CN101463089B

CN101463089B - Human serum albumin-interferon fusion protein, and encoding gene and use thereof

Info

Publication number: CN101463089B
Application number: CN2009100762413A
Authority: CN
Inventors: 刘志敏; 赵洪亮; 薛冲
Original assignee: Institute of Bioengineering Chinese Academy of Military Medical Sciences
Current assignee: Institute of Bioengineering Chinese Academy of Military Medical Sciences
Priority date: 2009-01-08
Filing date: 2009-01-08
Publication date: 2012-07-04
Anticipated expiration: 2029-01-08
Also published as: CN101463089A

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

Human serum albumin-interferon fusion protein and encoding sox thereof and application

Technical field

The present invention relates to human serum albumin-interferon fusion protein and encoding sox thereof and application.

Background technology

(interferon IFN) is one type of cytokine with antiviral, antiproliferative and immunoregulatory activity to 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 ' (like interferon-' alpha ' 2a, interferon-' alpha ' 2b and interferon-' alpha ' 1b) is widely used in treating several diseases viral disease (hepatitis B, hepatitis C) and tumour clinically.

Yet the transformation period of natural interferon-α is shorter, has only frequent drug administration (in clinical treatment, often adopt 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.Research show many protein drugs with human serum albumin is crosslinked or merge after its transformation period is prolonged, thereby minimizing 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 got into 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 through the phase place that changes fusion rotein.IFN-α 2b-HSA is improving the expression amount and the 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 sox 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 production cost high, awkward freeze-dried formulation.Less stable under the IFN-α 2b-HSA fusion rotein earthquake and the equipressure condition of being heated is prone to form 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 sox thereof and application.

Human serum albumin-interferon fusion protein provided by the present invention, called after IFN-α 2b-HSA (C34S) is (a) or protein (b) as follows:

(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
			FLAG	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 sox earlier, carry out biology again and express and to obtain.The encoding sox 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.

Sequence 1 in the sequence table is made up of 750 amino-acid residues; From N-terminal 1-165 position is the amino acid residue sequence of IFN-α 2b; From N-terminal 166-750 position is the amino acid residue sequence of human serum albumin (HSA), has been replaced the cysteine residues in the human serum albumin (HSA) by serine residue from the 199th of N-terminal.

The encoding sox of above-mentioned fusion rotein also belongs to protection scope of the present invention.The encoding sox 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) the dna fragmentation hybridization that limits and the dna molecular of the coding human serum albumin and the fusion rotein of Interferon, rabbit composition;

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 said step 3) is with 1) gene the 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, and 0.1% SDS and 1 * SSC, 0.1%SDS respectively wash film once.

Sequence 2 in the sequence table is made up of 2253 Nucleotide, the albumen shown in the sequence 1 in the code sequence tabulation.

The recombinant expression vector, transgenic cell line or the reorganization bacterium that contain the encoding sox of said fusion rotein all belong to protection scope of the present invention.

The above-mentioned fusion rotein encoding sox 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 said human serum albumin-interferon fusion protein.

The method of the said human serum albumin-interferon fusion protein of production provided by the present invention is that said fusion rotein encoding sox is imported host cell, expresses obtaining fusion rotein.

Wherein, said host can be yeast, intestinal bacteria, mammalian cell, insect cell or Bacillus subtilus etc., is preferably yeast.

Said yeast is preferably pichia pastoris (Pichia pastoris).Wherein, said pichia pastoris is preferably pichia pastoris GS115, KM71 or SMD1168 (can available from American I nvitrogen company).

Said intestinal bacteria can be E.coli JM109, E.coli HB101, E.coli Top10.

The encoding sox of fusion rotein of the present invention, said 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.Said carrier comprises the conventional thinner of pharmaceutical field, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier etc.

Medicine of the present invention can be processed 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 in every separated 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 is high, be prone to 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 compares the stability under IFN-α 2b-HSA and IFN-α 2b-HSA (C34S) the earthquake condition for gel permeation chromatography.

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

Be ordinary method like no specified otherwise method therefor among the following embodiment.

Agents useful for same all can obtain from commercial sources 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 ltd

2, pcr amplification test kit (Sweden Pharmacia Company products)

3, dna sequence analysis test kit (U.S. USB Company products)

4, casein hydrolysate (German MERK Company products)

5, Bacto-yeast extract (U.S. Difco Company products)

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(pH 7.4)。

(7) 1M PBS damping fluid: 132ml 1M K ₂HPO ₄, 868ml 1M KH ₂PO ₄, pH 6.0.

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 products) amplification HSA gene; IFN-α 2b gene to obtain in the step (1) is a template, under the guiding of primer I AF and primer NR, utilizes pcr amplification test kit (Sweden Pharmacia Company products) amplification IFN-α 2b gene.Amplified production with in above-mentioned two steps is a 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:

HSA gene 1 μ l

IFN-α 2b gene 1 μ l

Primer I AF 2 μ l

Primer I AR 2 μ l

dNTP 1μl

10x pfu enzyme buffer liquid 5 μ l

Pfu enzyme 1 μ l

Zero(ppm) water 37 μ l

TV 50 μ l.

Reaction conditions is: 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 4 minutes, carry out 30 circulations altogether.

After Yeast expression carrier pPIC9 used XhoI and EcoRI digestion with restriction enzyme respectively with the IFN-α 2b-HSA gene that obtains, 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 products) 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

Through 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 products) 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 products) amplification IFN-α 2b-HSA (C34S) antigen-4 fusion protein gene.Amplified production with 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

Primers F 2 μ l

Primer R 2 μ l

dNTP 1μl

10x pfu enzyme buffer liquid 5 μ l

Pfu enzyme 1 μ l

Zero(ppm) water 38 μ l

TV 50 μ l

After Yeast expression carrier pPIC9 used XhoI and EcoRI digestion with restriction enzyme respectively with IFN-α 2b-HSA (C34S) antigen-4 fusion protein gene that obtains, 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 products) 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 * 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 * 10 at random ^-5G/100ml vitamin H, 1g/100ml glycerine, 100mM pH6.0 potassium phosphate buffer) in the nutrient solution, whenever added 0.5ml/100ml methyl alcohol at a distance from 12 hours and carry out abduction delivering, at 30 ℃, 200rpm cultivation 36-48 hour down.Nutrient solution with the centrifugal 10min of 5000rpm, is respectively got 100 μ L supernatants, 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.Then these high-expression clones that screen are protected bacterium, slant culture with 15g/100ml glycerine low temperature respectively, be 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, again 18 hours nutrient solution of the above-mentioned cultivation of 40ml is inserted in the 1000ml BMGY nutrient solution, 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 * 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, every methyl alcohol (final concentration is 0.5ml/100ml) of adding at a distance from 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 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 pH 6.0 of SO4, elution buffer are the 20mM phosphate buffered saline buffer of pH 6.0.Use Sephadex G25 that buffer system is exchanged for through the sample of hydrophobic chromatography purifying and carry 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 pH 6.0, and elution buffer is the 20mM phosphate buffered saline buffer that contains the pH 6.0 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 pH 7.0.

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 recovery (25%) height of the 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%.

It is as shown in Figure 3 that the non-reduced SDS-PAGE result in back is handled in concussion; 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 is 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 is illustrated under the concussion and the equipressure condition of being heated, and 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

Utilize 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-(penicillium mould, 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 process 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 for use.

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), adds a cover to leave standstill and puts into CO2gas incubator and cultivated 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, observes the whole pathologies of hole inner cell that add standard substance and removes 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 appearance (at A 570nm) with enzyme and locate to read the result and calculate.Experiment repetition 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.

Embodiment 4, IFN-α 2b-HSA and IFN-α 2b-HSA (C34S) measure in pharmacokinetics in rats

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 with 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 with 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.Spinning 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 saline water and 200 μ l20g/100ml; (4min * 800g), cleer and peaceful deposition in the separation is measured deposition part and supernatant gamma activity partly respectively in centrifugal back.

Calculate AUC at Window ' s Excel version 7.0 by trapezoidal rule, non-other pharmacokinetic parameters of compartment model statistics moments method estimation is asked regression equation and ASSOCIATE STATISTICS parameter and mapping with SigmaPlot 2001 softwares.

It is as shown in Figure 6 that IFN-α 2b-HSA and IFN-α 2b-HSA (C34S) measure the result in pharmacokinetics in rats, and showing after the 34th non-matching halfcystine with the 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 sox thereof and application

<130>CGGNARW92013

<160>2

<210>1

<211>750

<212>PRT

< 213>artificial sequence

<220>

<223>

<400>1

Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met

1 5 10 15

Phe Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp

20 25 30

Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln

35 40 45

Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe

50 55 60

Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu

65 70 75 80

Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu

85 90 95

Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys

100 105 110

Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu

115 120 125

Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg

130 135 140

Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser

145 150 155 160

Leu Arg Ser Lys Glu Asp Ala His Lys Ser Glu Val Ala His Arg Phe

165 170 175

Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe

180 185 190

Ala Gln Tyr Leu Gln Gln Ser Pro Phe Glu Asp His Val Lys Leu Val

195 200 205

Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala

210 215 220

Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys

225 230 235 240

Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys

245 250 255

Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp

260 265 270

Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met

275 280 285

Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu

290 295 300

Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu

305 310 315 320

Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala

325 330 335

Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp

340 345 350

Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu

355 360 365

Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu

370 375 380

Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val

385 390 395 400

Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu

405 410 415

Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn

420 425 430

Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu

435 440 445

Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro

450 455 460

Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val

465 470 475 480

Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu

485 490 495

Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu

500 505 510

Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala

515 520 525

Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro

530 535 540

Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe

545 550 555 560

Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr

565 570 575

Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser

580 585 590

Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala

595 600 605

Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln

610 615 620

Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys

625 630 635 640

Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu

645 650 655

Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe

660 665 670

Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile

675 680 685

Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala

690 695 700

Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val

705 710 715 720

Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu

725 730 735

Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu

740 745 750

<210>2

<211>2253

<212>DNA

< 213>artificial sequence

<220>

<223>

<400>2

tgtgacttgc cacaaaccca ctccttgggt tccagaagaa ccttgatgtt tttggctcaa 60

atgagaagaa tctccttgtt ctcttgtttg aaggacagac acgacttcgg tttcccacaa 120

gaggagttcg gtaaccaatt ccaaaaggct gagaccatcc cagttttgca cgagatgatc 180

caacaaatct tcaacttgtt ctccaccaag gactcctccg ctgcttggga cgagaccttg 240

ttggacaagt tctacaccga gttgtaccaa caattgaacg acttggaggc ttgtgttatc 300

caaggtgttg gtgttaccga gaccccattg atgaaggagg actccatctt ggctgttaga 360

aagtacttcc aaagaatcac cttgtacttg aaggagaaga agtactcccc atgtgcttgg 420

gaggttgtta gagctgagat catgagatcc ttctccttgt ccaccaactt gcaagagtcc 480

ttgagatcca aggaggacgc tcacaagtct gaagttgctc acagattcaa ggacttgggt 540

gaagaaaact tcaaggcttt ggttttgatt gctttcgctc aatacttgca acaatctcca 600

ttcgaagacc acgttaagtt ggttaacgaa gttactgaat ttgctaagac ttgtgttgct 660

gacgaatctg ctgaaaactg tgacaagtct ttgcacactt tgttcggtga caagttgtgt 720

actgttgcta ctttgagaga aacttacggt gaaatggctg actgttgtgc taagcaagaa 780

ccagaaagaa acgaatgttt cttgcaacac aaggacgaca acccaaactt gccaagattg 840

gttagaccag aagtcgacgt tatgtgtact gctttccacg acaacgaaga aactttcttg 900

aagaagtact tgtacgaaat tgctagaaga cacccatact tctacgctcc agaattgttg 960

ttcttcgcta agagatacaa ggctgctttc actgaatgtt gtcaagctgc tgacaaggct 1020

gcttgtttgt tgccaaagtt ggacgaattg agagacgaag gtaaggcttc ttctgctaag 1080

caaagattga agtgtgcttc tttgcaaaag ttcggtgaaa gagctttcaa agcttgggct 1140

gttgctagat tgtctcaaag attcccaaag gctgaatttg ctgaagtttc taagttggtt 1200

actgacttga ctaaggttca cactgaatgt tgtcacggtg acttgttgga atgtgctgac 1260

gacagagctg acttggctaa gtacatttgt gaaaaccaag actctatttc ttctaagttg 1320

aaggaatgtt gtgaaaagcc attgttggaa aagtctcact gtattgctga agttgaaaac 1380

gacgaaatgc cagctgactt gccatctttg gctgctgact tcgttgaatc taaggacgtt 1440

tgtaagaact acgctgaagc taaggacgtt ttcttgggta tgttcttgta cgaatacgct 1500

agaagacacc cagactactc tgttgttttg ttgttgagat tggctaagac ttacgaaact 1560

actttggaaa agtgttgtgc ggccgctgac ccacacgaat gttacgctaa ggttttcgac 1620

gaatttaagc cattggttga agaaccacaa aacttgatta agcaaaactg tgaattgttc 1680

gaacaattgg gtgaatacaa gttccaaaac gctttgttgg ttagatacac taagaaggtt 1740

ccacaagttt ctactccaac tttggttgaa gtttctagaa acttgggtaa ggttggttct 1800

aagtgttgta agcacccaga agctaagaga atgccatgtg ctgaagacta cttgtctgtt 1860

gttttgaacc aattgtgtgt tttgcacgaa aagactccag tttctgacag agttactaag 1920

tgttgtactg aatctttggt taacagaaga ccatgtttct ctgctttgga agttgacgaa 1980

acttacgttc caaaggaatt taacgctgaa actttcactt tccacgctga catttgtact 2040

ttgtctgaaa aggaaagaca aattaagaag caaactgctt tggttgaatt ggttaagcac 2100

aagccaaagg ctactaagga acaattgaag gctgttatgg acgacttcgc tgctttcgtt 2160

gaaaagtgtt gtaaggctga cgacaaggaa acttgtttcg ctgaagaagg taagaagttg 2220

gttgctgctt ctcaagctgc tttgggtttg taa 2253

Claims

1. human serum albumin-interferon fusion protein, the protein of forming by the amino-acid residue of sequence in the sequence table 1.

2. the encoding sox of the described human serum albumin-interferon fusion protein of claim 1.

3. gene according to claim 2 is characterized in that: the encoding sox of said fusion rotein, its nucleotide sequence are sequences 2 in the sequence table.

4. the recombinant expression vector that contains the encoding sox of claim 2 or 3 said fusion roteins.

5. the transgenic cell line that contains the encoding sox of claim 2 or 3 said fusion roteins.

6. the reorganization bacterium that contains the encoding sox of claim 2 or 3 said fusion roteins.

7. a method of producing human serum albumin-interferon fusion protein is to obtain fusion rotein with expressing in claim 2 or the 3 said fusion rotein encoding soxs importing host cells.

8. method according to claim 7 is characterized in that: said host is yeast, intestinal bacteria, mammalian cell, insect cell or Bacillus subtilus.

9. method according to claim 8 is characterized in that: said host is a pichia pastoris (Pichiapastoris).

10. method according to claim 9 is characterized in that: said host is pichia pastoris GS115, KM71 or SMD1168.

11. the encoding sox of the said fusion rotein of claim 1, claim 2 or 3 said fusion roteins prevents and/or treats tumour in preparation, and/or the application in the medicine of inhibition virus.

12. one kind prevents and/or treats tumour, and/or suppresses the medicine of virus, its activeconstituents is the described human serum albumin-interferon fusion protein of claim 1.

13. medicine according to claim 12 is characterized in that: said medicine also comprises pharmaceutically acceptable carrier.