CN1884520A - Preparation method of fusion protein of human interleukin-2 and human serum albumin and product - Google Patents

Abstract

The method for preparation of molten protein of human interleukin-2 (IL-2) and human serum albumins (HSA) and its products belong to long-acting molten protein drug technical sphere. The invention uses overlapped PCR skill to connect IL-2cDNA and HSAcDNA, without adding any junction peptide in middle, and the obtained IL-2-HSA cDNA molten body expresses in host system and is integrated to host chromosome; the molten protein comprises the first area homologous with 85 % sequence of human interleukin-2 at least and the second area homologous with 85 % sequence of human serum albumins at least, and the said molten protein can perform substitution, deletion or admission to individual amino acid residue without changing molten protein specificity; the host expression system can be bacteria, yeast, insect cell, animal cell or plant cell. The molten protein of this invention can prolong half-decay time in vivo on base that human interleukin-2 physiological characteristic is kept, which has good application foreground in pharmic domain.

Description

The preparation method of the fusion rotein of human interleukin-2 and human serum albumin and product

Technical field

The preparation method of the fusion rotein of human interleukin-2 and human serum albumin and product belong to long-acting fusion rotein 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 Ha et al., Biochimica et Biophysica Acta, 2003 640:119-128).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., 198537:161-245).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.

(IL-2 is immune response and immunoregulatory core substance Interleukin-2) to interleukin-2, has effect antitumor, antiviral and the enhancing body immunological competence.It also is a class Hemopoietic factor, and the interleukin-2 of reorganization is used for the thrombopenia symptom that causes because of chemotherapy clinically.Simultaneously, it all plays vital regulating and controlling effect to the immunocompetence and the intensity of T cell.Interleukin II is that Morgan in 1976 etc. at first find in peripheral blood lymphocyte.Its molecular weight is 14.5KD, is made up of 133 amino-acid residues; Contain 1 pair of disulfide linkage (C58-C105), but at 125 a halfcystine is arranged equally, so the possibility of mispairing is arranged, if mispairing, the activity of interleukin-2 just has very large loss.The common interleukin-2 transformation period in vivo is very short, has only about 10 minutes, and easily by glomerular filtration.In order to reach result of treatment, generally need frequent heavy dose of medication, frequent heavy dose of medication has not only increased patient's misery and medical expense, and is easy to generate toxic side effect.In order to overcome above-mentioned shortcoming, can be modified interleukin-2, to prolong its transformation period.Main method is made slow release formulation, is utilized chemical means to modify (as using PEG, glucan-modified), utilize genetic engineering means, and itself and other high molecular weight protein is merged.So human interleukin-2 and human serum albumin are merged in the present invention.

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 interleukin-2 and human serum albumin.

Technical scheme of the present invention: by extracting human peripheral blood mononuclear cell mRNA, reverse transcription obtains IL-2 cDNA then, and IL-2 cDNA is cloned in the pMD19T carrier, PCR from the carrier IL-2-pMD19T that contains IL-2 cDNA directly amplification obtain IL-2 cDNA; Increase from contain HSA cDNA plasmid by PCR and to obtain HAS cDNA; Utilize overlapping pcr, connect IL-2 cDNA and HSAcDNA, the centre does not add any connection peptides, and the IL-2-HSA cDNA syzygy that obtains is inserted into cloning vector; IL-2-HAS cDNA syzygy is expressed at host system, and IL-2-HSA cDNA syzygy is integrated in host's the karyomit(e).

The extraction of template mRNA, the reverse transcription of IL-2-cDNA and clone, the amplification of IL-2 cDNA, the amplification of HSAcDNA, IL-2 cDNA is connected with HSA cDNA's, and the yeast expression system of fusion rotein IL-2-HSA makes up, the expression of fusion rotein IL-2-HSA, its operation steps sees embodiment for details.

With the human interleukin-2 of method for preparing and the fusion rotein of human serum albumin, comprise with first district of human interleukin-2 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 interleukin-2 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 interleukin-2 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 interleukin-2 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 or human serum albumin after structural domain is reset is formed human serum albumin part-structure territory, comprise outside the naturally occurring multiformity of its 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 interleukin-2 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 expression system is bacterium, yeast, insect cell, zooblast or vegetable cell expression system.Yeast preferably.Preferred yeast is a pichia spp.Preferred pichia spp is pichia spp KM71.

Another content of the present invention provides the host of expressed fusion protein coding region.IL-2 cDNA and HSAcDNA also can obtain by the method for synthetic.This method can be selected the codon of host's preference artificially, so that goal gene efficiently expresses.Utilize overlapping pcr, connect IL-2 cDNA and HSAcDNA, the centre does not add any connection peptides, and avoiding because of connection peptides adds the fusion rotein stability bring and the problem of immunogenicity aspect, IL-2 cDNA that obtains and HSA cDNA syzygy are inserted in the cloning vector preserves.

IL-2 cDNA and HSA cDNA syzygy can be expressed in a plurality of 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 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.Preferred pichia spp is pichia spp KM71.

The pichia yeast expression system secreted expression carrier mainly contains pPIC9, pPIC9k, pHIL-S1, pPICZa, pYAM75P6; The type expression vector mainly contains in the born of the same parents: pPIC3K, pPICZ, pHWO10, pGAPZ, pGAPZa (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.The two kinds of alcohol oxidases of in pichia spp, encoding altogether, 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 IL-2 antibody test nutrient solution supernatant behind abduction delivering.

Beneficial effect of the present invention: utilize overlapping pcr, connect IL-2 cDNA 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.

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.

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 saltout, the combination of technology such as precipitation, ultrafiltration, chromatography, preparation electrophoresis and these technology.

Description of drawings

The foundation of Fig. 1 typical curve.

Fig. 2 pPIC9K-IL-2-HSA makes up.

The SDS-PAGE of Fig. 3 fusion rotein analyzes, and 1, sample, applied sample amount 7 μ l; 2, Marker.

The HSA antibody test Western blot of Fig. 4 fusion rotein analyzes; 1, sample; 2, Marker.

Specific implementation method

Embodiment 1: the extraction of template mRNA

Take the human peripheral 10mL of EDTA anti-freezing, in 2 hours, carry out lymphocytic separation:, note not destroying the interface the careful centrifuge tube that fills the equivalent lymphocyte separation medium that adds of blood; After the centrifuge tube trim, it is centrifugal to put into whizzer, 1000rpm, 8 minutes; Take out centrifuge tube, with the careful taking-up of PBMC layer (peripheral blood lymphocytes cloud and mist layer), put into other clean tube with aseptic liquid-transfering gun; Add PBS and suspend, careful vibration for several times, 800rpm, 8 minutes, abandon supernatant, add 1640 substratum 15ml, add penicillin and Streptomycin sulphate all to final concentration be 100U/ μ l; The phytoh(a)emagglutinin gradient adds, and adds 0.5ml * 2 respectively, 0.3ml * 2,1.0ml * 2; Put into 37 ℃, 5%CO ₂In the incubator, 30～48 hours, according to the specification sheets extraction template mRNA of Trizol;

Reverse transcription and the clone of embodiment 2:IL-2 cDNA

RT system: template 1 μ g mRNA solution 10 μ L; 5 * Reverse Transcription Buffer, 4 μ L; The dNTP 2 μ L of 10mM; The RNase Inhibiter0.5 μ L of 20U; The Oligo of 50pmol (dT) ₁₈1 μ L; The Amv Rewerse Transcriptase 1 μ L of 5U; DEPC H ₂O is settled to 20 μ L.

Stir gently, on the PCR instrument, carry out the synthetic of cDNA: 42 ℃, 60min, room temperature is placed 10min, places 2min on ice, and-80 ℃ of Ultralow Temperature Freezers are preserved standby or direct use;

With aforesaid RT-PCR product is template, carries out following amplification, and primer is:

I-1：5′-TGCAACTCCTGTCTTGCATTGCACTAAG-3′

I-2：5′-GAAGGCCTGATATGTTTTAAGTGGGAAG-3′

Add in the PCR reaction system of 50 μ l: each 2 μ l of the I-1 of 10 μ mol/L and I-2, the dNTP5 μ l of 2mmol/L, 10 * Taq Buffer, 5 μ l, the Taq archaeal dna polymerase 0.5 μ l of 5U/ μ l, IL-2 cDNA 1 μ g, add distilled water polishing 50 μ l, the PCR condition is: 95 ℃ of abundant sex change 5 minutes, 94 ℃ of sex change 1 minute, 57 ℃ of annealing 1 minute, 72 ℃ were extended 90 seconds, and circulated 30 times.

The product of amplification carries out T-A is cloned among the pMD19T-Vector.

Embodiment 3:IL-2 cDNA and the segmental amplification of HSA cDNA, fusion, clone

The pcr amplification of IL-2 cDNA:

With the method amplification of PCR, primer is as follows from the plasmid IL-2-pMD19 that contains human IL-2 cDNA:

IH-15′-GGAATTCAAAAGAGCACCTTACTTCAAGTTCTAC-3′

IH-25′-ACCTCACTCTTGTGTGCATCAGTCAGTGTTGAGATGATGC-3′

PCR method is as follows: add in the reaction system of 50 μ l: each 2.5 μ l of the primer I H-1 of 10 μ mol/L and primer I H-2, 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 (dNTP, 10 * pfu Buffer and pfu archaeal dna polymerase are all available from Shanghai biotechnology service company), IL-2-pMD19 1 μ g adds distilled water polishing 50 μ l.PTC-100 in MJ Research company ^TMOn the PCR instrument, the PCR condition is: 95 ℃ of abundant sex change 5 minutes, and 94 ℃ of sex change 1 minute, 57 ℃ 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 stripe that test kit is purified into 0.4kb with PCR fragment glue, the target fragment that purifying is good is standby;

The pcr amplification of HSA cDNA:

Amplify HSA cDNA from contain HSA cDNA plasmid, the primer is as follows:

IH-3：5′-GCATCATCTCAACACTGACTGATGCACACAAGAGTGAGGT-3′

IH-4：5′-TTGCGGCCGCTTATAAGCCTAAGGCAGCTT-3′

PCR method is as follows: add in the reaction system of 50 μ l: each 2.5 μ l of the IH-3 of 10 μ mol/L and IH-4, 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 (dNTP, reaction buffer and pfu archaeal dna polymerase are all available from Shanghai biotechnology service company), pBlue-HSA1 μ g adds distilled water polishing 50 μ l.PTC-100 in MJ Research company ^TMOn the PCR instrument, the PCR condition is: 95 ℃ of abundant sex change 5 minutes, and 94 ℃ of sex change 1 minute, 60 ℃ of annealing 1 minute, 72 ℃ were extended 150 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 stripe that test kit is purified into 1.8kb with PCR fragment glue, the target fragment that purifying is good is standby;

IL-2 cDNA is connected with HSA cDNA's

Utilize overlapping PCR to merge IL-2 cDNA and HSA cDNA, concrete grammar is as follows:

Behind the pcr amplification product purifying with the pcr amplification product of IL-2 cDNA and HAS cDNA, according to after 1: 1 mixed in molar ratio as template.In the reaction system of 50 μ l, add: the dNTP 5 μ l of 2mmol/L, 10 * Taq Buffer, 5 μ l, the Taq archaeal dna polymerase 0.5 μ l of 5U/ μ l (dNTP, reaction buffering and Taq archaeal dna polymerase are all available from Shanghai biotechnology service company), the template 1 μ l that mixes adds distilled water polishing 45 μ l.PTC-100 in MJ Research company ^TMOn the PCR instrument, the PCR condition is: 95 ℃ of abundant sex change 5 minutes, 94 ℃ of sex change 1 minute, 60 ℃ of annealing 1 minute, 72 ℃ were extended 3 minutes, circulated after 10 times, in reaction system, add the IH-1 of 10 μ mol/L and each 2.5 μ l of primer of IH-4, continue to do 30 above-mentioned circulations.

By agarose gel electrophoresis analytical reaction product, the band of expection size (about 2.2kb) appears at the application of sample swimming lane.Reclaim the target fragment that test kit (available from vast Tyke, Beijing biological gene technology company limited) is purified into 2.2kb with PCR fragment glue.Target fragment IL-2-HSA that double digestion and purifying are good and carrier pMD19T carrier were according to 1: 7 mol ratio T again ₄Ligase enzyme connects.Connect product transformed into escherichia coli XL-1blue competent cell, conversion product is coated the LB agar plate that contains X-gal, IPTG and penbritin (100 μ g/ml), 37 ℃ of overnight incubation.The picking white colony 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, NotI double digestion, agarose gel electrophoresis is identified positive colony, and recombinant plasmid EcoRI-NotI district is carried out dna sequencing.Positive recombinant is kept among the LB that contains 20% glycerine, freezes in-80 ℃.Recombinant plasmid called after pMD19-IL-2-HSA, positive recombinant called after XL1-Blue-IL-2-HSA.

Embodiment 4:IL-2-HSA yeast expression system makes up

Get a frozen XL-1Blue-IL-2-HSA, be inoculated into 20ml and contain 37 ℃ of overnight incubation in the LB substratum of penbritin (100 μ g/ml), extract plasmid with ordinary method.Plasmid pMD19-IL-2-HSA and Yeast expression carrier pPIC9K are through EcoRI, NotI double digestion, and the agarose gel electrophoresis purifying reclaims test kit with PCR fragment glue and reclaims.Get the good pMD19-IL-2-HSA fragment 5 μ l of purifying behind the double digestion, pPIC9K 5 μ l add 10 * ligation buffer, 2 μ l, and T4 ligase enzyme 0.4 μ l adds distilled water polishing 20 μ l, and 15 ℃ of reactions are spent the night.Connect product transformed into escherichia coli XL-1Blue competent cell, conversion product is coated the LB agar plate that contains penbritin (100 μ g/ml), 37 ℃ of overnight incubation.Several white colonies of picking are inoculated in 37 ℃ of overnight incubation in the LB substratum of 20ml penbritin (100 μ g/ml), extract plasmid with ordinary method.By PCR and EcoRI, 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 pPIC-IL-2-HSA, positive recombinant called after XL-1Blue-pPIC-IL-2-HSA.Extract the plasmid among the XL-1Blue-pPIC-IL-2-HSA, after the SalI enzyme was cut, electric shock transformed pichia spp KM71.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 ℃, extracts the recombinant yeast pichia pastoris genomic dna with ordinary method, and by PCR, agarose gel electrophoresis is identified recon.Positive recombinant called after KM71-pPIC-IL-2-HSA.

Embodiment 5 IL-2-HSA Expression of Fusion Protein

KM71-pPIC-IL-2-HSA 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, it is 2～6 that 250rpm, 29 ℃ are cultured to OD600, centrifugal collection thalline.The thalline of collecting is seeded to 20mlBMMY (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%, induced 7 days in per 24 hours.Centrifugal collection supernatant according to different dilutions, is used for being ELLISA, to determine Expression of Fusion Protein amount and the antigenicity of IL-2 wherein; The immunogenicity of the HSA of fusion rotein IL-2-HSA molecule is identified in Western hybridization.

The IL-2 of Diaclone company test kit, operation steps following the instructions is established typical curve, sees Table 1 and Fig. 1.

Centrifugal collection supernatant, dilute sample 10 ^-5Doubly, the A value is 0.175, so calculate according to IL-2, should be 12200ng/mL, with fusion rotein, is the concentration of 68.9 μ g/mL.

The doubling dilution of table 1 standard substance and A value

Standard substance doubling dilution pg/mL	A	492
			1000 500 250 125 62.5 31.25 15.12	1.087 0.547 0.297 0.178 0.12 0.093 0.078

Claims (10)

1, the preparation method of the fusion rotein of human interleukin-2 and human serum albumin, it is characterized in that by extracting human peripheral blood mononuclear cell mRNA, reverse transcription obtains IL-2cDNA then, and IL-2cDNA is cloned in the pMD19T carrier, PCR from the carrier IL-2-pMD19T that contains IL-2cDNA directly amplification obtain IL-2cDNA; Increase from contain HSA cDNA plasmid by PCR and to obtain HSA cDNA; Utilize overlapping pcr, connect IL-2cDNA and HSA cDNA, the centre does not add any connection peptides, and the IL-2-HSA cDNA syzygy that obtains is inserted into cloning vector; IL-2-HSA cDNA syzygy is expressed at host system, and IL-2-HSA cDNA syzygy is integrated in host's the karyomit(e);

(1) extraction of template mRNA

Take the human peripheral 10mL of EDTA anti-freezing, in 2 hours, carry out lymphocytic separation:, note not destroying the interface the careful centrifuge tube that fills the equivalent lymphocyte separation medium that adds of blood; After the centrifuge tube trim, it is centrifugal to put into whizzer, 1000rpm, 8 minutes; Take out centrifuge tube, peripheral blood lymphocytes cloud and mist layer is carefully taken out, put into other clean tube with aseptic liquid-transfering gun; Add PBS and suspend, careful vibration for several times, 800rpm, 8 minutes, abandon supernatant, add 1640 substratum 15ml, add penicillin and Streptomycin sulphate all to final concentration be 100U/ μ l; The phytoh(a)emagglutinin gradient adds, and adds 0.5ml * 2 respectively, 0.3ml * 2,1.0ml * 2; Put into 37 ℃, 5%CO ₂In the incubator, 30～48 hours, according to the specification sheets extraction template mRNA of Trizol;

(2) reverse transcription of IL-2cDNA and clone

RT system: template 1 μ g mRNA solution 10 μ L; 5 * Reverse Transcription Buffer, 4 μ L; The dNTP 2 μ L of 10mM; The RNase Inhibiter0.5 μ L of 20U; The Oligo of 50pmol (dT) ₁₈1 μ L; The Amv Rewerse Transcriptase 1 μ L of 5U; DEPC H ₂O is settled to 20 μ L;

Stir gently, on the PCR instrument, carry out the synthetic of cDNA: 42 ℃, 60min, room temperature is placed 10min, places 2min on ice, and-80 ℃ of Ultralow Temperature Freezers are preserved standby or direct use;

With the RT-PCR product is template, carries out following amplification, and primer is:

I-1：5′-TGCAACTCCTGTCTTGCATTGCACTAAG-3′

I-2：5′-GAAGGCCTGATATGTTTTAAGTGGGAAG-3′

Add in the PCR reaction system of 50 μ l: each 2 μ l of the I-1 of 10 μ mol/L and I-2, the dNTP5 μ l of 2mmol/L, 10 * Taq Buffer, 5 μ l, the Taq archaeal dna polymerase 0.5 μ l of 5U/ μ l, IL-2cDNA 1 μ g, add distilled water polishing 50 μ l, the PCR condition is: 95 ℃ of abundant sex change 5 minutes, 94 ℃ of sex change 1 minute, 57 ℃ of annealing 1 minute, 72 ℃ were extended 90 seconds, and circulated 30 times;

The product of amplification carries out T-A is cloned among the pMD19 T-Vector;

(3) amplification of IL-2c DNA

With the method amplification of PCR, primer is as follows from the plasmid IL-2-pMD19 that contains human IL-2 cDNA:

IH-1：5′-GGAATTCAAAAGAGCACCTTACTTCAAGTTCTAC-3′

IH-2：5′-ACCTCACTCTTGTGTGCATCAGTCAGTGTTGAGATGATGC-3′

PCR method is as follows: add in the reaction system of 50 μ l: each 2.5 μ l of the primer I H-1 of 10 μ mol/L and primer I H-2, 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, IL-2-pMD19 1 μ g, add distilled water polishing 50 μ l, the PCR condition is: 95 ℃ of abundant sex change 5 minutes, 94 ℃ of sex change 1 minute, 57 ℃ 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 stripe that test kit is purified into 0.4kb with PCR fragment glue, the target fragment that purifying is good is standby;

(4) pcr amplification of HSA cDNA

Utilize PCR to amplify HSA cDNA from contain HSA cDNA plasmid, used primer is:

IH-3：5′-GCATCATCTCAACACTGACTGATGCACACAAGAGTGAGGT-3′

IH-4：5′-TTGCGGCCGCTTATAAGCCTAAGGCAGCTT-3′

Add in the reaction system of 50 μ l: each 2.5 μ l of the primer I H-3 of 10 μ mol/L and primer I H-4, 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, contain the plasmid pBlue-HSA 1 μ g of HSAcDNA, add distilled water polishing 50 μ l; The PCR condition is: 95 ℃ of abundant sex change 5 minutes, and 94 ℃ of sex change 1 minute, 60 ℃ of annealing 1 minute, 72 ℃ were extended 150 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 stripe that test kit is purified into 1.8kb with PCR fragment glue, the target fragment that purifying is good is standby;

(5) IL-2cDNA and HSA cDNA's is connected

Utilize overlapping PCR to merge IL-2cDNA and HSA cDNA, with behind the pcr amplification product purifying of IL-2cDNA and behind the pcr amplification product purifying of HSAcDNA according to after 1: 1 the mixed in molar ratio as template, in the reaction system of 50 μ l, add: the dNTP 5 μ l of 2mmol/L, 10 * Taq Buffer, 5 μ l, the Taq archaeal dna polymerase 0.5 μ l of 5U/ μ l, the template 1 μ l that mixes adds distilled water polishing 45 μ l; The PCR condition is: 95 ℃ of abundant sex change 5 minutes, and 94 ℃ of sex change 1 minute, 60 ℃ of annealing 1 minute, 72 ℃ were extended 3 minutes, circulates after 10 times, and the primer that adds the IH-1 of 10 μ mol/L and IH-4 in reaction system is 2.5 μ l respectively, continue to do 30 above-mentioned circulations;

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, the target fragment IL-2-HAS that double digestion and purifying are good is connected with the T4 ligase enzyme according to 1: 7 mol ratio with carrier pMD19T again; Connect product transformed into escherichia coli XL-1Blue 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 37 ℃ of overnight incubation in the LB substratum of 20ml100 μ g/ml penbritin, 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, freezes in-80 ℃; Recombinant plasmid called after pMD19-IL-2-HSA, positive recombinant called after XL-1-Blue-pMD19-IL-2-HSA;

(6) yeast expression system of fusion rotein IL-2-HSA makes up

Get a frozen XL-1Blue-pMD19-IL-2-HSA, be inoculated into 20ml and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/ml penbritins, extract plasmid, plasmid pMD19-IL-2-HSA and Yeast expression carrier pPIC9K are respectively through EcoRI and NotI double digestion, the agarose gel electrophoresis purifying, reclaim test kit with PCR fragment glue and reclaim, connect the good fragment of purifying behind two double digestions with the T4 ligase enzyme; Connect product transformed into escherichia coli XL-1Blue competent cell, conversion product is coated the LB agar plate that contains 100 μ g/ml penbritins, 37 ℃ of overnight incubation; The picking white colony is inoculated in 37 ℃ of overnight incubation in the LB substratum of 20ml 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 pPIC-IL-2-HSA, positive recombinant called after XL-1Blue-pPIC-IL-2-HSA; Extract the plasmid among the XL-1Blue-pPIC-IL-2-HSA, 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-pPIC-IL-2-HSA;

(7) expression of fusion rotein IL-2-HSA:

KM71-pPIC-IL-2-HSA is seeded to is equipped with in the 100ml BMGY500ml triangular flask, it is 2～6 that 250rpm, 29 ℃ are cultured to OD600, centrifugal collection thalline, the thalline of collecting is seeded in the 100ml triangular flask that 20mlBMMY is housed, added a methyl alcohol in per 24 hours to final concentration 0.5%, induced 7 days, centrifugal collection supernatant, filtration sterilization, the ELISA method is measured the fusion rotein IL-2-HSA content in the supernatant, and the immunogenicity of the HSA of fusion rotein IL-2-HSA molecule is identified in Western hybridization.

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

4, the fusion rotein of human interleukin-2 according to claim 2 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 structural domain after resetting is formed.

5, the fusion rotein of human interleukin-2 according to claim 2 and human serum albumin, it is characterized in that described fusion rotein comprises first district identical with the human interleukin-2 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.

6, the fusion rotein of human interleukin-2 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 expression system is bacterium, yeast, insect cell, zooblast or vegetable cell expression system.

8, method according to claim 7 is characterized in that preferred host expression 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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