CN101463089A - Human serum albumin-interferon fusion protein, and encoding gene and use thereof - Google Patents
Human serum albumin-interferon fusion protein, and encoding gene and use thereof Download PDFInfo
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Abstract
The invention discloses a proserum-interferon fusion protein and the encoding gene and the application thereof. The protein is formed by the amino acid residue sequences in the sequence 1 of a sequence table; the invention also discloses the encoding gene of the proserum-interferon fusion protein and the nucleotide sequence thereof is sequence 2 in the sequence table; the fusion protein of the invention has high steadiness, long half-life and high safety and good healing effect medically; the fusion protein has the advantages of high expression, easy purification, short production cycle, large production scale and low cost, thereby having important application prospect and practical significance.
Description
Technical field
The present invention relates to human serum albumin-interferon fusion protein and encoding gene thereof and application.
Background technology
(interferon, IFN) to be that a class has antiviral, the cytokine of antiproliferative and immunoregulatory activity for Interferon, rabbit.Active different with celliferous type according to it, Interferon, rabbit can be divided into interferon-' alpha ', interferon-beta and interferon-etc.Wherein interferon-' alpha ' (as interferon-' alpha ' 2a, interferon-' alpha ' 2b and interferon-' alpha ' 1b) is widely used in treating multiple virus disease (hepatitis B, hepatitis C) and tumour clinically.
Yet the transformation period of natural interferon-α is shorter, has only frequent drug administration (often adopt in clinical treatment and inject three times application method weekly) could obtain gratifying curative effect.So frequent application method add long treatment cycle (6-12 month) make patient difficulty bear.Therefore press for the recombined human interferon-alpha of developing long action time.
(human serum albumin HSA) is long albumen of a kind of stable and transformation period in the body to human serum albumin.Studies show that many protein drugs and human serum albumin are crosslinked or merge after its transformation period is prolonged, thereby reduce the medication number of times.
The Albuferon of U.S. HGSI (Human Genome Sciences Inc) company is one and does not contain the human serum albumin of joint and the fusion rotein of interferon-' alpha ' 2b (HSA-IFN-α 2b), Interferon, rabbit partly is in the C-terminal of fusion rotein in this fusion rotein, and human serum albumin partly is in the N-terminal of fusion rotein.Albuferon has entered III phase clinical study at present.Result of study shows that Albuferon has the transformation period in the long body, and higher security reaches curative effect preferably.But there are defectives such as unhomogeneity and unstable in HSA-IFN-α 2b.IFN-α 2b-HSA is a new fusion protein with higher homogeneity and stability that obtains by the phase place that changes fusion rotein.IFN-α 2b-HSA is improving the expression amount and the rate of recovery, and aspect such as reduce production costs has shown remarkable advantages, has the excellent development prospect.(Zhao HL, Xue C, Wang Y, Li XY, Xiong XH, Yao XQ, Liu ZM.Circumventing the heterogeneity and instabilityof human serum albumin-interferon-alpha2b fusion protein by altering itsorientation.J Biotechnol.2007 Sep 15; 131 (3): 245-52), (quick Zhao of Liu Zhi is loud and clear etc.: human serum albumin-interferon fusion protein and encoding gene thereof and application.Chinese invention patent application number: CN 200710099325).
Protein drug is being produced, deposit with use in unavoidably to run into the concussion and the equipressure condition of being heated, the protein drug of less stable must adopt the production cost height, awkward freeze-dried formulation.Less stable under the IFN-α 2b-HSA fusion rotein earthquake and the equipressure condition of being heated easily forms the covalency aggressiveness.
Summary of the invention
The purpose of this invention is to provide a kind of human serum albumin-interferon fusion protein and encoding gene thereof and application.
Human serum albumin-interferon fusion protein provided by the present invention, called after IFN-α 2b-HSA (C34S) is following (a) or protein (b):
(a) protein of forming by the amino-acid residue of sequence in the sequence table 1;
(b) with the amino-acid residue of sequence in the sequence table 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and to have an interferon alpha 2 b active by (a) deutero-protein.
In order to make the IFN-α 2b-HSA (C34S) in (a) be convenient to purifying, proteinic N end or C end that can the amino acid residue sequence of sequence 1 is formed in by sequence table connect label as shown in table 1.
The sequence of table 1. label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| |
8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 11 | EQKLISEEDL |
IFN-α 2b-HSA (C34S) in above-mentioned (b) but synthetic also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.The encoding gene of IFN-α 2b-HSA (C34S) in above-mentioned (b) can pass through SEQ ID № in the sequence table: the codon of one or several amino-acid residue of disappearance in 2 the dna sequence dna, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The encoding gene of above-mentioned fusion rotein also belongs to protection scope of the present invention.The encoding gene of IFN-α 2b-HSA (C34S) specifically can be following 1) or 2) or 3) gene:
1) its nucleotide sequence is a sequence 1 in the sequence table;
2) under stringent condition with 1) dna molecular of the fusion rotein formed of the dna fragmentation hybridization that limits and coding human serum albumin and Interferon, rabbit;
3) with 1) or 2) gene have the homology 90% or more and the dna molecular of the fusion rotein of coding human serum albumin and Interferon, rabbit composition.
Gene in the described step 3) is with 1) gene homology more than 95% is preferably arranged.
Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5% SDS, 65 ℃ of hybridization down, uses 2 * SSC then, 0.1%SDS and 1 * SSC, and 0.1% SDS respectively washes film once.
The recombinant expression vector, transgenic cell line or the reorganization bacterium that contain the encoding gene of described fusion rotein all belong to protection scope of the present invention.
The above-mentioned fusion rotein encoding gene total length that increases or its any segmental primer are to also within protection scope of the present invention.
Another object of the present invention provides a kind of method of producing described human serum albumin-interferon fusion protein.
The method of the described human serum albumin-interferon fusion protein of production provided by the present invention is that described fusion rotein encoding gene is imported host cell, expresses obtaining fusion rotein.
Wherein, described host can be yeast, intestinal bacteria, mammalian cell, insect cell or Bacillus subtilus etc., is preferably yeast.
Described yeast is preferably pichia pastoris (Pichia pastoris).Wherein, described pichia pastoris is preferably pichia pastoris GS115, KM71 or SMD1168 (can available from American I nvitrogen company).
Described intestinal bacteria can be E.coli JM109, E.coli HB101, E.coli Top10.
The encoding gene of fusion rotein of the present invention, described fusion rotein can be used to preparation and prevents and/or treats tumour, and/or suppresses the medicine of virus.
Prevent and/or treat tumour, and/or the activeconstituents of the medicine of inhibition virus is described human serum albumin-interferon fusion protein.
When needing, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises thinner, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier of pharmaceutical field routine etc.
Medicine of the present invention can be made various ways such as injection liquid, tablet, pulvis, granula, capsule, oral liquid.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.
The consumption of said medicine is generally the fusion rotein of each adult's per injection 500-1000 μ g, is administered once every 14-28 days, and be 3 to 12 months the course of treatment.
Experiment showed, that IFN-α 2b-HSA of the present invention (C34S) fusion rotein stability is high, the transformation period is longer, medically also have higher security and curative effect preferably; IFN-α 2b-HSA (C34S) Expression of Fusion Protein amount height, easy purifying, and tool is with short production cycle, and industrial scale is big, and cost is low, has important application prospects and practical significance.
Description of drawings
Fig. 1 is the purification result of IFN-α 2b-HSA and IFN-α 2b-HSA (C34S).
Fig. 2 is the stability under gel permeation chromatography comparison IFN-α 2b-HSA and IFN-α 2b-HSA (C34S) the earthquake condition.
Fig. 3 is that non-reduced SDS-PAGE compares the stability under IFN-α 2b-HSA and IFN-α 2b-HSA (C34S) the earthquake condition.
Fig. 4 compares IFN-α 2b-HSA and the stability of IFN-α 2b-HSA (C34S) under heating condition for gel permeation chromatography.
Fig. 5 is for utilizing non-reduced SDS-PAGE relatively IFN-α 2b-HSA and the stability of IFN-α 2b-HSA (C34S) under heating condition.
Fig. 6 is for comparing IFN-α 2b-HSA and IFN-α 2b-HSA (C34S) in pharmacokinetics in rats.
Embodiment
Among the following embodiment if no special instructions method therefor be ordinary method.
Agents useful for same all can obtain from commercial channels among the following embodiment.
Main agents:
1, DNA restriction enzyme, T
4Dna ligase, polysaccharase etc. are respectively available from GIBCO-BRL, Pharmacia, Bio-Labs and magnificent biotechnology company limited
2, pcr amplification test kit (Sweden Pharmacia company product)
3, dna sequence analysis test kit (U.S. USB company product)
4, casein hydrolysate (German MERK company product)
5, Bacto-yeast extract (U.S. Difco company product)
6, the required solution of yeast protoplast transformation method
(1) SED:1M sorbyl alcohol, 25mM EDTA, 50mM DTT, pH8.0.
(2) SCE:1M sorbyl alcohol, 10mM Trisodium Citrate, 10mM EDTA, pH5.8.
(3) CaS:1M sorbyl alcohol, 10mM CaCl
2
(4) SOS:1M sorbyl alcohol, 0.3 * YPD, 10mM CaCl
2
(5)CaT：20mM Tris-HCl，pH7.5，20mM CaCl
2。
(6)PEG：20％PEG-3350，10mM CaCl
2，10mM Tris-HCl(pH7.4)。
(7) 1M PBS damping fluid: 132ml 1M K
2HPO
4, 868ml 1M KH
2PO
4, pH6.0.
7, primer: it is synthetic to give birth to the worker by Shanghai
F：5＇-CCCTCGAGAAAAGATGTGACTTGCCACAAACCCACTCC-3＇；
R：5＇-GGGAATTCCTATTACAAACCCAAAGCAGCTTGAGAAGC-3＇。
C34SF：5＇-TACTTGCAACAATCTCCATTCGAAGAC-3＇；
C34SR：5＇-GTCTTCGAATGGAGATTGTTGCAAGTA-3＇。
IAR：5＇-GGGAATTCCTATTACAAACCCAAAGCAGCTTGAGAAGC--3＇；
NF：5＇-CAAGAGTCCTTGAGATCCAAGGAGGACGCTCACAAGTCTGAAGTTGCT-3＇。
IAF：5＇-CCCTCGAGAAAAGATGTGACTTGCCACAAACCCACTCC-3＇；
NR：5＇-AGCAACTTCAGACTTGTGAGCGTCCTCCTTGGATCTCAAGGACTCTTG-3＇。
The preparation of embodiment 1, IFN-α 2b-HSA and IFN-α 2b-HSA (C34S) fusion rotein
1.IFN-the acquisition of α 2b-HSA antigen-4 fusion protein gene
(1) interferon-' alpha ' 2b gene
Interferon-' alpha ' 2b gene is according to pichia pastoris phaff preference codon synthetic artificial gene.(Martinez et al:PCR-based gene synthesis as anefficient approach for expression of the A+T-rich malaria genome.ProteinEngineering.1999,12 (12): the method for describing 1113-1120) is carried out according to document for interferon-' alpha ' 2b gene synthetic.
(2) HSA gene
The HSA gene line by Liu Zhimin etc. according to pichia pastoris phaff preference codon synthetic artificial gene.(Liu Zhimin etc.: human serum albumin changes structure gene, expression vector and host thereof according to document for HSA gene synthetic.Chinese invention patent patent No. CN 99102794.9) method of describing in is carried out.
(3) acquisition of IFN-α 2b-HSA antigen-4 fusion protein gene
The structure of fusion rotein IFN-α 2b-HSA gene: with the HSA gene that obtains in the step (2) is template, under the guiding of primer NF and primer I AR, utilizes pcr amplification test kit (Sweden Pharmacia company product) amplification HSA gene; With the IFN-α 2b gene that obtains in the step (1) is template, under the guiding of primer I AF and primer NR, utilizes pcr amplification test kit (Sweden Pharmacia company product) amplification IFN-α 2b gene.With the amplified production in above-mentioned two steps is template, is that primer amplification goes out IFN-α 2b-HSA antigen-4 fusion protein gene with primer I AF and primer I AR.The amplification system of PCR reaction is:
IFN-α 2b gene 1 μ l
Primer I AR 2 μ l
dNTP 1μl
10x pfu enzyme buffer liquid 5 μ l
Distilled water 37 μ l
Reaction conditions is: 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 4 minutes, carry out 30 circulations altogether.
After Yeast expression carrier pPIC9 and the IFN-α 2b-HSA gene that obtains used XhoI and EcoRI digestion with restriction enzyme respectively, use T
4Dna ligase is gone into pPIC9 with IFN-α 2b-HSA gene clone, obtains recombinant expression vector IFN-α 2b-HSA/pPIC9.And with dna sequence analysis test kit (U.S. USB company product) this recombinant vectors is checked order, the result shows that IFN-α 2b-HSA gene order conforms to expection.
2.IFN-the acquisition of α 2b-HSA (C34S) antigen-4 fusion protein gene
By rite-directed mutagenesis the 34th the non-matching cysteine residues partly of HSA in the IFN-α 2b-HSA fusion rotein replaced with serine residue, and with improved fusion rotein called after IFN-α 2b-HSA (C34S).
Concrete implementation method is: with IFN-α 2b-HSA antigen-4 fusion protein gene is template, under the guiding of primers F and primer C34SR, utilize 5 ' parts of pcr amplification test kit (Sweden Pharmacia company product) amplification IFN-α 2b-HSA (C34S) antigen-4 fusion protein gene; With FN-α 2b-HSA antigen-4 fusion protein gene is template, under the guiding of primer C34SF and primer R, utilizes 3 ' part of pcr amplification test kit (Sweden Pharmacia company product) amplification IFN-α 2b-HSA (C34S) antigen-4 fusion protein gene.With the amplified production in above-mentioned two steps is that template is that primer amplification goes out IFN-α 2b-HSA (C34S) antigen-4 fusion protein gene with primers F and primer R.The amplification system of PCR reaction is:
5 ' part, the 1 μ l of IFN-α 2b-HSA (C34S) antigen-4 fusion protein gene
3 ' part, the 1 μ l of IFN-α 2b-HSA (C34S) antigen-4 fusion protein gene
dNTP 1μl
10x pfu enzyme buffer liquid 5 μ l
Distilled water 38 μ l
Reaction conditions is: 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 4 minutes, carry out 30 circulations altogether.
After Yeast expression carrier pPIC9 and IFN-α 2b-HSA (C34S) antigen-4 fusion protein gene that obtains used XhoI and EcoRI digestion with restriction enzyme respectively, use T
4Dna ligase is cloned into pPIC9 with IFN-α 2b-HSA (C34S) antigen-4 fusion protein gene, obtains recombinant expression vector IFN-α 2b-HSA (C34S)/pPIC9.With dna sequence analysis test kit (U.S. USB company product) this recombinant vectors is checked order, the result shows the nucleotide sequence of IFN-α 2b-HSA (C34S) gene shown in sequence in the sequence table 2, the albumen shown in the sequence 1 in the code sequence tabulation.
3. human serum albumin and the expression of interferon fusion protein gene in pichia pastoris phaff
IFN-α 2b-HSA (C34S)/pPIC9 and IFN-α 2b-HSA/pPIC9 are transformed pichia pastoris phaff host bacterium GS115 (Mut with yeast protoplast transformation method
+His
-), and converted product is coated on MD flat board (1.34g/100ml YNB, 2g/100ml glucose, 4 x 10
-5The g/100ml vitamin H, 1g/100ml agar) on, obtain transformant.24 transformants of picking are inoculated in 4ml BMGY substratum (1g/100ml yeast extract, 2g/100ml Tryptones, 1.34g/100ml YNB, 4 x 10 at random
-5G/100ml vitamin H, 1g/100ml glycerine, 100mM pH6.0 potassium phosphate buffer) in the nutrient solution, added 0.5ml/100ml methyl alcohol every 12 hours and carry out abduction delivering, at 30 ℃, 200rpm cultivated 36-48 hour down.Nutrient solution with the centrifugal 10min of 5000rpm, is respectively got 100 μ L supernatant liquors, and vacuum is drained, and sample is carried out the 10%SDS-PAGE electrophoresis respectively identify, compares with the unloaded vector expression thing of pPIC9, identifies high-expression clone.The high-expression clone that these are screened is protected bacterium, slant culture with 15g/100ml glycerine low temperature respectively then, is used for the engineering bacteria of expressing as further.
The above-mentioned engineering bacteria of picking is inoculated in the 4ml BMGY nutrient solution, and 30 ℃ of 100rpm shake training 12-24 hour.Getting the 1ml yeast liquid transfers in 50ml BMGY nutrient solution, continue to shake training 18 hours, 18 hours nutrient solution of the above-mentioned cultivation of 40ml is inserted in the 1000ml BMGY nutrient solution again, at 30 ℃, 200rpm continues to shake training 30 hours, the nutrient solution that obtains at the centrifugal 5min of 5000rpm, is abandoned supernatant, then with engineering bacteria with 200mlBMMY (1g/100ml yeast extract, 2g/100ml Tryptones, 1.34g/100mlYNB, 4 x 10
-5G/100ml vitamin H, 0.5ml/100ml methyl alcohol, 100mM pH6.0 potassium phosphate buffer) abduction delivering substratum suspension thalline, shake training 3 days at 30 ℃ of 100rpm, added methyl alcohol (final concentration is 0.5ml/100ml) every 24 hours.The cell density of pichia pastoris phaff engineering bacteria reaches OD
600Be 18, the fusion rotein great majority of expression are secreted in the nutrient solution.4 ℃ of centrifugal 20min of 10000rpm abandon thalline, obtain to contain the supernatant liquor of a large amount of fusion protein expression products.
4. the separation and purification of fusion rotein
The pH of the culture supernatant in the step 3 is transferred to 4.5, and carry out preliminary purification with S SepharoseF.F with behind 5 times of the distilled water dilutings.The used level pad of cation-exchange chromatography is the acetate buffer solution of 50mM pH4.5, and elution buffer is the 50mM pH4.5 acetate buffer solution that contains 1M NaCl.Behind cation-exchange chromatography, carry out purifying with Phenyl Sepharose HP hydrophobic chromatography again.The used level pad of hydrophobic chromatography is for containing 1M (NH
4)
2The 20mM phosphate buffered saline buffer of the pH6.0 of SO4, elution buffer are the 20mM phosphate buffered saline buffer of pH6.0.Buffer system is exchanged for Sephadex G25 through the sample of hydrophobic chromatography purifying and carries out purifying with Source 30Q anion-exchange chromatography after pH is 6.0 20mM phosphate buffered saline buffer.The used level pad of anion-exchange chromatography is the 20mM phosphate buffered saline buffer of pH6.0, and elution buffer is the 20mM phosphate buffered saline buffer that contains the pH6.0 of 200mM NaCl.Be further purified the acquisition final product with the Superdex200 molecular sieve at last, the used damping fluid of molecular sieve is the 20mM phosphate buffered saline buffer of pH7.0.
The purification result of IFN-α 2b-HSA and IFN-α 2b-HSA (C34S) is seen Fig. 1.Wherein 1 for expressing supernatant, and swimming lane 2-5 is respectively the sample through cation-exchange chromatography, hydrophobic chromatography, anion-exchange chromatography and sieve chromatography purifying.Electrophoresis result shows the rate of recovery (25%) height of the rate of recovery (33%) of IFN-α 2b-HSA (C34S) than IFN-α 2b-HSA.
The comparison of embodiment 2, IFN-α 2b-HSA and IFN-α 2b-HSA (C34S) fusion rotein stability
The protein concentration of IFN-α 2b-HSA and IFN-α 2b-HSA (C34S) fusion rotein is adjusted to utilizes gel permeation chromatography and non-reduced SDS-PAGE relatively (30 ℃ of IFN-α 2b-HSA and IFN-α 2b-HSA (C34S) earthquakes behind the 1mg/ml, 200rpm concussion 24,48 and 72 hours) and be heated (60 ℃, the 2h) stability under the condition.
Back gel permeation chromatography result such as Fig. 2 are handled in concussion, and among Fig. 2, I-IV represents IFN-α 2b-HSA albumen at 30 ℃, and 200rpm shakes 0,24,48 and 72 hour gel permeation chromatography result; V-VIII represents IFN-α 2b-HSA (C34S) albumen at 30 ℃, and 200rpm shakes 0,24,48 and 72 hour gel permeation chromatography result.Monomeric content is 100% before IFN-α 2b-HSA albumen and the earthquake of IFN-α 2b-HSA (C34S) albumen; Shook 24 o'clock, the content of IFN-α 2b-HSA protein monomer is 80%, and dimer content is 12%, and polymer content is 8%, and the content of IFN-α 2b-HSA (C34S) protein monomer is 95%, and dimer content is 5%, and polymer content is 0%; Shook 48 o'clock, the content of IFN-α 2b-HSA protein monomer is 67%, and dimer content is 20%, and polymer content is 13%, and the content of IFN-α 2b-HSA (C34S) protein monomer is 94%, and dimer content is 16%, and polymer content is 0%; Shook 72 o'clock, the content of IFN-α 2b-HSA protein monomer is 58%, and dimer content is 17%, and polymer content is 25%, and the content of IFN-α 2b-HSA (C34S) protein monomer is 92%, and dimer content is 8%, and polymer content is 0%.
Concussion is handled the non-reduced SDS-PAGE result in back as shown in Figure 3, IFN-α 2b-HSA albumen is at 30 ℃, 200rpm concussion after 24,48 and 72 hours non-reduced SDS-PAGE the big band of molecular weight ratio IFN-α 2b-HSA appears, and IFN-α 2b-HSA (C34S) albumen is at 30 ℃, the 200rpm concussion after 24,48 and 72 hours non-reduced SDS-PAGE have only the band of IFN-α 2b-HSA (C34S) protein monomer.
1. gel permeation chromatography result such as Fig. 4 after the heat-treated represent IFN-α 2b-HSA albumen at 60 ℃ among Fig. 4, handle 2h gel permeation chromatography result; 2. represent IFN-α 2b-HSA (C34S) albumen at 60 ℃, handle 2h gel permeation chromatography result.60 ℃ of content of handling 2h IFN-α 2b-HSA protein monomer are 69%, and dimer content is 31%, and the content of IFN-α 2b-HSA (C34S) protein monomer is 97%, and dimer content is 3%.
Non-reduced SDS-PAGE result as shown in Figure 5 after the heat-treated, the big band of molecular weight ratio IFN-α 2b-HSA appearred in non-reduced SDS-PAGE after IFN-α 2b-HSA protein 60 ℃ was handled 2h, and IFN-α 2b-HSA (C34S) protein 60 ℃ is handled the band that non-reduced SDS-PAGE behind the 2h has only IFN-α 2b-HSA (C34S) protein monomer.
Above-mentioned experimental result show earthquake and the equipressure condition of being heated under, IFN-α 2b-HSA (C34S) has the higher stability than IFN-α 2b-HSA.
The mensuration of embodiment 3, IFN-α 2b-HSA and IFN-α 2b-HSA (C34S) fusion rotein antiviral activity utilizes cytopathic-effect inhibition assay to measure the activity of IFN-α 2b-HSA and IFN-α 2b-HSA (C34S).
Cell cultures: Wish cell (available from Chinese medicine and biological products assay institute) is an attached cell, adopt 0.25% (quality percentage composition) trysinization, RPMI 1640 substratum that contain the 10g/100ml calf serum add two anti-(penicillin, each 100 unit of Streptomycin sulphate), 37 ℃, 5% carbonic acid gas is cultivated and is gone down to posterity every other day.
VSV viral suspension: the chicken embryo of getting 9～10 ages in days, decaptitate, wing, internal organ make cell suspension, be seeded in the Tissue Culture Flask to cultivate and formed fine and close monolayer in 24 hours, inoculation VSV virus (available from Chinese medicine and biological products assay institute) cultivates in the cell bottle that to collect culture supernatant-45 ℃ preservation in 30 hours stand-by.
Micro plate staining: will handle clean micro plate and be placed under the UV-lamp irradiation 30 minutes, every hole adds 100 μ l cell culture fluids, add standard substance (IFN-α 2b) (Recombinant Interferon respectively, available from Tianjin Hualida Biological Engineering Co., Ltd.), the IFN-α 2b-HSA fusion rotein of different doubling dilutions and IFN-α 2b-HSA (C34S) fusion rotein of different doubling dilutions, every then hole adds 100 μ l cell suspensions again, and (cell concn is about 400,000/ml), add a cover to leave standstill and put into CO2gas incubator cultivation 6～8 hours, the microscopically observation of cell has been grown to serve as individual layer, every hole adds 100 μ l VSV viral suspensions and began to attack poison 20～30 hours, observe the whole pathologies of hole inner cell that add standard substance and remove nutrient solution, dye (about 30～40 minutes), go dye liquor to rinse well and dry, every hole adds 200 μ l destainers decolouring back and joins instrument (at A570nm) with enzyme and locate to read the result and calculate.Experiment repeats 3 times.
The EC of IFN-α 2b, IFN-α 2b-HSA fusion rotein and IFN-α 2b-HSA (C34S) fusion rotein
50Be respectively 1.52 ± 0.34ng/ml, 132 ± 15.2ng/ml and 128 ± 13.1ng/ml.
Above-mentioned experimental result shows that the 34th with human serum albumin part do not match the antiviral activity that cysteine residues replaces with does not influence this fusion rotein behind the serine residue.
1.
125The preparation and the evaluation of I mark IFN-α 2b-HSA and IFN-α 2b-HSA (C34S) fusion protein sample
With Iodogen method mark IFN-α 2b-HSA and IFN-α 2b-HSA (C34S), concrete grammar is with reference to Fraker, PJ, Speck JC Jr.Protein and cell membrane iodinations with a sparinglysoluble chloroamide, 1,3,4,6-tetrachloro-3a, 6a-diphrenylglycoluril.Biochem Biophys Res Commun 1978,80:849-57.Identify with size-exclusion HPLC
125I-IFN-α 2b-HSA and
125The radiochemical purity of I-IFN-α 2b-HSA (C34S) is 98.5%, is 36.8kBq μ g than radiation activity
-1, protein content is 0.74mgml
-1Can satisfy SD rat internal metabolism and bioavailability requirement.
2. laboratory animal
The SD rat, available from Military Medical Science Institute's Experimental Animal Center, credit number SCXK-(army) 2002-001 raises in Animal House, regularly air draft, illumination is good, normal temperature.Male and female are divided cage, raise with feed, freely drink water.
3.
125I-IFN-α 2b-HSA and
125The pharmacokinetic of I-IFN-α 2b-HSA (C34S)
(body weight 250 ± 20g) is divided into two groups to 10 SD rats for 5 of ♂, 5 of ♀, 5 every group (3 of ♂, 2 of ♀).Every subcutaneous injection administration
125I-IFN-α 2b-HSA or
125(7.5 μ g are 36.8kBq μ g to I-IFN-α 2b-HSA (C34S) 276kBq than living
-1), dosage is equivalent to 30 μ gkg approximately
-10.5h, 2h, 12h, 24h, 48h, 72h, 120h, 144h and 192h are from tail vein blood after administration.Centrifugation serum.Get the serum 50 μ L of each time point, each pipe is taken up in order of priority the trichloroacetic acid solution that adds 200 μ l injection physiological saline and 200 μ l20g/100ml, (4min * 800g), cleer and peaceful precipitation in the separation is measured precipitation part and supernatant gamma activity partly respectively in centrifugal back.
Calculate AUC at Window ' s Excel version 7.0 by trapezoidal rule, non-compartment model statistics moments method is estimated other pharmacokinetic parameters, asks regression equation and ASSOCIATE STATISTICS parameter and mapping with SigmaPlot2001 software.
IFN-α 2b-HSA and IFN-α 2b-HSA (C34S) in the pharmacokinetics in rats measurement result as shown in Figure 6, showing after the 34th non-matching halfcystine with human serum albumin part replaces with Serine does not influence this fusion rotein in the intravital transformation period of rat.
Sequence table
＜110〉BIO ENGINEERING INST MILITARY
＜120〉human serum albumin-interferon fusion protein and encoding gene thereof and application
<130>CGGNARW92013
<160>2
<210>1
<211>750
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>1
<210>2
<211>2253
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
Claims (10)
1, human serum albumin-interferon fusion protein is following (a) or protein (b):
(a) protein of forming by the amino-acid residue of sequence in the sequence table 1.
(b) with the amino-acid residue of sequence in the sequence table 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and to have an interferon alpha 2 b active by (a) deutero-protein.
2, the encoding gene of the described human serum albumin-interferon fusion protein of claim 1.
3, gene according to claim 2 is characterized in that: the encoding gene of described fusion rotein is following 1) or 2) or 3) gene:
1) its nucleotide sequence is a sequence 2 in the sequence table;
2) under stringent condition with 1) dna molecular of the fusion rotein formed of the dna fragmentation hybridization that limits and coding human serum albumin and Interferon, rabbit;
3) with 1) or 2) gene have the homology 90% or more and the dna molecular of the fusion rotein of coding human serum albumin and Interferon, rabbit composition.
4, the recombinant expression vector, transgenic cell line or the reorganization bacterium that contain the encoding gene of claim 2 or 3 described fusion roteins.
5, amplification claim 2 or 3 described fusion rotein encoding gene total lengths or its any segmental primer are right.
6, a kind of method of producing human serum albumin-interferon fusion protein is to obtain fusion rotein with expressing in claim 2 or the 3 described fusion rotein encoding genes importing host cells.
7, method according to claim 6 is characterized in that: described host is yeast, intestinal bacteria, mammalian cell, insect cell or Bacillus subtilus; Described host is preferably pichia pastoris (Pichiapastoris); Described pichia pastoris is preferably pichia pastoris GS115, KM71 or SMD1168.
8, the encoding gene of the described fusion rotein of claim 1, claim 2 or 3 described fusion roteins prevents and/or treats tumour in preparation, and/or the application in the medicine of inhibition virus.
9, a kind of tumour that prevents and/or treats, and/or suppress viral medicine, its activeconstituents is the described human serum albumin-interferon fusion protein of claim 1.
10, method according to claim 9 is characterized in that: described medicine also comprises pharmaceutically acceptable carrier.
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