CN102093480A - Recombinant human serum albumin-interferon alpha 1b fusion protein and preparation method thereof - Google Patents
Recombinant human serum albumin-interferon alpha 1b fusion protein and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a recombinant human serum albumin-interferon alpha 1b fusion protein, a DNA sequence which encodes the fusion protein, and a production process for purifying rhSA-IFN alpha 1b in pichia pastoris fermentation liquor. On the basis of keeping the physiological property of the interferon alpha 1b, the half-life period of the interferon alpha 1b is prolonged in vivo by the fusion protein; the purification operation is simple; and industrial production is convenient to expand.
Description
Technical field
The present invention is general relates to recombined human serum albumin fusion proteins and preparation method thereof, relates to recombination human serum albumin-Interferon, rabbit and preparation method thereof especially.
Background technology
(interferon is the important familial cytokine of a class IFN) to Interferon, rabbit, has the opposing virus infection, suppresses the effect of tumor growth and adjusting body's immunity.The IFN protein family is divided into 3 types based on their gene order, chromosomal localization and receptor-specific: the I type comprises IFN-α ,-β ,-ω ,-ε, hypotypes such as-κ; II type Interferon, rabbit is made of the IFN-γ of single-gene family, is called type II interferon again; The III type is a kind of newfound cytokine, is called IFN-λ.Wherein the antiviral activity of IFN-α is the strongest, and the different hypotypes of IFN-alpha molecule are made up of 165-172 amino acid, about the about 19kD of molecular weight.Along with the development of genetic engineering technique, IFN-α becomes first and is used for clinical gene recombination cytokine, has been applied to multiple treatment of diseases such as viral hepatitis, cancer and multiple sclerosis by drugs approved by FDA.But the interferon molecule amount is less, and is unstable in the body easily by glomerular filtration, and easily by the serum protein enzyme liberating, plasma half-life is short and treatment cycle is long, needs frequent drug administration by injection, the reduction of patient's compliance.
In order to overcome above-mentioned shortcoming, the long-acting interferon drug development has become an important directions.There is following several effective means the prolongation recombinant interferon transformation period: 1) sustained-release preparation, Interferon, rabbit is encapsulated in the polymeric layer, by subcutaneous or muscle administration, polymeric layer reduces in time, make medicine from micro-capsule, slowly continue to discharge, this helps stablizing medicine, reduces the destruction of gastrointestinal enzyme, the transformation period in the prolong drug body; 2) utilize site-directed mutagenesis technique, make as methods such as the rite-directed mutagenesis of Oligonucleolide primers, PCR mediation and cassette mutagenesises that protease site changes over insensitive other amino acid of proteolytic enzyme in the Interferon, rabbit, thereby the enzyme in the minimizing blood is degraded to it, the transformation period in the extension body; 3) chemical modification technology is linked to the Interferon, rabbit surface with covalency such as glycosyl or PEG, and the albumen relative molecular weight is increased, and has reduced the filtration of renal glomerulus, and protected protein blocks the antigenic determinant of protein surface simultaneously not by proteasome degradation, reduces immunogenicity; 4) albumen integration technology merges interferon gene and specific carrier protein gene by the gene recombination means, by same regulating and controlling sequence controlling gene expression product, with the artificial recruit who creates of genetic engineering means.Be HSA and antibody Fc fragment for making albumen obtain the maximum carrier of long lasting purpose employing at present.
Because Atrigel exists encapsulation rate low, prominently release and discharge not exclusively and, prepare ideal Interferon, rabbit sustained release preparation and also need big quantity research in the problems such as IFN sex change inactivation that preparation process intensive physics, chemical transformation cause; Present two kinds of PEGization Interferon, rabbit of FDA approved are used for clinical, are respectively the PEG-Intron (branchedPEG40kD-IFN-α 2b) of Schering Plough and the Pegasys (linear PEG12kD-IFN-α 2b) of Luo Shi.Yet these two kinds of PEGization Interferon, rabbit are non-homogeneous product.The mixture that PEG-Intron IFN α-2b mono-modified by 14 kinds and a small amount of unmodified forms, and Pegasys is at least by 6 kinds of mono-modified mixtures of forming, this numerous isomer mixture causes different physiological responses, has also caused difficult problems such as production quality control, suitability for industrialized production purifying simultaneously.Site-directed mutagenesis technique requires quite high to protein engineering, the effect that is similar to combinatorial chemical library triage techniques and its prolong half-life is unsatisfactory.Therefore the long-acting protein drug based on the genetic engineering means development more and more receives people's concern, and this is wherein outstanding with the human serum albumin integration technology, and this method is carrier with the human serum albumin.Human serum albumin (HSA) is a natural substance delivery vehicles in the blood of human body, content is the highest in the serum, its relative molecular weight is about 65kDa, the strand ball-type protein of forming by 585 amino acid, no zymetology and immunologic competence, the transformation period reaches 19 days in the body, is the medicine fusion rotein that ideal improves drug half-life.
Summary of the invention
Primary and foremost purpose of the present invention provides a kind of human serum albumin-interferon alpha 1b, and it has the sequence shown in the SEQ ID No.1.
Second purpose of the present invention provides the preparation method of human serum albumin shown in the SEQ ID No.1-interferon alpha 1b, and this method comprises the steps: at least
(a) the codon optimized and chemosynthesis of recombination human serum albumin-interferon alpha 1b expressing fusion protein gene;
(b) structure that contains the expression plasmid of yeast of (a) described expressing gene;
(c) contain the structure and the screening of the pichia spp transformant of (b) described expression plasmid;
(d) fermentation culture of transformant engineering bacteria;
(e) purifying of engineering bacterium expression fusion rotein.
The codon of the codon optimized preferred yeast preference in the step in the aforesaid method (a).
The preferred pichia spp GS115 of pichia spp in the step (c).
The preferred secretion type expression of fusion rotein in the step (e) is preferably realized by the natural signals peptide of human serum albumin.
Step (e) preferably includes:
(1) (d) gained engineering bacteria is carried out after the centrifugal treating gained centrifuged supernatant being carried out ultrafiltration and concentration;
(2) the Blue Sepharose FF dye affinity chromatography purifying of ultrafiltration and concentration liquid;
(3) the CM Sepharose FF cation-exchange chromatography of preceding step gained elutriant is further purified.
The ultra-filtration membrane preferred 30kDa in aperture during ultrafiltration in the step (1) wherein; 20mM PB damping fluid is preferred in the step (2), the 2M NaCl that wash-out uses, and pH7.0 is preferred; The 25mmol/L sodium-acetate buffer is preferred in the step (3), the 0.3mol/LNaCl that wash-out uses, and pH4.4-5.0 is preferred.
Last purpose of the present invention provides the purposes of aforementioned human serum albumin-interferon alpha 1b in the medicine for preparing the relevant disease of prevention or treatment hepatitis B and hepatitis B virus infection.
Description of drawings
Fig. 1 represents the pcr analysis electrophorogram of GS115 transformant, and each swimming lane sample is followed successively by DNA Marker from left to right among the figure, positive transformant, positive transformant, positive transformant, positive transformant, negative transformant, positive transformant, negative control.
Fig. 2 represents the Western blot detected result of fusion rotein purified product, albumen Marker from left to right among the figure, fusion rotein purified product.
Fig. 3 represents the irreducibility SDS-PAGE detected result of fusion rotein purified product, each swimming lane sample is followed successively by albumen Marker from left to right among the figure, the ultrafiltration and concentration sample, Blue Sepharose FF dye affinity chromatography purification of samples, CM Sepharose FF cation-exchange chromatography purification of samples.
Fig. 4 represents the MALDI-TOF mass spectrum molecular weight detection result of fusion rotein purified product.
Embodiment
Embodiment 1: the structure of expression vector
The worker synthetic pBluescript II KS of company limited (+)-HAS-NIFN is given birth to BamHI, EcoRI double digestion in Shanghai, enzyme is cut product with 0.8% agarose gel electrophoresis, reclaim goal gene with Agarose Gel DNA Purification Kit, be connected with the yeast expression vector pPIC3.5 that cuts through same enzyme, construct pPIC3.5-HSA-IFN α 1b recombinant plasmid.
Embodiment 2: the electric shock of pichia spp GS115 transforms and screening
Recombinant plasmid pPIC3.5-HSA-IFN α 1b electric shock transformed competence colibacillus pichia spp GS115 with 2-4 ug SalI linearization process.Electric shock adds 1mL 1mol/L sorbyl alcohol after finishing immediately, gets 200uL behind the mixing and coats MD flat board (1.34%YNB, 4 * 10
-5The % vitamin H, 2% glucose, 1.5% agar), 30 ℃ are cultured to bacterium colony and occur.Screening positive clone.
Embodiment 3: the PCR of recombinant yeast pichia pastoris identifies
Extract recon GS115/pPIC3.5-HSA-IFN α 1b genomic dna with TIANamp Yeast DNA Kit.In the PCR of 50uL reaction system, add: upstream primer (5 ' TCAAAGATTCCCAAAGGC, 3 ' 20 μ M) 1 μ L, downstream primer (5 ' CAGCGAAGTCGTCCATAA3 ', 20 μ M) 1 μ L, the recon genome 2.5ng that extracts, 4 * dNTP mixed solution (each 2.5mM), 4 μ L, Pyrobest archaeal dna polymerase (0.5U/ μ L) 2.5 μ L, 10 * Pyrobest damping fluid, 5 μ L, distilled water H
2O to 50 μ L.The PCR condition is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 1min, 53 ℃ of annealing 1min, 72 ℃ are extended 90s, 30 circulations, 72 ℃ are extended 5min.Detected through gel electrophoresis PCR product.
Embodiment 4: abduction delivering and the purifying of recon GS115/pPIC3.5-HSA-IFN α 1b
The higher recon of expression amount that screening is obtained is inoculated in 50ml BMGY substratum (1% yeast extract, 2% peptone, 1.34%YNB, 4 * 10 is housed
-5% vitamin H, 1% glycerine) in the 500ml triangular flask, 30 ℃, 300r/min is cultured to A
600Value is 2.0-6.0, centrifugal collection thalline, and BMMY (with the glycerine among the 0.5% methyl alcohol replacement BMGY) re-suspended cell is to A
600Value is 1.0,30 ℃, carries out abduction delivering under the 300r/min, adds methyl alcohol, abduction delivering 96h one time every 24h.
The centrifugal 5min of 8000rpm collects supernatant.Utilize the ultrafiltration cup, the ultra-filtration membrane aperture is 30kDa, with A liquid (20mM PBpH7.0) balance ultra-filtration membrane, then with 10 times of 1L supernatant concentration.Concentrated solution adds 2 times of damping fluids and continues ultrafiltration, finally collects sample solution 50ml.To with the abundant equilibrated Blue of A liquid Sepharose FF chromatography column,, use B liquid (20mM PB+2M NaCl pH7.0) to carry out wash-out then in sample on the concentrated solution, collect the elution peak component with the A liquid flushing of two column volumes; Sample on the elution peak component of BlueSepharose FF affinity chromatography is arrived with the abundant good CM Sepharose FF cation-exchange chromatography post of balance of C liquid (25mmol/L sodium-acetate buffer pH4.5), with D liquid (25mmol/L sodium-acetate buffer+0.3mol/L NaCl pH4.5) flushing, collect the elution peak component.
Embodiment 5: the Western blot of fusion rotein purified product identifies
The fusion rotein purified product is behind SDS-PAGE, electrotransfer albumen is to nitrocellulose filter, film is changed among the 5%BSA of TBST preparation, 4 ℃ of sealings are spent the night, clean monoclonal antibody (1: 2000) the incubated at room 1h that the back adds mouse anti human HSA with TBST, clean two anti-(1: 2000) incubated at room 1h of the goat anti-mouse IgG of back adding horseradish peroxidase-labeled with TBS, clean the back with TBS and develop the color with HRP-DAB Kit.
Embodiment 6:rHSA-IFN α 1b molecular weight detection
Adopt substance assistant laser desorpted ionization time-of-flight mass spectrometry (TOFMS), matrix is sinapinic acid; N
2Laser source; Wavelength is 337nm; Flight pipe range 1.5M; Acceleration voltage 20KV.
Embodiment 7: the determination of activity of fusion rotein rHSA-IFN α 1b extracorporeal antivirus effect
According to the 3rd one of Pharmacopoeia of the People's Republic of China version in 2010, adopt cytopathic-effect inhibition assay (CPE), utilize the activity of WISH-VSV systems measurement HSA-NIFN.Measure its protein content with the Lowry method, calculate specific activity.
Attached sequence table
Sequence table 1: the aminoacid sequence of recombination human serum albumin-interferon alpha 1b fusion rotein
＜110〉Beijing Bio-Tech Development Co., Ltd. of Beijing Sanyuan Gene Engineering Co. Ltd.
＜120〉recombination human serum albumin interferon alpha 1b fusion rotein and preparation method thereof
<130>
<160>1
<170>PatentIn?version?3.3
<210>1
<211>786
<212>PRT
＜213〉artificial sequence
<400>1
Met?Lys?Trp?Val?Thr?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Arg?Gly?Val?Phe?Arg?Arg?Asp?Ala?His?Lys?Ser?Glu?Val?Ala
20 25 30
His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu
35 40 45
Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val
50 55 60
Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp
65 70 75 80
Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp
85 90 95
Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala
100 105 110
Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln
115 120 125
His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val
130 135 140
Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys
145 150 155 160
Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro
165 170 175
Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys
180 185 190
Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu
195 200 205
Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys
210 215 220
Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val
225 230 235 240
Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser
245 250 255
Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly
260 265 270
Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile
275 280 285
Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu
290 295 300
Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp
305 310 315 320
Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser
325 330 335
Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly
340 345 350
Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val
355 360 365
Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys
370 375 380
Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu
385 390 395 400
Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys
405 410 415
Glu?Leu?Phe?Lys?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu
420 425 430
Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val
435 440 445
Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His
450 455 460
Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val
465 470 475 480
Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg
485 490 495
Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe
500 505 510
Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala
515 520 525
Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu
530 535 540
Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys
545 550 555 560
Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala
565 570 575
Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe
580 585 590
Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly
595 600 605
Leu?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Met?Cys?Asp?Leu?Pro
610 615 620
Glu?Thr?His?Ser?Leu?Asp?Asn?Arg?Arg?Thr?Leu?Met?Leu?Leu?Ala?Gln
625 630 635 640
Met?Ser?Arg?Ile?Ser?Pro?Ser?Ser?Cys?Leu?Met?Asp?Arg?His?Asp?Phe
645 650 655
Gly?Phe?Pro?Gln?Glu?Glu?Phe?Asp?Gly?Asn?Gln?Phe?Gln?Lys?Ala?Pro
660 665 670
Ala?Ile?Ser?Val?Leu?His?Glu?Leu?Ile?Gln?Gln?Ile?Phe?Asn?Leu?Phe
675 680 685
Thr?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Asp?Leu?Leu?Asp?Lys
690 695 700
Phe?Cys?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu?Ala?Cys?Ala
705 710 715 720
Met?Gln?Glu?Glu?Arg?Val?Gly?Glu?Thr?Pro?Leu?Met?Asn?Ala?Asp?Ser
725 730 735
Ile?Leu?Ala?Val?Lys?Lys?Tyr?Phe?Arg?Arg?Ile?Thr?Leu?Tyr?Leu?Thr
740 745 750
Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg?Ala?Glu?Ile
755 760 765
Met?Arg?Ser?Leu?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Arg?Leu?Arg?Arg
770 775 780
Lys?Glu
785
Sequence table 2: the nucleotide sequence of recombination human serum albumin-interferon alpha 1b fusion rotein
＜110〉Beijing Bio-Tech Development Co., Ltd. of Beijing Sanyuan Gene Engineering Co. Ltd.
＜120〉recombination human serum albumin-interferon alpha 1b fusion rotein and preparation method thereof
<130>
<160>1
<170>PatentIn?version?3.3
<210>2
<211>2358
<212>DNA
＜213〉artificial sequence
<400>1
ttcgaaacga?tgaagtgggt?tactttcatt?tctttgttgt?tcttgttctc?ttctgcttac 60
tccagaggtg?ttttccgtag?agacgctcac?aagtctgaag?ttgctcacag?attcaaggac 120
ttgggtgaag?aaaacttcaa?ggctttggtt?ttgattgctt?tcgctcaata?cttgcaacaa 180
tgtccattcg?aggaccacgt?taagttggtt?aacgaagtta?ctgaatttgc?taagacttgt 240
gttgctgacg?aatctgctga?aaactgtgac?aagtctttgc?acactttgtt?cggtgacaag 300
ttgtgtactg?ttgctacttt?gagagaaact?tacggtgaaa?tggctgactg?ttgtgctaag 360
caagaaccag?aaagaaacga?atgtttcttg?caacacaagg?acgacaaccc?aaacttgcca 420
agattggtta?gaccagaagt?tgacgttatg?tgtactgctt?tccacgacaa?cgaagaaact 480
ttcttgaaga?agtacttgta?cgaaattgct?agaagacacc?catacttcta?cgctccagaa 540
ttgttgttct?tcgctaagag?atacaaggct?gctttcactg?aatgttgtca?agctgctgac 600
aaggctgctt?gtttgttgcc?aaagttggac?gaattgagag?acgaaggtaa?ggcttcttct 660
gctaagcaaa?gattgaagtg?tgcttctttg?caaaagttcg?gtgaaagagc?tttcaaagct 720
tgggctgttg?ctagattgtc?tcaaagattc?ccaaaggctg?aatttgctga?agtttctaag 780
ttggttactg?acttgactaa?ggttcacact?gaatgttgtc?acggtgactt?gttggaatgt 840
gctgacgaca?gagctgactt?ggctaagtac?atttgtgaaa?accaagactc?tatttcttct 900
aagttgaagg?aatgttgtga?aaagccattg?ttggaaaagt?ctcactgtat?tgctgaagtt 960
gaaaacgacg?aaatgccagc?tgacttgcca?tctttggctg?ctgacttcgt?tgaatctaag 1020
gacgtttgta?agaactacgc?tgaagctaag?gacgttttct?tgggtatgtt?cttgtacgaa 1080
tacgctagaa?gacacccaga?ctactctgtt?gttttgttgt?tgagattggc?taagacttac 1140
gaaactactt?tggaaaagtg?ttgtgcggcc?gctgacccac?acgaatgtta?cgctaaggtt 1200
ttcgacgaat?ttaagccatt?ggttgaagaa?ccacaaaact?tgattaagca?aaactgtgaa 1260
ttgttcgagc?aattgggtga?atacaagttc?caaaacgctt?tgttggttag?atacactaag 1320
aaggttccac?aagtttctac?tccaactttg?gttgaagttt?ctagaaactt?gggtaaggtt 1380
ggttctaagt?gttgtaagca?cccagaagct?aagagaatgc?catgtgctga?agactacttg 1440
tctgttgttt?tgaaccaatt?gtgtgttttg?cacgaaaaga?ctccagtttc?tgacagagtt 1500
actaagtgtt?gtactgaatc?tttggttaac?agaagaccat?gtttctctgc?tttggaagtt 1560
gacgaaactt?acgttccaaa?ggaatttaac?gctgaaactt?tcactttcca?cgctgacatt 1620
tgtactttgt?ctgaaaagga?aagacaaatt?aagaagcaaa?ctgctttggt?tgaattggtt 1680
aagcacaagc?caaaggctac?taaggaacaa?ttgaaggctg?ttatggacga?cttcgctgct 1740
ttcgttgaaa?agtgttgtaa?ggctgacgac?aaggaaactt?gtttcgctga?agaaggtaag 1800
aagttggttg?ctgcttctca?agctgctttg?ggtttgggag?gtggcggttc?ttgtgattta 1860
cccgagacac?atagtcttga?taaccgtaga?actttgatgt?tgctggctca?aatgtcaaga 1920
atttcccctt?cttcctgctt?gatggatagg?catgacttcg?gattcccaca?agaagagttc 1980
gacggtaatc?aattccaaaa?ggccccagcc?attagtgttc?tgcacgagtt?gattcagcag 2040
atatttaatc?tttttaccac?taaggactca?tctgccgctt?gggacgaaga?ccttctagat 2100
aagttttgca?ctgaattgta?ccagcagctg?aacgatttag?aggcttgtgc?tatgcaagaa 2160
gaaagagttg?gtgagactcc?tcttatgaac?gcagattcaa?ttctggctgt?caagaagtac 2220
tttagacgta?tcacattata?tctaaccgaa?aagaagtatt?ccccatgtgc?atgggaagta 2280
gtgagagcag?agatcatgag?gtctttatct?ttgagtacga?atttgcaaga?aagactacga 2340
cgaaaagaat?aagaattc 2358
Claims (10)
1. recombination human serum albumin-interferon alpha 1b fusion rotein is characterized in that having sequence shown in the sequence table SEQ ID No.1.
2. the preparation method of recombination human serum albumin as claimed in claim 1-interferon alpha 1b fusion rotein is characterized in that this method may further comprise the steps at least:
(a) the codon optimized and chemosynthesis of recombination human serum albumin-interferon alpha 1b expressing fusion protein gene;
(b) structure that contains the expression plasmid of yeast of (a) described expressing gene;
(c) contain the structure and the screening of the pichia spp transformant of (b) described expression plasmid;
(d) fermentation culture of transformant engineering bacteria;
(e) purifying of engineering bacterium expression fusion rotein.
3. method as claimed in claim 2 is characterized in that: the codon of the described codon optimized selection yeast preference of step (a).
4. method as claimed in claim 2 is characterized in that: the pichia spp of selecting in the step (c) is pichia spp GS115.
5. method as claimed in claim 2 is characterized in that: the fusion rotein described in the step (e) is a secretion type expression.
6. method as claimed in claim 2 is characterized in that step (e) further comprises:
(1) (d) gained engineering bacteria is carried out after the centrifugal treating gained centrifuged supernatant being carried out ultrafiltration and concentration;
(2) the Blue Sepharose FF dye affinity chromatography purifying of ultrafiltration and concentration liquid;
(3) the CM Sepharose FF cation-exchange chromatography of preceding step gained elutriant is further purified.
7. method as claimed in claim 6 is characterized in that: the ultra-filtration membrane aperture is 30kDa in the step (1).
8. method as claimed in claim 6 is characterized in that: select sample on the damping fluid in the step (2), wash-out is selected 20mMPB+2M NaCl, pH7.0.
9. method as claimed in claim 6 is characterized in that: select sample on the damping fluid in the step (3), wash-out is selected 25mmol/L sodium-acetate buffer+0.3mol/L NaCl pH4.4-5.0.
10. the described recombination human serum albumin of claim 1-interferon alpha 1b fusion rotein prevents and/or treats purposes in the medicine of the relevant disease of hepatitis B and hepatitis B virus infection in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101893551A CN102093480A (en) | 2010-06-02 | 2010-06-02 | Recombinant human serum albumin-interferon alpha 1b fusion protein and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103641905A (en) * | 2013-12-12 | 2014-03-19 | 北京三元基因工程有限公司 | Interferon alpha1b mutant and fusion protein containing interferon alpha1b and human serum albumin |
| CN109485719A (en) * | 2018-11-28 | 2019-03-19 | 深圳市利云德生物技术有限公司 | A kind of new forms of interferon α 1 and preparation method thereof, composition and purposes |
| CN111995686A (en) * | 2019-05-27 | 2020-11-27 | 兰州大学 | Medicine with anti-angiogenesis activity and preparation method thereof |
| CN112279924A (en) * | 2020-10-27 | 2021-01-29 | 广州源博医药科技有限公司 | Long-acting canine alpha interferon fusion protein and preparation method and application thereof |
| CN114539426A (en) * | 2022-03-02 | 2022-05-27 | 山东仙普爱瑞科技股份有限公司 | Fusion protein containing interferon alpha, recombinant strain expressing fusion protein and preparation method thereof |
-
2010
- 2010-06-02 CN CN2010101893551A patent/CN102093480A/en not_active Withdrawn
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103641905A (en) * | 2013-12-12 | 2014-03-19 | 北京三元基因工程有限公司 | Interferon alpha1b mutant and fusion protein containing interferon alpha1b and human serum albumin |
| CN103641905B (en) * | 2013-12-12 | 2015-04-01 | 北京三元基因工程有限公司 | Interferon alpha1b mutant and fusion protein containing interferon alpha1b and human serum albumin |
| CN109485719A (en) * | 2018-11-28 | 2019-03-19 | 深圳市利云德生物技术有限公司 | A kind of new forms of interferon α 1 and preparation method thereof, composition and purposes |
| CN111995686A (en) * | 2019-05-27 | 2020-11-27 | 兰州大学 | Medicine with anti-angiogenesis activity and preparation method thereof |
| CN111995686B (en) * | 2019-05-27 | 2022-06-14 | 兰州大学 | Medicine with anti-angiogenesis activity and preparation method thereof |
| CN112279924A (en) * | 2020-10-27 | 2021-01-29 | 广州源博医药科技有限公司 | Long-acting canine alpha interferon fusion protein and preparation method and application thereof |
| CN114539426A (en) * | 2022-03-02 | 2022-05-27 | 山东仙普爱瑞科技股份有限公司 | Fusion protein containing interferon alpha, recombinant strain expressing fusion protein and preparation method thereof |
| CN114539426B (en) * | 2022-03-02 | 2023-07-07 | 山东仙普爱瑞科技股份有限公司 | Fusion protein containing interferon alpha, recombinant strain expressing fusion protein and preparation method of recombinant strain |
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