CN101063123A - Preparation method for fused protein of human C-type diuresis promotion sodium peptide and human serum albumins and its product - Google Patents

Abstract

The invention discloses a preparing method of blend protein of human C-type diuretic sodium peptide (CNP) and human blood serum albumin (HSA) and product in long-acting recombinant blend protein medical technical domain, which comprises the following steps: adopting overlapping PCR technique; splitting HSA cDNA and CNP cDNA; non-adding into any connecting peptide; getting HSA-CNP cDNA fuse gene; integrating into colorant layer of host; expressing in host system; getting the product. This product incorporates the first zone with at least 85% sequence isogenesis with human blood serum albumin and the second zone with at least 85% sequence isogenesis with human C-type diuretic sodium peptide, which possesses good application prospect in medical domain.

Description

The preparation method and the product of the fusion rotein of people C-type natriuretic peptide and human serum albumin

Technical field

The preparation method and the product of the fusion rotein of people C-type natriuretic peptide and human serum albumin belong to long-acting recombination fusion protein technical field of pharmaceuticals.

Background technology

Human serum albumin (Human serum albumin, HSA) be major protein composition in the blood plasma, concentration in blood plasma is 40mg/ml (Phillip P.Minghettis et al., THE JOURNAL OFBIOLOGICAL CHEMISTRY, 1986 261:6747-6757), it can also comprise hormone, toxic metabolite product, medicine etc. in conjunction with endogenous and/or exogenous part except having the infiltration of plasma of keeping compression functions.By in conjunction with these parts, HSA can regulate the toxicity of hormonal activity, endogenous and/or exogenous material and the availability of medicine (Ji-Sook Haet al., Biochimica et Biophysica Acta, 20031640:119-128).The HalfHSA of structures such as David S.Park (preceding 297 amino-acid residues of intercepting HSA) has the similar secondary structure of wild-type HSA respective area, kept ability (the David S.Park et al. of wild-type HSA simultaneously in conjunction with thyroxine and thyroxine analogues, IUBMB Life, 1999 48:169-174), the human serum albumin that translation is just come out in the cell has typical pre-pro-protein structure, comprises the signal peptide of 18 amino-acid residues and the former peptide of 6 amino-acid residue propetides.Signal peptide and propetide are cut in transhipment and excretory process, and sophisticated HSA is made up of 585 amino-acid residues, contains 17 pairs of disulfide linkage, and molecular weight is about 66.5KDa.Generally, HSA is a non-glycosylated protein, yet also exists N-to connect glycosylation (seeing the protein ALBU_HUMAN among the Uniprot) in the mutant that has.The viewpoint of Peters think evolve out the advantage of high molecular weight protein be reduce its when internal recycle through renal excretion (Peters T.Jr., Adv.Protein Chem., 1985 37:161-245).The molecular weight of HSA is relatively large, under normal circumstances, is difficult for by glomerular filtration, and its plasma half-life is about 20 days.

People C-type natriuretic peptide (typc C natriutetic peptides, CNP) mainly be present in central nervous system, synthetic and the secretion by vascular endothelial cell, by with the vascular smooth muscle cell of target cell on specificity NP-B receptors bind, the guanylate cyclase that activation links to each other with plasma membrane, promote second messenger cGMP accumulation, cause cGMP dependent kinases phosphorylation, by reducing intracellular calcium concentration, make vascular smooth muscle relaxation, reach and regulate the effect of local vascular tensile, and stop the vascular smooth muscle hyperplasia.CNP is a kind of peripheral vein expander of non-endothelium-dependent relaxation by force, and the latent effect in many nervous system disorderss and other disease is worth and more and more is subjected to extensive attention.CNP concentration is very low in the blood plasma, exists with pg concentration, and the transformation period is shorter, is about 2.6 minutes, and these characteristics have caused very big obstacle for the medicineization of CNP, and the medicineization of research CNP at first will be improved its stability, improves its bioavailability.In order to overcome above-mentioned shortcoming, can be modified CNP, to prolong its transformation period.Main method is made slow release formulation, utilizes chemical means to modify (as using PEG, glucan-modified), is utilized genetic engineering means that itself and other high molecular weight protein is merged.The present invention will carry out amalgamation and expression to CNP and HSA, by the long-actingization technology of research and development people CNP, improve pharmacokinetics proterties in the CNP body, develop a kind of long-acting new drug with independent intellectual property right.

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 people C-type 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 people C-type natriuretic peptide, at pharmaceutical field good prospects for application is arranged.

Technical scheme of the present invention: from human vascular endothelial, extract RNA, obtain CNP cDNA by the RT-PCR reverse transcription; Increase from people's tire liver cDNA library by PCR and to obtain HSA cDNA; Utilize overlapping pcr, connect HSA cDNA and CNP cDNA, the centre does not add any connection peptides, and the HSA-CNP cDNA fusion gene that obtains is inserted in the cloning vector to be preserved; HSA-CNP cDNA fusion gene is expressed at host system, and HSA-CNP cDNA fusion gene is integrated in host's the karyomit(e).

The clone of CNP cDNA, the clone of HSA cDNA, the clone of HSA cDNA and CNP cDNA fusion gene, the step of the recombination yeast structure of fusion rotein HSA-CNP and the expression of fusion rotein HSA-CNP sees embodiment for details.

With the people C-type natriuretic peptide of method for preparing and the fusion rotein of human serum albumin, comprise with first district of human serum albumin at least 85% sequence homology and with second district of people C-type natriuretic peptide at least 85% sequence homology; Described and human serum albumin homologous first district is positioned at the N-terminal of fusion rotein, and described and people C-type natriuretic peptide homologous second district is positioned at the C-terminal of fusion rotein, and the centre does not add any connection peptides; Or described and people C-type natriuretic peptide homologous second district is positioned at the N-terminal of fusion rotein, and described and human serum albumin 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 serum albumin at least 95% sequence homology and with second district of people C-type natriuretic peptide at least 95% sequence homology.

First district of described fusion rotein can be made up of or human serum albumin after structural domain is reset is formed human serum albumin part-structure territory, 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 comprise first district identical with the human serum albumin amino acid residue sequence and with the second identical district of people C-type natriuretic peptide 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.

Another content of the present invention provides the host of expressed fusion protein coding region.Host system is expression systems such as bacterium, yeast, insect cell, zooblast or vegetable cell.Preferred host system is a yeast.Preferred yeast is a pichia spp.Preferred pichia spp is pichia spp KM71.

CNP cDNA 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.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 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.

The pichia yeast expression system secreted expression carrier mainly contains 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 spp 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.

Have the expression vector that merges cDNA and can transform host system by transforming lithium salts method, PEG method, protoplasm body or electroporation.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 CNP antibody test nutrient solution supernatant behind abduction delivering.

Beneficial effect of the present invention:

Utilize overlapping pcr, connect HSA cDNA and CNP cDNA, the centre does not add any connection peptides, to avoid adding the fusion rotein stability (avoiding the proteasome degradation at connection peptides) brought 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 system of the DNA of the present invention's structure by cultivation, produce fusion rotein of the present invention.Host system is cultivated and is preferably carried out on bio-reactor, cultivates and divides two stages, and the fs is mainly used in the host system 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 saltout, the combination of technology such as precipitation, ultrafiltration, chromatography, preparation electrophoresis and these technology.

Description of drawings

Fig. 1. plasmid pPIC9K-HSA-CNP makes up

Fig. 2. the agarose electrophoretic analysis Lane1:DNA Mark of antigen-4 fusion protein gene; The Lane2:PCR product

Fig. 3. the SDS-PAGE of fusion rotein analyzes Lane1: protein Mark; Lane2:HSA-CNP

Fig. 4. the HSA antibody test Lane1 of fusion rotein: protein Mark; Lane2:HSA-CNP

Specific implementation method

The clone of embodiment 1:CNP cDNA

Extract RNA from human vascular endothelial, CNP cDNA first chain that obtains by the RT-PCR reverse transcription is a template, goes out CNP cDNA by pcr amplification, and used primer is as follows:

p1：5’C GGAATTCCACCATGCATCTCTCCCA-3’

p2：5’AAT GCGGCCGCCTAACATCCCAGGCCGCT-3’

PCR method is as follows: add in the reaction system of 50 μ l: each 1.5 μ l of the p1 of 10 μ mol/L and the primer of p2, the dNTP 5 μ l of 2mmol/L, 10 * pfu Buffer, 5 μ l, the pfu archaeal dna polymerase 0.5 μ l of 5U/ μ l, CNPcDNA first chain 1 μ g, add distilled water polishing 50 μ l, the PCR condition is: 95 ℃ of sex change 10 minutes, 94 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, 72 ℃ were extended 90 seconds, and circulated 35 times;

By agarose gel electrophoresis analytical reaction product, target stripe appears at the application of sample swimming lane, reclaim the target fragment that test kit is purified into 0.5kb with PCR fragment glue, target fragment that purifying is good and carrier pBluescriptII KS (+) are respectively through EcoR I and NotI double digestion; The agarose gel electrophoresis purifying reclaims test kit with PCR fragment glue and reclaims, and uses T ₄Ligase enzyme connects the good fragment of purifying behind two double digestions; Connect product transformed into escherichia coli DH5 α competent cell, 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 37 ℃ of overnight incubation in the LB substratum of 100 μ g/ml penbritins, extracts plasmid; By PCR, and EcoR I and NotI double digestion, agarose gel electrophoresis is identified positive colony, and dna sequencing is carried out in zone between recombinant plasmid EcoR I and NotI double enzyme site; Positive recombinant is kept among the LB that contains 20% glycerine, and is frozen in-80 ℃; Recombinant plasmid called after pBlue-CNP, positive recombinant called after DH5 α/pBlue-CNP;

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’

PCR method is as follows: add in the reaction system of 50 μ l: each 1.5 μ l of the PH1 of 10 μ mol/L and the primer of PH2, the dNTP 5 μ l of 2mmol/L, 10 * pfu Buffer, 1 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; The PCR condition is: 95 ℃ of sex change 5 minutes, and 94 ℃ of sex change 1 minute, 60 ℃ of annealing 1 minute, 72 ℃ were extended 90 seconds, and circulated 30 times;

By agarose gel electrophoresis analytical reaction product, target stripe appears at the application of sample swimming lane, reclaim the target fragment that test kit is purified into 1.8kb with PCR fragment glue, target fragment that purifying is good and carrier pBluescriptII KS (+) are respectively through SalI and HindIII double digestion; The agarose gel electrophoresis purifying reclaims test kit with PCR fragment glue and reclaims, and uses T ₄Ligase enzyme connects the good fragment of purifying behind two double digestions; Connect product transformed into escherichia coli DH5 α competent cell, 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 37 ℃ of overnight incubation in the LB substratum of 100 μ g/ml penbritins, extracts plasmid; By PCR, and SalI and HindIII double digestion, agarose gel electrophoresis is identified positive colony, and dna sequencing is carried out in zone between recombinant plasmid SalI and HindIII double enzyme site; Positive recombinant is kept among the LB that contains 20% glycerine, and is frozen in-80 ℃; Recombinant plasmid called after pBlue-HSA, positive recombinant called after DH5 α/pBlue-HSA;

The clone of embodiment 3:HSA cDNA and CNP cDNA fusion gene

The pcr amplification of HSA cDNA, the primer is as follows:

P1：5’-CG GAATTCAAAAGAGATGCACACAAGAGTGAGGTTGCTCATCG-3’

P2：5’-AGCAGCTGGGAGAGATGCATGCCTAAGGCAGCTTGACTTGCAG-3’

PCR method is as follows: add in the reaction system of 50 μ l: each 1.5 μ l of the primer P1 of 10 μ mol/L and P2, the dNTP 5 μ l of 2mmol/L, 10 * pfu Buffer, 5 μ l, the pfu archaeal dna polymerase 0.5 μ l of 5U/ μ l, pBlue-HSA 1 μ g adds distilled water polishing 50 μ l; The PCR condition is: 95 ℃ of sex change 5 minutes, and 65 ℃ of annealing 1 minute, 72 ℃ were extended 4 minutes, and 94 ℃ of sex change 1 minute circulate 30 times;

The pcr amplification of CNP cDNA, the primer is as follows:

P3：5’-CAAGTCAAGCTGCCTTAGGCATGCATCTCTCCCAGCTGCT-3’

P4：5’-AAT GCGGCCGCCTAACATCCCAGGCCGCTCATGGAGCC-3’

PCR method is as follows: add in the reaction system of 50 μ l: each 1.5 μ l of the P3 of 10 μ mol/L and the primer of P4, the dNTP 5 μ l of 2mmol/L, 10 * pfu Buffer, 5 μ l, the pfu archaeal dna polymerase 0.5 μ l of 5U/ μ l, pBlue-CNP 1 μ g adds distilled water polishing 50 μ l; The PCR condition is: 95 ℃ of sex change 10 minutes, and 56 ℃ of annealing 45 seconds, 72 ℃ were extended 90 seconds, and 94 ℃ of sex change 1 minute circulate 30 times;

Utilize overlapping PCR to merge CNPcDNA and HSA cDNA:

With the pcr amplification product of the pcr amplification product of CNP and HSA with 1: 1 mixed of volume ratio after as template, in the PCR reaction system of 50 μ l, add: the dNTP4 μ l of 2.5mmol/L, 10 * pfuBuffer, 5 μ l, 5U/ μ l pfu archaeal dna polymerase 1 μ l, the template 2 μ l that mix, distilled water 36 μ l; The PCR condition is: 95 ℃ of abundant sex change 10min, and 65 ℃ of annealing 1min, 72 ℃ are extended 4min30s, 94 ℃ of sex change 1min.Circulate after 8 times, in reaction system, add the P1 of 10 μ mol/L and each 1 μ l of primer of P4, circulate 30 times;

By agarose gel electrophoresis analytical reaction product, target stripe appears at the application of sample swimming lane, reclaim the target stripe that test kit is purified into 2.2kb with PCR fragment glue, target fragment that purifying is good and carrier pBluescriptII KS (+) are respectively through EcoRI and NotI double digestion; The agarose gel electrophoresis purifying reclaims test kit with PCR fragment glue and reclaims, and gets the good pBlue-HSA-CNP fragment 5 μ l of purifying behind the double digestion, pPIC9K 5 μ l add 10 * ligation buffer, 2 μ l, T4 ligase enzyme 0.4 μ l, add distilled water polishing 20 μ l, 15 ℃ of reactions are spent the night; Connect product transformed into escherichia coli DH5 α competent cell, conversion product is coated the LB agar plate that contains 100 μ g/ml penbritins, 37 ℃ of overnight incubation; Picking colony is inoculated in 20ml and contains 37 ℃ of overnight incubation in the LB substratum of 100 μ g/ml penbritins, extracts plasmid; By PCR, and EcoRI and NotI double digestion, agarose gel electrophoresis is identified positive colony, and dna sequencing is carried out in zone between recombinant plasmid EcoRI and NotI restriction enzyme site; Positive recombinant is kept among the LB that contains 20% glycerine, and is frozen in-80 ℃; Recombinant plasmid called after pBlue-HSA-CNP, positive recombinant called after DH5 α/pBlue-HSA-CNP;

Embodiment 4:HSA-CNP expression of recombinant yeast system constructing and Expression of Fusion Protein:

The recombination yeast of fusion rotein HSA-CNP makes up:

Get a frozen DH5 α/pBlue-HSA-CNP, be inoculated into 20ml and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/ml penbritins, extract plasmid, the plasmid pBlue-HSA-CNP and the Yeast expression carrier pPIC9K that extract, respectively through EcoRI and NotI double digestion, the agarose gel electrophoresis purifying reclaims test kit with PCR fragment glue and reclaims, and uses T ₄Ligase enzyme connects the good fragment of purifying behind two double digestions, gets the good pBlue-HSA-CNP fragment 5 μ l of purifying behind the double digestion, pPIC9K 5 μ l, add 2 μ l, 10 * ligationbuffer, 0.4 μ l T4 ligase enzyme adds distilled water polishing 20 μ l, 15 ℃ of reactions are spent the night.Connect product transformed into escherichia coli DH5 α competent cell, 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 37 ℃ of overnight incubation in the LB substratum of 20ml penbritin (100 μ g/ml), extracts plasmid with ordinary method; By PCR, and EcoRI and NotI double digestion, agarose gel electrophoresis is identified positive colony, positive recombinant is kept among the LB that contains 20% glycerine, and is frozen in-80 ℃; Recombinant plasmid called after pPIC9K-HSA-CNP, positive recombinant called after DH5 α/pPIC9K-HSA-CNP;

Extract the plasmid among DH5 α/pPIC9K-HSA-CNP, after the SalI enzyme was cut, electric shock transformed pichia spp KM71, and conversion product is coated on the MD flat board that contains the 1mol/L sorbyl alcohol, in 30 ℃, cultivates 6 days.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.Picking grows bacterium colony on the YPD of maximum concentration G418 flat board, be inoculated in the 20mlMD substratum, cultivates 24 hours for 30 ℃, extract the recombinant yeast pichia pastoris genomic dna with ordinary method, by PCR, agarose gel electrophoresis is identified, positive recombinant called after KM71/pPIC9K-HSA-CNP;

The expression of fusion rotein HSA-CNP:

KM71/pPIC9K-HSA-CNP is seeded to 100mlBMGY (100mM potassiumphosphate, pH 6.0,1.34% no amino acid whose yeast nitrogen base, 4 * 10 are housed ^-5% vitamin H, 1% glycerine) in the 500ml triangular flask, 300rpm, 29 ℃ are cultured to the A600nm value is 2～6, centrifugal collection thalline.The thalline of collecting is seeded to 20ml BMMY (100mM potassiumphosphate, pH 6.0,1.34% no amino acid whose yeast nitrogen base, 4 * 10 are housed ^-5% vitamin H, 0.5% methyl alcohol) the 100ml triangular flask in, added a methyl alcohol to final concentration 0.5% (V/V), induced 3 days in per 24 hours.Centrifugal collection supernatant, the SDS-PAGE checking is carried out in filtration sterilization, and the immunogenicity of the CNP of fusion rotein HSA-CNP molecule is identified in Western hybridization.

With the HSA-CNP in the radioimmunology evaluation supernatant.Illustration method with reference to C-type natriuretic peptide radioimmunological kit (HY-044) is measured.Get the good fermented supernatant fluid 200 μ l of dilution, add anti-100 μ l, shake up and be positioned over 37 ℃ of water-baths, taking-up adds after 6 hours ¹²⁵I-CNP mark 100 μ l shake up in 4 ℃ of placements and spend the night, and add 500 μ l separating agents (two is anti-) afterwards, and the mixing room temperature was placed 20 minutes, and centrifugal 25 minutes of 3500rpm removes supernatant, are measured precipitation cpm value and are read the result according to typical curve by γ-numeration instrument.Western hybridization shows that product has the immunogenicity of CNP and HSA.

Claims (10)

1, the preparation method of the fusion rotein of people C-type natriuretic peptide CNP and human serum albumin HSA is characterized in that extracting RNA from human vascular endothelial, obtain CNP cDNA by the RT-PCR reverse transcription; Increase from people's tire liver cDNA library by PCR and to obtain HSA cDNA; Use overlapping pcr, connect HSA cDNA and CNP cDNA, the centre does not add any connection peptides, the HSA-CNP cDNA fusion gene that obtains is inserted into cloning vector, HSA-CNP cDNA fusion gene is expressed at host system, and HSA-CNP cDNA fusion gene is integrated in host's the karyomit(e);

(1) clone of CNP cDNA:

CNP cDNA first chain that obtains with the RT-PCR reverse transcription is a template, goes out CNPcDNA by pcr amplification, and used primer is as follows:

p1：5’CG GAATTCCACCATGCATCTCTCCCA-3’

p2：5’AAT GCGGCCGCCTAACATCCCAGGCCGCT-3’

PCR method is as follows: add in the reaction system of 50 μ l: each 1.5 μ l of the p1 of 10 μ mol/L and the primer of p2, the dNTP 5 μ l of 2mmol/L, 10 * pfu Buffer, 5 μ l, the pfu archaeal dna polymerase 0.5 μ l of 5U/ μ l, CNP cDNA first chain 1 μ g, add distilled water polishing 50 μ l, the PCR condition is: 95 ℃ of sex change 10 minutes, 94 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, 72 ℃ were extended 90 seconds, and circulated 35 times;

By agarose gel electrophoresis analytical reaction product, target stripe appears at the application of sample swimming lane, reclaim the target fragment that test kit is purified into 0.5kb with PCR fragment glue, target fragment that purifying is good and carrier pBluescriptIIKS (+) are respectively through EcoRI and NotI double digestion; The agarose gel electrophoresis purifying reclaims test kit with PCR fragment glue and reclaims, and uses T ₄Ligase enzyme connects the good fragment of purifying behind two double digestions; Connect product transformed into escherichia coli DH5 α competent cell, 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 37 ℃ of overnight incubation in the LB substratum of 100 μ g/ml penbritins, extracts plasmid; By PCR, and EcoR I and NotI double digestion, agarose gel electrophoresis is 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, and is frozen in-80 ℃; Recombinant plasmid called after pBlue-CNP, positive recombinant called after DH5 α/pBlue-CNP;

(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’

PCR method is as follows: add in the reaction system of 50 μ l: each 1.5 μ l of the PH1 of 10 μ mol/L and the primer of PH2, the dNTP 5 μ l of 2mmol/L, 10 * pfu Buffer, 1 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; The PCR condition is: 95 ℃ of sex change 5 minutes, and 94 ℃ of sex change 1 minute, 60 ℃ of annealing 1 minute, 72 ℃ were extended 90 seconds, and circulated 30 times;

By agarose gel electrophoresis analytical reaction product, target stripe appears at the application of sample swimming lane, reclaim the target fragment that test kit is purified into 1.8kb with PCR fragment glue, target fragment that purifying is good and carrier pBluescriptIIKS (+) are respectively through SalI and HindIII double digestion; The agarose gel electrophoresis purifying reclaims test kit with PCR fragment glue and reclaims, and uses T ₄Ligase enzyme connects the good fragment of purifying behind two double digestions; Connect product transformed into escherichia coli DH5 α competent cell, 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 37 ℃ of overnight incubation in the LB substratum of 100 μ g/ml penbritins, extracts plasmid; By PCR, and SalI and HindIII double digestion, agarose gel electrophoresis is identified positive colony, and dna sequencing is carried out in zone between recombinant plasmid SalI and HindIII double enzyme site; Positive recombinant is kept among the LB that contains 20% glycerine, and is frozen in-80 ℃; Recombinant plasmid called after pBlue-HSA, positive recombinant called after DH5 α/pBlue-HSA;

(3) clone of HSA cDNA and CNP cDNA fusion gene:

The pcr amplification of HSA cDNA, the primer is as follows:

P1：5’-CG GAATTCAAAAGAGATGCACACAAGAGTGAGGTTGCTCATCG-3’

P1：5’-AGCAGCTGGGAGAGATGCATGCCTAAGGCAGCTTGACTTGCAG-3’

PCR method is as follows: add in the reaction system of 50 μ l: each 1.5 μ l of the primer P1 of 10 μ mol/L and P2, the dNTP 5 μ l of 2mmol/L, 10 * pfu Buffer, 5 μ l, the pfu archaeal dna polymerase 0.5 μ l of 5U/ μ l, pBlue-HSA 1 μ g adds distilled water polishing 50 μ l; The PCR condition is: 95 ℃ of sex change 5 minutes, and 65 ℃ of annealing 1 minute, 72 ℃ were extended 4 minutes, and 94 ℃ of sex change 1 minute circulate 30 times;

The pcr amplification of CNP cDNA, the primer is as follows:

P3：5’-CAAGTCAAGCTGCCTTAGGCATGCATCTCTCCCAGCTGCT-3’

P4：5’-AAT GCGGCCGCCTAACATCCCAGGCCGCTCATGGAGCC-3’

PCR method is as follows: add in the reaction system of 50 μ l: each 1.5 μ l of the P3 of 10 μ mol/L and the primer of P4, the dNTP 5 μ l of 2mmol/L, 10 * pfu Buffer, 5 μ l, the pfu archaeal dna polymerase 0.5 μ l of 5U/ μ l, pBlue-CNP 1 μ g adds distilled water polishing 50 μ l; The PCR condition is: 95 ℃ of sex change 10 minutes, and 56 ℃ of annealing 45 seconds, 72 ℃ were extended 90 seconds, and 94 ℃ of sex change 1 minute circulate 30 times;

Utilize overlapping PCR to merge CNPcDNA and HSA cDNA:

With the pcr amplification product of the pcr amplification product of CNP and HSA with 1: 1 mixed of volume ratio after as template, in the PCR reaction system of 50 μ l, add: the dNTP 4 μ l of 2.5mmol/L, 10 * pfuBuffer, 5 μ l, 5U/ μ l pfu archaeal dna polymerase 1 μ l, the template 2 μ l that mix, distilled water 36 μ l; The PCR condition is: 95 ℃ of sex change 10min, and 65 ℃ of annealing 1min, 72 ℃ are extended 4min30s, and 94 ℃ of sex change 1min circulates after 8 times, and the primer that adds the P1 of 10 μ mol/L and P4 in reaction system is 1 μ l respectively, circulates 30 times;

By agarose gel electrophoresis analytical reaction product, target stripe appears at the application of sample swimming lane, reclaim the target stripe that test kit is purified into 2.2kb with PCR fragment glue, target fragment that purifying is good and carrier pBluescriptIIKS (+) are respectively through EcoRI and NotI double digestion; The agarose gel electrophoresis purifying reclaims test kit with PCR fragment glue and reclaims, and gets the good pBlue-HSA-CNP fragment 5 μ l of purifying behind the double digestion, pPIC9K 5 μ l add 10 * ligation buffer, 2 μ l, T4 ligase enzyme 0.4 μ l, add distilled water polishing 20 μ l, 15 ℃ of reactions are spent the night; Connect product transformed into escherichia coli DH5 α competent cell, conversion product is coated the LB agar plate that contains 100 μ g/ml penbritins, 37 ℃ of overnight incubation; Picking colony is inoculated in 20ml and contains 37 ℃ of overnight incubation in the LB substratum of 100 μ g/ml penbritins, extracts plasmid; By PCR, and EcoRI and NotI double digestion, agarose gel electrophoresis is identified positive colony, and dna sequencing is carried out in zone between recombinant plasmid EcoRI and NotI restriction enzyme site; Positive recombinant is kept among the LB that contains 20% glycerine, and is frozen in-80 ℃; Recombinant plasmid called after pBlue-HSA-CNP, positive recombinant called after DH5 α/pBlue-HSA-CNP;

(4) recombination yeast of fusion rotein HSA-CNP makes up:

Get a frozen DH5 α/pBlue-HSA-CNP, be inoculated into 20ml and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/ml penbritins, extract plasmid, the plasmid pBlue-HSA-CNP and the Yeast expression carrier pPIC9K that extract, respectively through EcoRI and NotI double digestion, the agarose gel electrophoresis purifying reclaims test kit with PCR fragment glue and reclaims, and uses T ₄Ligase enzyme connects the good fragment of purifying behind two double digestions; Get the good pBlue-HSA-CNP fragment 5 μ l of purifying behind the double digestion, pPIC9K 5 μ l, add 2 μ l, 10 * ligationbuffer, 0.4 μ l T4 ligase enzyme, add distilled water polishing 20 μ l, 15 ℃ of reactions are spent the night, and connect product transformed into escherichia coli DH5 α competent cell, conversion product is coated the LB agar plate that contains 100 μ g/ml penbritins, 37 ℃ of overnight incubation; Picking grows bacterium colony, is inoculated in 20ml and contains 37 ℃ of overnight incubation in the LB substratum of 100 μ g/ml penbritins, extracts plasmid; By PCR, and EcoRI and NotI double digestion, agarose gel electrophoresis is identified positive colony, positive recombinant is kept among the LB that contains 20% glycerine, freezes in-80 ℃; Recombinant plasmid called after pPIC9K-HSA-CNP, positive recombinant called after DH5 α/pPIC9K-HSA-CNP;

Extract the plasmid among DH5 α/pPIC9K-HSA-CNP, after the SalI enzyme was cut, electric shock transformed pichia spp KM71, extracted the recombinant yeast pichia pastoris genomic dna, and by PCR, agarose gel electrophoresis is identified, positive recombinant called after KM71/pPIC9K-HSA-CNP;

(5) expression of fusion rotein HSA-CNP:

KM71/pPIC9K-HSA-CNP is seeded in the 500ml triangular flask that 100ml BMGY is housed, 300rpm, 29 ℃ are cultured to A _600nmBe 2～6, centrifugal collection thalline, the thalline of collecting is seeded in the 100ml triangular flask that 20ml BMMY is housed, added a methyl alcohol in per 24 hours to final concentration 0.5% (V/V), induced 3 days, centrifugal collection supernatant carries out the SDS-PAGE checking, and Western hybridization and radioimmunity are identified the immunogenicity of the CNP of fusion rotein HSA-CNP molecule.

2, with the people C-type natriuretic peptide of the described method of claim 1 preparation and the fusion rotein of human serum albumin, it is characterized in that comprising with first district of human serum albumin at least 85% sequence homology and with second district of people C-type natriuretic peptide at least 85% sequence homology; Described and human serum albumin homologous first district is positioned at the N-terminal of fusion rotein, and described and people C-type natriuretic peptide homologous second district is positioned at the C-terminal of fusion rotein, and the centre does not add any connection peptides; Or described and people C-type natriuretic peptide homologous second district is positioned at the N-terminal of fusion rotein, and described and human serum albumin homologous first district are positioned at the C-terminal of fusion rotein, and the centre does not add any connection peptides.

3, the fusion rotein of people C-type natriuretic peptide according to claim 2 and human serum albumin, it is characterized in that described fusion rotein comprise with first district of human serum albumin at least 95% sequence homology and with second district of people C-type natriuretic peptide at least 95% sequence homology.

4, the fusion rotein of people C-type natriuretic peptide according to claim 2 and human serum albumin, first 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.

5, the fusion rotein of people C-type natriuretic peptide according to claim 2 and human serum albumin, it is characterized in that described fusion rotein comprise first district identical with the human serum albumin amino acid residue sequence and with the second identical district of people C-type natriuretic peptide amino acid residue sequence, or the function equivalent in above-mentioned two districts.

6, the fusion rotein of people C-type natriuretic peptide according to claim 5 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.

7, method according to claim 1 is characterized in that host system is bacterium, yeast, insect cell, zooblast or vegetable cell expression system.

8, method according to claim 7 is characterized in that preferred host system 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 KM71.

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