CN101063125A - Preparation method for fused protein of human parathyroid hormone and human serum albumins and its product - Google Patents

Abstract

The invention discloses a preparing method of blend protein of human parathormone (PTH) 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 PTH cDNA; non-adding into any connecting peptide; getting PTH-HSA 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 parathormone, which possesses good application prospect in medical domain.

Description

The preparation method of the fusion rotein of human parathyroid hormone and human serum albumin and product

Technical field

The preparation method of the fusion rotein of human parathyroid hormone and human serum albumin and product 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 Ha et al., Biochimica et BiophysicaActa, 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, 199948:169-174), 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.

Human parathyroid hormone (human parathyroid hormone, PTH) by with receptors bind after the activity of activated adenyl cyclase and protein kinase C, change bone resorption and bone synthetic speed, influence bone metabolism.If the active fragments of PTH long duration of action under the concentration of lower or median dose then can stimulate the formation of bone in bone, and treats osteoporosis effectively by inducing the Synthesis in the bone.Because the PTH molecular weight is less, easily by glomerular filtration, thereby the transformation period in vivo is shorter, and the transformation period of subcutaneous administration or intramuscular injection is generally about 12h.In order to reach result of treatment, generally need frequent heavy dose of medication, in order to overcome above-mentioned shortcoming, people are by being modified Rat parathyroid hormone 1-34, to prolong its transformation period.The present invention will carry out amalgamation and expression to PTH and HSA, by the long-actingization technology of research and development people PTH, improve pharmacokinetics proterties in the PTH 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 human parathyroid hormone 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 parathyroid hormone, improve its solubleness, at pharmaceutical field good prospects for application is arranged.

Technical scheme of the present invention: separating monocytic cell from Freshman parathyroid gland tissue, stimulate and cultivate, extract RNA, obtain PTH 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 PTH cDNA, the centre does not add any connection peptides, and the PTH-HAS cDNA fusion gene that obtains is inserted in the cloning vector to be preserved; PTH-HAS cDNA fusion gene is expressed at host system, and the PTH-HSAcDNA fusion gene is integrated in host's the karyomit(e).

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

With the human parathyroid hormone 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 human parathyroid hormone at least 85% sequence homology; Described and human serum albumin homologous first district is positioned at the C-terminal of fusion rotein, and described and human parathyroid hormone homologous second district is positioned at the N-terminal of fusion rotein, and the centre does not add any connection peptides; Or described and human parathyroid hormone homologous second district is positioned at the C-terminal of fusion rotein, and described and human serum albumin homologous first district are positioned at the N-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 human parathyroid hormone 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 comprises first district identical with the human serum albumin amino acid residue sequence and second district identical with the human parathyroid hormone 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 bacterium, yeast, insect cell, zooblast or vegetable cell etc.Preferred host system is a yeast.Preferred yeast is a pichia spp.Preferred pichia spp is pichia spp KM71.

Because the PTH gene contains intron, separating monocytic cell from Freshman parathyroid gland tissue stimulates and cultivates, and extracts RNA, obtains PTH cDNA by the RT-PCR reverse transcription.HAS cDNA can be by the PCR acquisition of increasing from people's tire liver cDNA library.The PTH cDNA and the HSA cDNA that obtain are inserted into respectively in the cloning vector, order-checking.Utilize overlapping pcr, connect PTH cDNA and HSA cDNA, 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, PTH cDNA that obtains and HSA cDNA fusion gene are inserted in the cloning vector to be preserved.

PTH 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, more preferably the pichia yeast expression system.

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 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.

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 G-CSF antibody test nutrient solution supernatant behind abduction delivering.

Beneficial effect of the present invention: utilize overlapping pcr, connect HSA cDNA and PTH 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 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-PTH-HSA

Fig. 2. the enzyme of plasmid pPIC9K-PTH-HSA is cut checking Lane1:DNA molecular weight standard; Lane2:pPIC9K-PTH-HSA NcoI enzyme is cut; Lane3:pPIC9K-PTH-HSA Sal I enzyme is cut.

Fig. 3. the SDS-PAGE of fusion rotein analyzes Lane1:KM71/pPIC9K; Lane2: molecular weight of albumen standard; Lane3～6:KM71/PTH-HSA

Fig. 4. the HSA antibody test Lane1 of fusion rotein: protein molecular weight standard; Lane2:PTH-HAS.

Specific implementation method

The clone of embodiment 1:PTH cDNA:

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

p1：5’CG GAATTCATGACCCCCCTGGGCCCTGC-3’

p2：5’-CGG CGGCCGCTCAGGGCTGGGCAAGGTGGC-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, PTHcDNA 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, 66 ℃ 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 0.27kb with PCR fragment glue, target fragment that purifying is good and carrier pBluescriptII KS (+) are respectively through EcoR I 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 100 μ g/ml penbritins, 37 ℃ of overnight incubation; 5 bacterium colonies of picking at random are inoculated in 20ml and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/ml penbritins, extract plasmid; By PCR, and EcoR I and HindIII double digestion, agarose gel electrophoresis is identified positive colony, and dna sequencing is carried out in zone between recombinant plasmid EcoR I and HindIII double enzyme site; Positive recombinant is kept among the LB that contains 15% glycerine, and is frozen in-80 ℃; Recombinant plasmid called after pBlue-PTH, positive recombinant called after DH5 α/pBlue-PTH.

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-HAS.

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

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

P1：5’-CG GAATTCAAAAGAGATGCACACAAGAGTGAGGTTGCTCATCG-3’

P2：5’-CAGGGGGGTGCCTAAGGCAGCTTGACTTGCAGCAACAA-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 PTHcDNA, the primer is as follows:

P3：5’ CTGCCTTAGGCACCCCCCTGGGCCCTG-3’

P4：5’CG GCGGCCGCTCAGGGCTGGGCAAGGTGGCGTAGAAC-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-PTH 1 μ g adds distilled water polishing 50 μ l; The PCR condition is: 95 ℃ of sex change 5 minutes, and 66.5 ℃ of annealing 1 minute, 72 ℃ were extended 90 seconds, and 94 ℃ of sex change 1 minute circulate 30 times.

Utilize overlapping PCR to merge PTH cDNA and HSA cDNA:

The pcr amplification product of PTH and the pcr amplification product of HSA are diluted 10 times respectively, mix the back as template with 4: 6 volume ratios again, in the reaction system of 50 μ l, add: 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, the template 1 μ l that mixes adds distilled water polishing 50 μ l; The PCR condition is: 95 ℃ of sex change 5 minutes, and 60 ℃ of annealing 1 minute, 72 ℃ were extended 4 minutes, and 94 ℃ of sex change 1 minute 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 2kb with PCR fragment glue, target fragment that purifying is good and carrier pUC19 are respectively through EcoRI and HindIII double digestion; The agarose gel electrophoresis purifying, reclaiming test kit with PCR fragment glue reclaims, get the good PTH-HSA fragment 5 μ l of purifying behind the double digestion, pBluescript II KS (+) 5 μ l, add 2 μ l, 10 * ligation buffer, 0.4 μ l T4 ligase enzyme adds distilled water polishing 20 μ l, 15 ℃ of reactions are spent the night; 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; 5 single bacterium colonies of picking at random are inoculated in 20ml and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/ml penbritins, extract plasmid; By PCR and EcoRI and HindIII double digestion, agarose gel electrophoresis is identified positive colony, and dna sequencing is carried out in zone between recombinant plasmid EcoRI and HindIII restriction enzyme site; Positive recombinant is kept among the LB that contains 20% glycerine, and is frozen in-80 ℃; Recombinant plasmid called after pBlue-PTH-HAS, positive recombinant called after XL-1/pBlue-HSA-PTH.

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

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

Get a frozen XL-1/pBlue-HSA-PTH, 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-PTH-HAS and the Yeast expression carrier pPIC9K that extract, through the EcoRI single endonuclease digestion, the agarose gel electrophoresis purifying reclaims test kit with PCR fragment glue and reclaims.Get enzyme and cut the good pBlue-PTH-HSA fragment 5 μ l of back purifying, pPIC9K 5 μ l add 2 μ l10 * ligation buffer, and 0.4 μ l T4 ligase enzyme adds distilled water polishing 20 μ l, and 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; Several bacterium colonies that grow 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, reach Nco I enzyme and cut, agarose gel electrophoresis is identified positive colony, positive recombinant is kept among the LB that contains 15% glycerine, and is frozen in-80 ℃; Recombinant plasmid called after pPIC9K-PTH-HSA, positive recombinant called after DH5 α/pPIC9K-PTH-HAS; Extract the plasmid among DH5 α/pPIC9K-PTH-HSA, 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 20ml MD 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-PTH-HAS.

The PTH-HSA Expression of Fusion Protein

KM71/pPIC9K-PTH-HSA is seeded to 100mlBMGY (100mM potassiumphosphate, pH6.0,1.34% no amino acid whose yeast nitrogen base, 4 * 10 are housed ^-5% vitamin H, 1% glycerine) the 500ml triangular flask in, 300rpm, 29 ℃ are cultured to A _600nmValue 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 PTH of fusion rotein PTH-HSA molecule is identified in Western hybridization.

With the fusion rotein PTH-HSA activity in the enzyme linked immunosorbent assay mensuration supernatant.The UMR2106 cell is in the DMEM2F12 that contains 10% calf serum (U.S. GIBCO2BRL company product) nutrient solution (pH7.2), at 37 ℃, 5%CO ₂Condition under cultivate.During determination of activity, the cell in the vegetative period of taking the logarithm is diluted to 1.5 * 105 cell/ml with nutrient solution behind 0.02% tryptic digestion.In 96 porocyte culture plates, every hole adds 100 μ l cell diluents, cultivates 5h, changes the DMEM2F12 solution (not containing bovine serum) of every hole 100 μ l then into, continues overnight incubation.Get each 1 of Rat parathyroid hormone 1-34 international standard substance and trial-product, be dissolved in respectively in the DMEM2F12 nutrient solution, after the pre-dilution, carry out serial doubling dilution by 1: 2 volume ratio again.Dilution is DMEM2F12 solution (not containing bovine serum) with solution.After cell abandoned nutrient solution, every hole adds 170 μ l DMEM2F12 nutrient solutions, and 10 μ l/ hole IBMX (isobutyl-xanthine, Sigma company product), add above-mentioned serial dilution good Rat parathyroid hormone 1-34 international standard substance and trial-product then respectively successively, every hole 20 μ l, each extent of dilution do 3 multiple holes.All have the PTH international standard substance on every block of plate, and the cAMP standard, establish gross activity contrast (TA), non-specific binding contrast (NSB), maximum combined (B0), substrate blank (SB) hole simultaneously, place 37 ℃ of cultivations.Carry out the cAMP extracting behind the 45min.Method for extracting: remove substratum, every hole adds 200 μ l 0.1mol/L HCl, and behind 37 ℃ of incubation 30min, lysing cell is got supernatant and is used to measure cAMP.CAMP measures: with cAMP (low p H) kit measurement.Detect the absorbance value of each hole with the microtiter plate microplate reader at the 405nm place.The activation analysis result proves that product has the immunologic competence of PTH.Western hybridization shows that product has the immunogenicity of PTH and HSA.

Claims (10)

1, the preparation method of the fusion rotein of human parathyroid hormone and human serum albumin is characterized in that separating monokaryon parathyroid gland cell from Freshman parathyroid gland tissue, stimulates and cultivates, and extracts RNA; Obtain PTH 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 PTH cDNA, the centre does not add any connection peptides, the PTH-HAS cDNA fusion gene that obtains is inserted into cloning vector, PTH-HAS cDNA fusion gene is expressed in host system, and PTH-HAS cDNA fusion gene is integrated in host's the karyomit(e);

(1) clone of PTH cDNA:

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

p1：5’CG GAATTCATGACCCCCCTGGGCCCTGC-3’

p2：5’CG GCGGCCGCTCAGGGCTGGGCAAGGTGGC-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, PTHcDNA 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 0.27kb with PCR fragment glue, target fragment that purifying is good and carrier pBluescriptII KS (+) are respectively through EcoRI and HindIII double digestion; The agarose gel electrophoresis purifying reclaims test kit with PCR fragment glue and reclaims, and uses T ₄Ligase enzyme connects two enzymes and cuts the good fragment of back purifying; 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; 5 single bacterium colonies of picking at random are inoculated in 20ml and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/ml penbritins, extract plasmid; By PCR, and EcoRI and HindIII double digestion, agarose gel electrophoresis is identified positive colony, and dna sequencing is carried out in zone between recombinant plasmid EcoRI and HindIII double enzyme site; Positive recombinant is kept among the LB that contains 15% glycerine, and is frozen in-80 ℃; Recombinant plasmid called after pBlue-PTH, positive recombinant called after DH5 α/pBlue-PTH;

(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 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-HAS;

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

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

P1：5’-CG GAATTCAAAAGAGATGCACACAAGAGTGAGGTTGCTCATCG-3’

P2：5’-CAGGGGGGTGCCTAAGGCAGCTTGACTTGCAGCAACAA-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 PTHcDNA, the primer is as follows:

P3：5’- CTGCCTTAGGCACCCCCCTGGGCCCTG-3’

P4：5’-CG GCGGCCGCTCAGGGCTGGGCAAGGTGGCGTAGAAC-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-PTH 1 μ g adds distilled water polishing 50 μ l; The PCR condition is: 95 ℃ of sex change 5 minutes, and 66.5 ℃ of annealing 1 minute, 72 ℃ were extended 90 seconds, and 94 ℃ of sex change 1 minute circulate 30 times;

Utilize overlapping PCR to merge PTH cDNA and HSA cDNA:

The pcr amplification product of PTH and the pcr amplification product of HSA are diluted 10 times respectively, mix the back as template with 4: 6 volume ratios again, in the reaction system of 50 μ l, add: the dNTP 5 μ l of 2mmol/L, 10 * pfuBuffer, 5 μ l, the pfu archaeal dna polymerase 0.5 μ l of 5U/ μ l, the template 1 μ l that mixes adds distilled water polishing 50 μ l; The PCR condition is: 95 ℃ of sex change 5 minutes, and 60 ℃ of annealing 1 minute, 72 ℃ were extended 4 minutes, and 94 ℃ of sex change 1 minute 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 2kb with PCR fragment glue, target fragment that purifying is good and carrier pBluescript IIKS (+) are through EcoRI and HindIII double digestion; The agarose gel electrophoresis purifying, reclaiming test kit with PCR fragment glue reclaims, get the good PTH-HSA fragment 5 μ l of purifying behind the double digestion, pBluescript IIKS (+) 5 μ l, add 2 μ l, 10 * ligation buffer, 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 XL-1Blue competent cell, conversion product is coated the LB agar plate that contains 100 μ g/ml penbritins, 37 ℃ of overnight incubation; 5 single bacterium colonies of picking at random are inoculated in 20ml and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/ml penbritins, extract plasmid; By PCR, and EcoRI and HindIII double digestion, agarose gel electrophoresis is identified positive colony, and dna sequencing is carried out in zone between recombinant plasmid EcoRI and HindIII double enzyme site; Positive recombinant is kept among the LB that contains 15% glycerine, and is frozen in-80 ℃; Fusion gene called after PTH-HSA, recombinant plasmid called after pBlue-PTH-HAS contains the recombination bacillus coli called after XL-1/pBlue-HSA-PTH of recombinant plasmid;

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

Get a frozen XL-1/pBlue-HSA-PTH, 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-PTH-HSA and the Yeast expression carrier pPIC9K that extract, respectively through EcoRI 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 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 HindIII double digestion, agarose gel electrophoresis is identified positive colony, positive recombinant is kept among the LB that contains 15% glycerine, and is frozen in-80 ℃; Recombinant plasmid called after pPIC9K-PTH-HSA, positive recombinant called after DH5 α/pPIC9K-PTH-HAS; Extract the plasmid among DH5 α/pPIC9K-PTH-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/pPIC9K-PTH-HSA;

(5) expression of fusion rotein PTH-HSA:

KM71/pPIC9K-PTH-HSA 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%, induced 3 days, centrifugal collection supernatant is with the content of the fusion rotein PTH-HAS in the enzyme linked immunosorbent assay mensuration supernatant liquor, carry out the SDS-PAGE checking, the PTH immunogenicity of fusion rotein PTH-HSA molecule is identified in Western hybridization.

2, with the human parathyroid hormone 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 human parathyroid hormone at least 85% sequence homology; Described and human serum albumin homologous first district is positioned at the C-terminal of fusion rotein, and described and human parathyroid hormone homologous second district is positioned at the N-terminal of fusion rotein, and the centre does not add any connection peptides; Or described and human parathyroid hormone homologous second district is positioned at the C-terminal of fusion rotein, and described and human serum albumin homologous first district are positioned at the N-terminal of fusion rotein, and the centre does not add any connection peptides.

3, the fusion rotein of human parathyroid hormone 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 human parathyroid hormone at least 95% sequence homology.

4, the fusion rotein of human parathyroid hormone 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 human parathyroid hormone according to claim 2 and human serum albumin, it is characterized in that described fusion rotein comprises first district identical with the human serum albumin amino acid residue sequence and second district identical with the human parathyroid hormone amino acid residue sequence, or the function equivalent in above-mentioned two districts.

6, the fusion rotein of human parathyroid hormone 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.

Images