brand)name newly live plain) that also gone on the market.
As widely used other recombinant polypeptide/protein medicaments clinically, reorganization BNP drug solubility is low, the transformation period only is 22min in the poor stability, body, and must continue the high dosage administration can prove effective.Such pharmacological agent expense height, drug utilization degree are low, and the patient frequently injects, and causes its actual amount far to be lower than demand.Prompting is necessary to pay attention to the research and development of long-acting BNP medicine, satisfies the needs of clinical application.
HSA has non-enzymatic activity and immunogenicity, big, the long half time advantages such as (about 23 days) of molecular weight in vivo as the main component of human plasma.The fusion protein technology (Albumin Fusion Technology) that with HSA is carrier comes into one's own.With HSA gene and human interferon-alpha-2 b (IFN α 2b) gene fusion, the fusion rotein HSA-IFN α 2b mean half-life in vivo of expression reaches 140h, and good antiviral activity, security and tolerance are arranged.This fused protein has increased the drug molecule amount on the one hand, has reduced the discharge rate of kidney; On the other hand, the protein molecular of fusion surface produces space steric effect, and proteolytic ferment stays the time to the hydrolytic action of common IFN α 2b thereby prolonged the Chu of protein molecular medicine in the recycle system effectively in the attenuating blood.By changing the pharmacokinetic properties of medicine, make that the plasma concentration of medicine is more stable, the time that medicine is kept in vivo is longer, thereby reaches the effect that improves curative effect of medication and reduce side effect.
Summary of the invention
The preparation method and the product that the purpose of this invention is to provide the fusion rotein of a kind of human brain natriuretic peptide and human serum albumin.Fusion rotein of the present invention prolongs its transformation period in vivo on the basis of the physiological property that has kept human brain natriuretic peptide, at pharmaceutical field good prospects for application is arranged.
Technical scheme of the present invention: synthetic (BNP)
2CDNA; Increase from people's tire liver cDNA library by PCR and to obtain HSA cDNA; Utilize overlapping pcr, connect (BNP)
2CDNA and HSA cDNA, the centre does not add any connection peptides, (BNP) that obtains
2-HSA cDNA fusion gene is inserted among the cloning vector pBlu2KSP to be preserved; (BNP)
2-HSA cDNA fusion gene is integrated in host's the karyomit(e), expresses in host cell.
(BNP)
2The clone of cDNA, the clone of HSA cDNA, (BNP)
2The clone of cDNA and HSA cDNA fusion gene, fusion rotein (BNP)
2The recombination yeast of-HSA makes up and fusion rotein (BNP)
2The step of the expression of-HSA sees specific implementation method for details.
With the human brain natriuretic peptide of method for preparing and the fusion rotein of human serum albumin, comprise with first district of human brain natriuretic peptide at least 85% sequence homology and with second district of human serum albumin at least 85% sequence homology or partial amino-acid series second district of human serum albumin; Described and human brain natriuretic peptide homologous first district is positioned at the N-terminal of fusion rotein, and described and human serum albumin homologous second district is positioned at the C-terminal of fusion rotein, and the centre does not add any connection peptides; Or described and human serum albumin homologous second district is positioned at the N-terminal of fusion rotein, and described and human brain natriuretic peptide homologous first district are positioned at the C-terminal of fusion rotein, and the centre does not add any connection peptides.
Preferably described fusion rotein comprise with first district of human brain natriuretic peptide at least 95% sequence homology and with second district of human serum albumin at least 95% sequence homology.
Second district of described fusion rotein can be made up of human serum albumin part-structure territory, the human serum albumin after structural domain is reset is formed, comprise outside the naturally occurring multiformity of all HSA, the part fragment that also comprises HSA, described in EP322094 [HSA (1-n) by name, n is 369-419].
Described fusion rotein comprises first district identical with the human brain natriuretic peptide amino acid residue sequence and second district identical with the human serum albumin amino acid residue sequence, or the function equivalent in above-mentioned two districts.
Described function equivalent is under the prerequisite that does not change described fusion rotein characteristic, the polypeptide that replacement, disappearance or the adding of the indivedual amino-acid residues of fusion rotein obtained.
Host cell is bacterium, yeast, insect cell, zooblast or vegetable cell etc.Preferred host cell is a yeast.Preferred yeast is a pichia spp.Preferred pichia spp is pichia spp GS115.
Because (BNP)
2Gene is shorter, adopts this gene of synthetic.HSA cDNA can be by the PCR acquisition of increasing from people's tire liver cDNA library.Utilize overlapping pcr, connect (BNP)
2CDNA and HSA cDNA, the centre does not add any connection peptides, to avoid adding the fusion rotein stability bring and the problem of immunogenicity aspect, (BNP) that obtains because of connection peptides
2CDNA and HSA cDNA fusion gene are inserted in the cloning vector to be preserved.
(BNP)
2CDNA and HSA cDNA fusion gene can be expressed in a plurality of host systems, as bacterium, yeast, insect cell, zooblast, vegetable cell, and organism (as vertebrates, insect etc.) etc.Goal gene is integrated in host's the karyomit(e) or is inserted in the plasmid in these expression systems.The present invention is yeast expression system preferably, this system except easy to operate, the growth that possesses prokaryotic expression system rapidly, the nutritional requirement characteristics simple, that easily industry is amplified, also have modification system behind the eukaryotic albumen, help the high-quality recombinant protein of mass production.More preferably the pichia yeast expression system is compared with the yeast saccharomyces cerevisiae expression system, and the pichia yeast expression system has remarkable advantages aspect protein excretion and the glycosylation.Pichia yeast self excretory albumen is few, very helps purifying; Degree of glycosylation is low, the immunogenicity problem of having avoided the super glycosylation of yeast saccharomyces cerevisiae to bring.
Pichia yeast expression system secreted expression carrier mainly contains pPIC9, pPIC9K, pHIL-S1, pPICZ α, pYAM75P6; The type expression vector mainly contains in the born of the same parents: pPIC3K, pPICZ, pHWO10, pGAPZ, pGAPZ α (invitrogen) etc., pichia spp secreted expression carrier pPIC9K preferably, it is a yeast integration plasmid, has selected marker HIS4, AOX1 promotor, MF α secreting signal peptide and the G418 resistant gene elements such as (can be used as the mark of screening recon) of His defective type.The AOX1 promotor is a strong promoter, can start the expression of goal gene efficiently.Encode altogether in pichia yeast two kinds of alcohol oxidase AOX1 and AOX2, the vigor of most alcohol oxidases is to be provided by AOX1 in the cell.At methyl alcohol is in the sole carbon source cultured cells, and this enzyme can account for more than 30% of total protein of cell.Though the homology of AOX2 and AOX1 is up to 97%, enzyme is lived very low.When AOX1 genetically deficient, when only having AOX2, the forfeiture of most oxidation of ethanol enzyme activity, cell utilizes the methyl alcohol ability to reduce, and cell is that growth is very slow on the substratum of sole carbon source at methyl alcohol.
Having the expression vector that merges cDNA can be by transforming lithium salts method, PEG method, protoplasm body or electroporation transformed host cell.The success cell transformed, the cell that promptly has the DNA of the present invention's structure, can be identified by method well known in the art, as collecting cell, extract DNA after the cracking, carry out Southern hybridization and PCR then and identify, also can be with HSA antibody and/or BNP antibody test nutrient solution supernatant behind abduction delivering.
Beneficial effect of the present invention:
Utilize overlapping pcr, connect (BNP)
2CDNA and HSA cDNA, the centre does not add any connection peptides, to avoid adding the fusion rotein stability (avoiding the proteasome degradation at connection peptides) bring and the problem of immunogenicity aspect because of connection peptides.
Preferred yeast expression system except easy to operate, the growth that possesses prokaryotic expression system rapidly, the nutritional requirement characteristics simple, that easily industry is amplified, also have modification system behind the eukaryotic albumen, help the high-quality recombinant protein of mass production.More preferably pichia yeast expression system is compared with the yeast saccharomyces cerevisiae expression system, and pichia yeast expression system has remarkable advantages aspect protein excretion and the glycosylation.Pichia spp self excretory albumen is few, very helps purifying; Degree of glycosylation is low, the immunogenicity problem of having avoided the super glycosylation of yeast saccharomyces cerevisiae to bring.
Can have the host cell of the DNA of the present invention's structure by cultivation, produce fusion rotein of the present invention.Host cell is cultivated and is preferably carried out on bio-reactor, cultivates and divides two stages, and the fs is mainly used in the host cell growth, and it is synthetic that subordinate phase is mainly used in product.Can in the cell culture that contain DNA construct of the present invention, separate with the method for various albumen sepn, purified fusion protein.As centrifugal, saltout, the combination of technology such as ultrafiltration, chromatography, preparation electrophoresis and these technology.
Description of drawings
Fig. 1. plasmid pPIC9K-(BNP)
2-HSA structure
Fig. 2. plasmid pPIC9K-(BNP)
2-HSA makes up
Fig. 3. (BNP)
2The overlapping PCR product M:DNA molecular weight standard of-HSA; Lane 1:(BNP)
2The PCR product; The PCR product of Lane 2:HSA; Lane 3:(BNP)
2The overlapping PCR product of-HSA; The M:DNA molecular weight standard
Fig. 4. (BNP)
2The SDS-PAGE of-HSA fusion rotein analyzes M: protein molecular weight standard; Lane 1: empty plasmid pPIC9K transformation fermentation supernatant; Lane 1: plasmid pPIC9K-(BNP)
2-HSA transformation fermentation supernatant
Fig. 5. (BNP)
2The BNP of-HSA fusion rotein and HSA antibody test (Western blot) Lane 1:(BNP)
2-HSA fusion rotein and the hybridization of HSA antibody; Lane 2: contrast ((GLP-1)
2-HSA fusion rotein and the hybridization of BNP antibody) Lane 3:(BNP)
2-HSA fusion rotein and the hybridization of BNP antibody; M: protein molecular weight standard
Specific implementation method
Embodiment 1:(BNP)
2The clone of cDNA
By Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's synthetic (BNP)
2CDNA (195bp) is cloned into it on carrier pBlu2KSP, and inserting the site is Sma I, and recipient bacterium is the e. coli jm109 bacterial strain.
The clone of embodiment 2:HSA cDNA
Utilize PCR to amplify HSA cDNA from people's tire liver cDNA library, used primer is:
PH1：5’-AG
GTCGACGATGCACACAAGAGTGAGGTTGCTC-3’
PH2：5’-GCC
AAGCTTTTATAAGCCTAAGGCAGCTTGACTT-3’
The PCR reaction system: each 1.5 μ l of the PH1 of 10 μ mol/L and PH2 primer, the dNTP 4 μ l of 2.5mmol/L, 10 * pfu Buffer, 5 μ l, the pfu archaeal dna polymerase 0.5 μ l of 5U/ μ l, people's tire liver cDNA library 1 μ g adds distilled water polishing 50 μ l.PCR response procedures: 95 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 1 minute, 60 ℃ of annealing 1 minute, 72 ℃ were extended 3 minutes, and circulated 30 times; 72 ℃ were extended 10 minutes.
Agarose gel electrophoresis analytical reaction product target stripe occurs at the application of sample swimming lane, reclaims the purpose segment that test kit is purified into 1.8kb with PCR fragment glue.Purpose segment that purifying is good and carrier pBlu2KSP are respectively through Sal I and Hind III double digestion, and agarose gel electrophoresis identifies that PCR fragment glue reclaims test kit and reclaims the purpose segment.T
4Dna ligase connects through the HSA of double digestion cDNA and carrier pBlu2KSP, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains X-gal, IPTG and 100 μ g/ml penbritins, 37 ℃ of overnight incubation.The picking white colony is inoculated in 20ml and contains in the LB substratum of 100 μ g/ml penbritins, and 37 ℃ of overnight incubation are extracted the conversion plasmid with the plasmid extraction test kit.Adopt pcr amplification, Sal I and Hind III double digestion are identified positive colony, and dna sequencing is carried out in zone between recombinant plasmid Sal I and Hind III double enzyme site.Positive recombinant is kept among the LB that contains 20% glycerine, freezes in-80 ℃.Recombinant plasmid called after pBlu2KSP-HSA, positive recombinant called after JM109/pBlu2KSP-HSA.
Embodiment 3:(BNP)
2The clone of cDNA and HSA cDNA fusion gene
(1) (BNP)
2The pcr amplification of cDNA, the primer is as follows:
PB1：5’-GCGCGC
GAATTCAAAAGATCTCCAAAGATGGTCCAAGG-3’
PB2：5’-ACCTCACTCTTGTGTGCATCGTGACGTCTCAGGACCTTGC-3’
PCR reaction system: each 1.5 μ l of the PB1 of 10 μ mol/L and PB2 primer, the dNTP 4 μ l of 2.5mmol/L, 10 * pfu Buffer, 5 μ l, the pfu archaeal dna polymerase 0.5 μ l of 5U/ μ l, plasmid template pBlu2KSP-(BNP)
21ng adds distilled water polishing 50 μ l.PCR response procedures: 95 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, 72 ℃ were extended 30 seconds, and circulated 30 times; 72 ℃ were extended 10 minutes.
(2) pcr amplification of HSA cDNA, the primer is as follows:
PH3：5’-GCAAGGTCCTGAGACGTCACGATGCACACAAGAGTGAGGT-3’
PH4：5’-CATAAG
GCGGCCGCTTATTATAAGCCTAAGGCAGCTTG-3’
PCR reaction system: each 1.5 μ l of the PH 3 of 10 μ mol/L and PH 4 primers, 2.5mmol/L dNTP 4 μ l, 10 * pfu Buffer, 5 μ l, the pfu archaeal dna polymerase 0.5 μ l of 5U/ μ l, plasmid template pBlu2KSP-HSA 1ng adds distilled water polishing 50 μ l.PCR response procedures: 95 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, 72 ℃ were extended 3 minutes, and circulated 30 times; 72 ℃ were extended 10 minutes.
(3) overlapping pcr merges (BNP)
2CDNA and HSA cDNA
With (BNP)
2Pcr amplification product and the pcr amplification product of HSA dilute 10 times respectively, again with after 1: 10 mixed as template, in the 50ul reaction system, add 2 * Pfu PCR MasterMix 25ul, the template 25 μ l that mix.PCR response procedures: 95 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, 72 ℃ were extended 4 minutes, and circulated 15 times; 72 ℃ were extended 10 minutes.
With this PCR product is template, adds primer PB1 and PH 4 each 1.5 μ l of 10 μ mol/L in the reaction system of 50 μ l, 2 * Pfu PCRMasterMix 25ul, and template 4ul adds distilled water polishing 50 μ l.PCR response procedures: 95 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, 72 ℃ were extended 4 minutes, and circulated 35 times; 72 ℃ were extended 10 minutes.
Agarose gel electrophoresis analytical reaction product target stripe occurs at the application of sample swimming lane, reclaims the purpose fragment that test kit is purified into 2kb with PCR fragment glue.The overlapping PCR product and the carrier pBlu2KSP of purifying, respectively through EcoR I and Not I double digestion, agarose gel electrophoresis is identified, reclaims test kit with PCR fragment glue and reclaims (BNP)
2-HSA gene fragment and carrier pBlu2KSP.Use T
4Dna ligase connects two and reclaims fragment, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains X-gal, IPTG and 100 μ g/ml penbritins, 37 ℃ of overnight incubation.The picking white colony is inoculated in 20ml and contains in the LB substratum of 100 μ g/ml penbritins, and 37 ℃ of overnight incubation are extracted the conversion plasmid with the plasmid extraction test kit.Adopt pcr amplification, EcoR I and Not I double digestion are identified positive colony, and dna sequencing is carried out in zone between recombinant plasmid EcoR I and Not I double enzyme site.Positive recombinant is kept among the LB that contains 20% glycerine, freezes in-80 ℃.Recombinant plasmid called after pBlu2KSP-(BNP)
2-HSA, positive recombinant called after JM109/pBlu2KSP-(BNP)
2-HSA.
Embodiment 4:(BNP)
2-HSA yeast expression system makes up and Expression of Fusion Protein
(1) fusion rotein (BNP)
2The structure of the yeast expression system of-HSA
Get a frozen JM109/pBlu2KSP-(BNP)
2-HSA is inoculated into 20ml and contains in the LB substratum of 100 μ g/ml penbritins, and 37 ℃ of overnight incubation are extracted plasmid with the plasmid extraction test kit.The plasmid pBlu2KSP-(BNP) that extracts
2-HSA and Yeast expression carrier pPIC9K, respectively through EcoR I and Not I double digestion, agarose gel electrophoresis is identified, reclaims test kit with PCR fragment glue and reclaims (BNP)
2-HSA gene fragment and carrier pPIC9K.Use T
4Dna ligase connects two and reclaims fragment, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 100 μ g/ml penbritins, 37 ℃ of overnight incubation.The bacterium colony that picking grows is inoculated in 20ml and contains in the LB substratum of 100 μ g/ml penbritins, and 37 ℃ of overnight incubation are extracted plasmid with the plasmid extraction test kit.Adopt pcr amplification, EcoR I and Not I double digestion are identified positive colony.Positive recombinant is kept among the LB that contains 20% glycerine, freezes in-80 ℃.Recombinant plasmid called after pPIC9K-(BNP)
2-HSA, positive recombinant called after JM109/pPIC9K-(BNP)
2-HSA.
Extract JM109/pPIC9K-(BNP)
2Plasmid among the-HSA, through the SalI single endonuclease digestion, after DNA glue reclaimed the test kit recovery, electric shock transformed pichia spp GS115, and conversion product is coated on the MD flat board that contains the 1mol/L sorbyl alcohol, cultivates 6 days in 30 ℃.Use the 2ml water washing on the every flat board, collect washings.Get the washings that part is collected, separate application contains the YPD flat board of 1mg/ml, 2mg/ml, 3mg/ml, 4mg/mlG418, in 30 ℃, cultivates 3～6 days.The bacterium colony that picking grows on maximum concentration G418 YPD flat board is inoculated in the 20ml YPD substratum, cultivates 24 hours for 30 ℃, extracts the recombinant yeast pichia pastoris genomic dna with ordinary method, and by PCR, agarose gel electrophoresis is identified recon.Positive recombinant called after GS115/pPIC9K-(BNP)
2-HSA.
(2) fusion rotein (BNP)
2The expression of-HSA
With GS115/pPIC9K-(BNP)
2-HSA is seeded to 10ml BMGY (2% Tryptones, 1% yeast extract, 100mmol/L potassiumphosphate (pH 6.0), 1.34% no amino acid whose yeast nitrogen base (YNB), 4 * 10 is housed
-5The % vitamin H, 1% glycerine) in the 50ml triangular flask, 250rpm, cultivated 24 hours for 30 ℃, centrifugal collection thalline is seeded to the thalline of collecting 3ml BMMY (2% Tryptones is housed, 1% yeast extract, 100mmol/L potassiumphosphate (pH 6.0), 1.34% no amino acid whose yeast nitrogen base (YNB), 4 * 10
-5% vitamin H, 2% methyl alcohol) in the 50ml triangular flask, added a methyl alcohol to final concentration 2% in per 24 hours, induced 3 days, centrifugal collection supernatant carries out the SDS-PAGE checking, and fusion rotein (BNP) is identified in Western hybridization
2The BNP of-HSA molecule and HSA immunogenicity.
Embodiment 5: fusion rotein (BNP)
2The biological activity assay of-HSA
Artery bar assay method exsomatizes: get the stripped artery bar of rabbit and be cut into about 1.5cm length, the wide spiral ring of 2～3mm, hang on the 37 ℃ of tyrode's solutions (NaCl 8.0g, KCl 0.2g, the CaCl that contain the logical oxygen of 10ml
20.2g, NaH
2PO
40.05g, MgSO
47H
2O 0.1g, NaHCO
31.0g, Glucose1.0g, be made into the solution of 1000ml with distilled water, transfer pH to 7.4 with 1mol/LNaOH solution) Magnus' bath in, application of load 1g stablizes 1h, during every 20min change tyrode's solution one time.Connect polygraph, regulate sensitivity, the tension variation of record blood vessel.After treating the tensammetric curve baseline stability, the AK-Nefrin 0.05ml of adding 1.25mg/ml makes its final concentration in Magnus' bath be 6.25 * 10
-5Mg/ml, curve be increased to the highest and stable after, begin that (the stable back of curve to doubly increasing sample concentration is: 6.25 * 10 by accumulative total concentration dose regimen
-4～8.0 * 10
-2RU/ml) add recombinant human brain natriuretic peptide reference substance, the tension variation of record blood vessel.After finishing above-mentioned administration, wash-out also stablize 1h, during every 20min change tyrode's solution one time, treat baseline stability after, measure testing sample by the method for measuring reference substance, write down measurement result.That calculates reference substance and each laboratory sample partly imitates extension rate, is calculated as follows sample and tires: and sample tires=reference substance tires * and sample partly imitates extension rate/reference substance and partly imitates extension rate (RU/mL).
Claims (10)
1, the human brain natriuretic peptide diad [(BNP)
2] with the fusion rotein of human serum albumin (HSA).It is characterized in that comprising with first district of human brain natriuretic peptide at least 85% sequence homology and with second district of human serum albumin at least 85% sequence homology or partial amino-acid series second district of human serum albumin; Described and human brain natriuretic peptide homologous first district is positioned at the N-terminal of fusion rotein, and described and human serum albumin homologous second district is positioned at the C-terminal of fusion rotein, and the centre does not add any connection peptides; Or described and human serum albumin homologous second district is positioned at the N-terminal of fusion rotein, and described and human brain natriuretic peptide homologous first district are positioned at the C-terminal of fusion rotein, and the centre does not add any connection peptides.
2, the fusion rotein of human brain natriuretic peptide according to claim 1 and human serum albumin.It is characterized in that described fusion rotein comprise with first district of human brain natriuretic peptide at least 95% sequence homology and with second district of human serum albumin at least 95% sequence homology.
3, the fusion rotein of human brain natriuretic peptide according to claim 1 and human serum albumin.Second district that it is characterized in that described fusion rotein is made up of human serum albumin part-structure territory or the human serum albumin after structural domain is reset is formed.
4, the fusion rotein of human brain natriuretic peptide according to claim 1 and human serum albumin.It is characterized in that described fusion rotein comprises first district identical with the human brain natriuretic peptide amino acid residue sequence and second district identical with the human serum albumin amino acid residue sequence, or the function equivalent in above-mentioned two districts.
5, the fusion rotein of human brain natriuretic peptide according to claim 4 and human serum albumin.It is characterized in that described function equivalent is under the prerequisite that does not change described fusion rotein characteristic, the polypeptide that replacement, disappearance or the adding of the indivedual amino-acid residues of fusion rotein obtained.
6, human brain natriuretic peptide diad [(BNP according to claim 1
2)] with the preparation of the fusion rotein of human serum albumin (HSA).It is characterized in that synthetic (BNP)
2CDNA; Increase from people's tire liver cDNA library by PCR and to obtain HSA cDNA; Utilize overlapping pcr, connect (BNP)
2CDNA and HSA cDNA, the centre does not add any connection peptides, (BNP) that obtains
2-HSA cDNA fusion gene is inserted in the cloning vector to be preserved; Utilize the pichia spp secreted expression carrier with (BNP)
2-HSA cDNA fusion gene is incorporated in host's the karyomit(e) and expresses.
(1) (BNP)
2The clone of cDNA
By Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's synthetic (BNP)
2CDNA (195bp) is cloned into it on carrier pBlu2KSP, and inserting the site is Sma I, and recipient bacterium is the e. coli jm109 bacterial strain.
(2) clone of HSA cDNA
Utilize PCR to amplify HSA cDNA from people's tire liver cDNA library, used primer is:
PH1：5’-AG
GTCGACGATGCACACAAGAGTGAGGTTGCTC-3’
PH2：5’-GCC
AAGCTTTTATAAGCCTAAGGCAGCTTGACTT-3’
The PCR reaction system: each 1.5 μ l of the PH1 of 10 μ mol/L and PH2 primer, the dNTP 4 μ l of 2.5mmol/L, 10 * pfu Buffer, 5 μ l, the pfu archaeal dna polymerase 0.5 μ l of 5U/ μ l, people's tire liver cDNA library 1 μ g adds distilled water polishing 50 μ l.PCR response procedures: 95 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 1 minute, 60 ℃ of annealing 1 minute, 72 ℃ were extended 3 minutes, and circulated 30 times; 72 ℃ were extended 10 minutes.
Agarose gel electrophoresis analytical reaction product target stripe occurs at the application of sample swimming lane, reclaims the purpose segment that test kit is purified into 1.8kb with PCR fragment glue.Purpose segment that purifying is good and carrier pBlu2KSP are respectively through Sal I and Hind III double digestion, and agarose gel electrophoresis identifies that PCR fragment glue reclaims test kit and reclaims the purpose segment.T
4Dna ligase connects through the HSA of double digestion cDNA and carrier pBlu2KSP, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains X-gal, IPTG and 100 μ g/ml penbritins, 37 ℃ of overnight incubation.The picking white colony is inoculated in 20ml and contains in the LB substratum of 100 μ g/ml penbritins, and 37 ℃ of overnight incubation are extracted the conversion plasmid with the plasmid extraction test kit.Adopt pcr amplification, Sal I and Hind III double digestion are identified positive colony, and dna sequencing is carried out in zone between recombinant plasmid Sal I and Hind III double enzyme site.Positive recombinant is kept among the LB that contains 20% glycerine, freezes in-80 ℃.Recombinant plasmid called after pBlu2KSP-HSA, positive recombinant called after JM109/pBlu2KSP-HSA.
(3) (BNP)
2The clone of cDNA and HSA cDNA fusion gene
1. (BNP)
2The pcr amplification of cDNA, the primer is as follows:
PB1：5’-GCGCGC
GAATTCAAAAGATCTCCAAAGATGGTCCAAGG-3’
PB2：5’-ACCTCACTCTTGTGTGCATCGTGACGTCTCAGGACCTTGC-3’
PCR reaction system: each 1.5 μ l of the PB1 of 10 μ mol/L and PB2 primer, the dNTP 4 μ l of 2.5mmol/L, 10 * pfu Buffer, 5 μ l, the pfu archaeal dna polymerase 0.5 μ l of 5U/ μ l, plasmid template pBlu2KSP-(BNP)
21ng adds distilled water polishing 50 μ l.PCR response procedures: 95 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, 72 ℃ were extended 30 seconds, and circulated 30 times; 72 ℃ were extended 10 minutes.
2. the pcr amplification of HSAcDNA, the primer is as follows:
PH3：5’-GCAAGGTCCTGAGACGTCACGATGCACACAAGAGTGAGGT-3’
PH4：5’-CATAAG
GCGGCCGCTTATTATAAGCCTAAGGCAGCTTG-3’
PCR reaction system: each 1.5 μ l of the PH 3 of 10 μ mol/L and PH 4 primers, 2.5mmol/L dNTP 4 μ l, 10 * pfu Buffer, 5 μ l, the pfu archaeal dna polymerase 0.5 μ l of 5U/ μ l, plasmid template pBlu2KSP-HSA 1ng adds distilled water polishing 50 μ l.PCR response procedures: 95 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, 72 ℃ were extended 3 minutes, and circulated 30 times; 72 ℃ were extended 10 minutes.
3. overlapping pcr merges (BNP)
2CDNA and HSA cDNA
With (BNP)
2Pcr amplification product and the pcr amplification product of HSA dilute 10 times respectively, again with after 1: 10 mixed as template, in the 50ul reaction system, add 2 * Pfu PCR MasterMix 25ul, the template 25 μ l that mix.PCR response procedures: 95 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, 72 ℃ were extended 4 minutes, and circulated 15 times; 72 ℃ were extended 10 minutes.
With this PCR product is template, adds primer PB1 and PH 4 each 1.5 μ l of 10 μ mol/L in the reaction system of 50 μ l, 2 * Pfu PCRMasterMix 25ul, and template 4ul adds distilled water polishing 50 μ l.PCR response procedures: 95 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, 72 ℃ were extended 4 minutes, and circulated 35 times; 72 ℃ were extended 10 minutes.
Agarose gel electrophoresis analytical reaction product target stripe occurs at the application of sample swimming lane, reclaims the purpose fragment that test kit is purified into 2kb with PCR fragment glue.The overlapping PCR product and the carrier pBlu2KSP of purifying, respectively through EcoR I and Not I double digestion, agarose gel electrophoresis is identified, reclaims test kit with PCR fragment glue and reclaims (BNP)
2-HSA gene and carrier pBlu2KSP.Use T
4Dna ligase connects two and reclaims fragment, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains X-gal, IPTG and 100 μ g/ml penbritins, 37 ℃ of overnight incubation.The picking white colony is inoculated in 20ml and contains in the LB substratum of 100 μ g/ml penbritins, and 37 ℃ of overnight incubation are extracted the conversion plasmid with the plasmid extraction test kit.Adopt pcr amplification, EcoR I and Not I double digestion are identified positive colony, and dna sequencing is carried out in zone between recombinant plasmid EcoR I and Not I double enzyme site.Positive recombinant is kept among the LB that contains 20% glycerine, freezes in-80 ℃.Recombinant plasmid called after pBlu2KSP-(BNP)
2-HSA, positive recombinant called after JM109/pBlu2KSP-(BNP)
2-HSA.
(4) fusion rotein (BNP)
2The structure of the yeast expression system of-HSA
Get a frozen JM109/pBlu2KSP-(BNP)
2-HSA is inoculated into 20ml and contains in the LB substratum of 100 μ g/ml penbritins, and 37 ℃ of overnight incubation are extracted plasmid with the plasmid extraction test kit.The plasmid pBlu2KSP-(BNP) that extracts
2-HSA and Yeast expression carrier pPIC9K, respectively through EcoR I and Not I double digestion, agarose gel electrophoresis is identified, reclaims test kit with PCR fragment glue and reclaims (BNP)
2-HSA gene and carrier pPIC9K.Use T
4Dna ligase connects two and reclaims fragment, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 100 μ g/ml penbritins, 37 ℃ of overnight incubation.The bacterium colony that picking grows is inoculated in 20ml and contains in the LB substratum of 100 μ g/ml penbritins, and 37 ℃ of overnight incubation are extracted plasmid with the plasmid extraction test kit.Adopt pcr amplification, EcoR I and Not I double digestion are identified positive colony.Positive recombinant is kept among the LB that contains 20% glycerine, freezes in-80 ℃.Recombinant plasmid called after pPIC9K-(BNP)
2-HSA, positive recombinant called after JM109/pPIC9K-(BNP)
2-HSA.
Extract JM109/pPIC9K-(BNP)
2Plasmid among the-HSA, through Sal I single endonuclease digestion, after DNA glue reclaimed the test kit recovery, electric shock transformed pichia spp GS115, extracts the recombinant yeast pichia pastoris genomic dna, and pcr amplification is identified, positive recombinant called after GS115/pPIC9K-(BNP)
2-HSA.
(5) fusion rotein (BNP)
2The expression of-HSA
With GS115/pPIC9K-(BNP)
2-HSA is seeded in the 50ml triangular flask that 10ml BMGY is housed, 250rpm, cultivated 24 hours for 30 ℃, centrifugal collection thalline is seeded to the thalline of collecting in the 50ml triangular flask that 3ml BMMY is housed, and adds a methyl alcohol to final concentration 2% in per 24 hours, induced 3 days, centrifugal collection supernatant carries out the SDS-PAGE checking, and fusion rotein (BNP) is identified in Western hybridization
2The BNP of-HSA molecule and HSA immunogenicity.
7, method according to claim 6 is characterized in that host cell is bacterium, yeast, insect cell, zooblast or vegetable cell.
8, method according to claim 7 is characterized in that preferred host cell is a yeast.
9, method according to claim 8 is characterized in that preferred yeast is a pichia spp.
10, method according to claim 9 is characterized in that preferred pichia spp is pichia spp GS115 and KM71.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102391376A (en) * | 2011-03-15 | 2012-03-28 | 江苏省原子医学研究所 | Fusion protein of human somatostatin tetradecapeptide and human serum albumin, and coding gene and preparation method thereof |
| CN104231088A (en) * | 2014-09-24 | 2014-12-24 | 上海交通大学医学院 | Fusion protein of human vasonatrin peptide and human serum albumin and preparation thereof |
| CN104861075A (en) * | 2015-05-08 | 2015-08-26 | 成都金凯生物技术有限公司 | Long-acting recombinant human brain natriuretic peptide fusion protein and preparation method and thereof and application |
| CN112608385A (en) * | 2020-12-18 | 2021-04-06 | 杭州贤至生物科技有限公司 | Preparation of canine Brain Natriuretic Peptide (BNP) monoclonal antibody |
| CN113278059A (en) * | 2021-07-14 | 2021-08-20 | 天津奇云诺德生物医学有限公司 | Recombinant human BNP protein and application thereof |
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2008
- 2008-05-23 CN CNA2008100249860A patent/CN101280017A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102391376A (en) * | 2011-03-15 | 2012-03-28 | 江苏省原子医学研究所 | Fusion protein of human somatostatin tetradecapeptide and human serum albumin, and coding gene and preparation method thereof |
| CN104231088A (en) * | 2014-09-24 | 2014-12-24 | 上海交通大学医学院 | Fusion protein of human vasonatrin peptide and human serum albumin and preparation thereof |
| CN104861075A (en) * | 2015-05-08 | 2015-08-26 | 成都金凯生物技术有限公司 | Long-acting recombinant human brain natriuretic peptide fusion protein and preparation method and thereof and application |
| CN104861075B (en) * | 2015-05-08 | 2018-06-08 | 成都金凯生物技术有限公司 | A kind of long-acting recombinant human brain natriuretic peptide fusion protein and preparation method thereof and purposes |
| CN112608385A (en) * | 2020-12-18 | 2021-04-06 | 杭州贤至生物科技有限公司 | Preparation of canine Brain Natriuretic Peptide (BNP) monoclonal antibody |
| CN112608385B (en) * | 2020-12-18 | 2022-05-20 | 杭州贤至生物科技有限公司 | Preparation of canine Brain Natriuretic Peptide (BNP) monoclonal antibody |
| CN113278059A (en) * | 2021-07-14 | 2021-08-20 | 天津奇云诺德生物医学有限公司 | Recombinant human BNP protein and application thereof |
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