CN107936111A - A kind of preparation method of high activity HIP/PAP albumen - Google Patents
A kind of preparation method of high activity HIP/PAP albumen Download PDFInfo
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- CN107936111A CN107936111A CN201711355675.8A CN201711355675A CN107936111A CN 107936111 A CN107936111 A CN 107936111A CN 201711355675 A CN201711355675 A CN 201711355675A CN 107936111 A CN107936111 A CN 107936111A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The invention discloses a kind of preparation method of high activity HIP/PAP albumen, comprise the following steps：(1) gene order of purpose of design albumen；(2) synthesis is used for the genetic fragment F for expressing HIP/PAP albumenHIP/PAP；(3) construction of expression vector；(4) transfect；(5) purifying of destination protein；(6) determination of activity of destination protein.This method is solubility expression, overcomes conventional Escherichia coli and is unable to solubility expression HIP/PAP, can only inclusion body expression limitation；The present invention prepares the method for HIP/PAP albumen with easy to operate, it is not necessary to renaturation, and activity is significantly higher than the advantages that conventional inclusion body is expressed, and has broad application prospects.
Description
Technical field
The present invention relates to biological technical field, is specifically a kind of preparation method of high activity HIP/PAP albumen.
Background technology
People's HIP/PAP albumen (Hepatocarcinoma-Intestine-Pancreas/Pancreatitis-
Associated Protein), also known as 3 α of regenerated protein (Regenerating islet-derived protein III-
Alpha), the protein being made of 165 amino acid of pancreatic secretion.
1984, KEIM et al. was found that a kind of new secreted protein in rat acute pancreatitis model pancreatic juice first
Matter, since the occurrence and development of this protein and acute pancreatitis are closely related, therefore is referred to as pancreatitis associated protein
(pancreatitis-associated protein, PAP).LASSERE et al. is by grinding human hepatocellular carcinoma cDNA library
To study carefully, successful clone goes out liver tumour-enteron aisle-pancreatic gene (hepatocarcinoma-intestine-pancreas, HIP), and
Confirm that HIP genes and PAP genes are homologous gene, be referred to as HIP/PAP.HIP/PAP can defend liver cell from neoplasm necrosis
The programmed death of factor mediation, while be also a kind of mitogen, liver cell mitosis can be promoted, so as to promote liver
Cytothesis.Meanwhile HIP/PAP participates in a variety of physiology such as cellular immunity, cell proliferation and differentiation, anti-apoptotic, inflammation, antibacterial
Or pathologic process, hold promise for the treatment of a variety of diseases such as acute hepatic failure, ulcerative colitis.HIP/PAP albumen is also joined
With a variety of physiology and pathological metabolism, have now been found that it has expression in the diseased tissues such as inflammation, diabetes, tumour.
The major way of recombination expression HIP/PAP albumen is expressed for inclusion bodies of colibacillus at present, and Liu Tong et al. is described
In E. coli BL21-CodonPlus (DE3)-RIL HIP/PAP albumen, Ran Houtong are expressed with inclusion bodies
Cross renaturation, cation exchange column and solvent resistant column purification of recombinant proteins (expression, purifying and the mirror of restructuring antibacterial protein Reg3 γ
Determine Chinese experimental diagnostics, 2016,20 (4)：536-539).US20110229435A1, which is described, to be made with Escherichia coli
For host cell expression HIP/PAP albumen, HIP/PAP protein expressions are inclusion bodies, and pass through renaturation and cation exchange
Column purification recombinant protein.Why inactive inclusion body expression-form is used, be due to that HIP/PAP albumen has antibacterial in itself
Activity, toxicity can be produced according to soluble form expression to host e. coli.But inclusion body expression has complicated, renaturation
The shortcomings of efficiency is low, protein active is poor.CN1835764B is disclosed produces and purifies HIP/PAP in the milk of transgenic mice
Albumen, but expression has the shortcomings that cumbersome, low output, of high cost in transgenic animals.So far, this area does not have still
It is related to the report that mammalian cell is host's recombination expression HIP/PAP albumen.
The content of the invention
It is an object of the invention to provide a kind of preparation method of high activity HIP/PAP albumen, to solve above-mentioned background skill
The problem of being proposed in art.
To achieve the above object, the present invention provides following technical solution：
A kind of preparation method of high activity HIP/PAP albumen, comprises the following steps：
(1) gene order of purpose of design albumen：According to amino acid sequence (the SEQ ID NO of HIP/PAP albumen：1) and
The codon preference of mammal, the nucleotide sequence of coding HIP/PAP albumen is obtained by codon optimization, and is designed accordingly
For expressing the genetic fragment F of HIP/PAP albumenHIP/PAP(SEQ ID NO：2)；
(2) synthesis is used for the genetic fragment F for expressing HIP/PAP albumenHIP/PAP；
(3) construction of expression vector：The genetic fragment FHIP/PAP for being used to express HIP/PAP albumen of synthesis is utilized into EcoR
After I and Hind, III double digestions, the pTT5 plasmids of same double digestion, construction of expression vector pTT5-HIP/PAP are inserted into；
(4) transfect：By expression vector pTT5-HIP/PAP transfection mammalian cells, culture supernatant is obtained；
(5) purifying of destination protein：Expression supernatant is obtained after centrifugation, is purified using ion-exchange chromatography；
(6) determination of activity of destination protein：Using kit, the anti-apoptotic activities of testing goal albumen.
As the further scheme of the present invention：Genetic fragment F described in step (1)HIP/PAPIncluding EcoRI sites-Kozak
Sequence-signal peptide-HIP/PAP (SEQ ID NO：2)-terminator codon-HindIII sites.
As the further scheme of the present invention：Mammalian cell is Chinese hamster ovary celI described in step (4), preferably
CHO3E7 cells.
As the further scheme of the present invention：Transfection described in step (4) includes transiently transfecting and stable transfection.
As the further scheme of the present invention：Ion-exchange chromatography described in step (5) uses ion-exchange chromatography layer
Analyse Q Sepharose.
As the further scheme of the present invention：Kit described in step (6) is 3/7 kits of Caspase Glo.
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention establishes a kind of preparation method of high activity HIP/PAP albumen, and mammalian cell expression system can
The correct of protein is instructed to fold and glycosylation modified, thus expression product is in molecular structure, physicochemical property and biological function
Convenient closest natural HIP/PAP albumen.The expression of exocytosis type is the current state-of-the-art expression suitable for industrialized production
Mode, extracellular expression can intracellular accumulation forms inclusion body again to avoid albumen, makes the downstream purification of destination protein simpler
Just, easy to large-scale industrial production.The advantage of the invention is that preparation method is easy to operate, it is not necessary to expression product into
Row renaturation, and expression quantity is high, is easy to purify using serum free medium and the expression of exocytosis form, it is only necessary to using once
Ion-exchange chromatogram purification, product purity reaches more than 94%, and because using serum free medium, product is immunized
Originality is low, and obtained HIP/PAP albumen has significantly higher biology compared with the HIP/PAP albumen of prokaryotic expression
Activity.
Brief description of the drawings
After Fig. 1 is transfection, the expression schematic diagram of destination protein in cell culture supernatant, swimming lane " M " is albumen
Marker, swimming lane " D1-D6 " are the expression supernatant of the 1-6 days after transfection, and swimming lane " CHO is empty " is that the CHO of untransfected expresses supernatant.
Fig. 2 is that purpose albumen analyzes diagram through the SDS-PAGE of Q Sepharose after purification, and swimming lane " M " is albumen
Marker, for 4 μ g, HIP/PAP albumen, swimming lane 2 are 2 μ g HIP/PAP albumen after purification to swimming lane 1 after purification, and swimming lane 3 is that 1 μ g are purified
HIP/PAP albumen afterwards, swimming lane 4 are 0.5 μ g HIP/PAP albumen after purification.
Fig. 3 is the anti-apoptotic expression activitiy figure of the HIP/PAP albumen of separate sources, and HIP/PAP-E represents large intestine bar
The HIP/PAP albumen of bacterium expression, HIP/PAP-C represent the HIP/PAP albumen prepared using the method for the present invention.
Embodiment
The technical solution of this patent is described in more detail with reference to embodiment.Following embodiments will
Those skilled in the art is contributed to further understand the present invention, but do not limit the invention in any way.It should be understood that, it is right
For those skilled in the art, without departing from the inventive concept of the premise, many changes and improvements can be made, these are all
Belong to protection scope of the present invention.
A kind of preparation method of high activity HIP/PAP albumen, comprises the following steps：
First, the expressed sequence of HIP/PAP albumen is designed
The gene order of purpose of design albumen：According to amino acid sequence (the SEQ ID NO of HIP/PAP albumen：And lactation 1)
The codon preference of animal, the nucleotide sequence of coding HIP/PAP albumen is obtained by codon optimization, and is designed for accordingly
Express the genetic fragment F of HIP/PAP albumenHIP/PAP(SEQ ID NO：2)；It includes EcoRI sites-Kozak sequences-signal
Peptide-HIP/PAP (SEQ ID NO：2)-terminator codon-HindIII sites, it is specific as follows shown：
FHIP/PAP
GAATTCCCGCCGCCACCATGGGTTGGTCCTGTATCATCCTGTTTCTGGTCGCCACTGCCACTGGGGTCCACTCC TGATAAGCTT(SEQ
ID NO：2)
Wherein：
Italic GAATTC is EcoRI sites,
Runic GCCGCCACCATGG is Kozak sequences,
ATG is initiation codon,
Single underscore " _ _ _ _ " sign for signal peptide sequence,
Double underlineSign for HIP/PAP nucleotide sequences,
TGATAA is terminator codon,
Italic AAGCTT is HindIII sites.
2nd, synthesis is used for the genetic fragment F for expressing HIP/PAP albumenHIP/PAP；The genetic fragment FHIP/PAP(SEQ ID
NO：2) synthesized by Nanjing Genscript Biotechnology Co., Ltd..
3rd, the structure of expression vector
By synthesis be used for express HIP/PAP albumen genetic fragment and pTT5 plasmids at the same time with EcoRI and HindIII into
Row double digestion, is attached after endonuclease bamhi is recovered with T4 DNA ligases, obtains expression vector pTT5-HIP/PAP.
4th, the culture of Chinese hamster ovary celI
CHO 3E7 cells (being purchased from ATCC) are with serum-free expression culture medium FreeStyleTM CHO Expression
Medium (Life Technologies) is incubated in conical flask in (Corning), and in 37 DEG C, 5%CO2Condition of culture
Under, in shaken cultivation on orbit shaker (VWR Scientific).
5th, the transfection of HIP/PAP, including transient transfection and stable transfection；
In the day before transfection, CHO 3E7 cells are inoculated with appropriate density, approach its cell density on the day of transfection
90%.On the day of transfection, 3 μ l transfection reagents lipofectamine 2000 (Thermo Fisher) are with 50 μ l free serum cultures
Base dilutes, and 1 μ g expression vectors pTT5-HIP/PAP dilute with 50 μ l serum free mediums, by diluted expression vector and diluted
Transfection reagent mixes, and then adds in the flask comprising cell to be transfected, cultivates 6 days, collects 50 μ l cultures daily, carries out
SDS-PAGE electrophoresis, the expression of testing goal albumen, and collected all cell culture supernatants in the 6th day and be used to analyze
And purifying.
The expression of destination protein is shown in Fig. 1, it can be seen that using the method for the present invention, is successfully realized HIP/PAP albumen
Expression in Chinese hamster ovary celI, and expression quantity increases as expression time increases, after transfecting 6 days, expression quantity 22mg/L.
6th, the purifying of IP/PAP albumen
Centrifugation：After culture 6 days, culture centrifuges 30min in 4 DEG C with 15000rpm, obtains culture supernatant, supernatant is with 0.22
μm membrane filtration, removes insoluble impurities and particle.
Purification system：The purifying of HIP/PAP albumen, its corresponding chromatographic column and buffer solution are carried out using ion-exchange chromatography
It is as follows：
Chromatographic column：Q Sepharose FF (GE), 5ml；
Buffer A：50mM Gly-NaOH, 8M urea, 10mM DTT, pH9.0；
Buffer B1：50mM Gly-NaOH, 8M urea, 10mM DTT, 200mM NaCl, pH9.0；
Buffer B2：50mM Gly-NaOH, 8M urea, 10mM DTT, 2M NaCl, pH9.0.
Loading：With 100%Buffer A loadings after culture supernatant after centrifugation is concentrated.
Gradient elution：100%Buffer A elute 15 column volumes, 75%Buffer A+25%Buffer B1 elutions 12
A column volume, 75-0%Buffer A+25-100%Buffer B1 elute 10 column volumes, and 100%Buffer B1 elute 5
Column volume, 100%Buffer B2 elute 4 column volumes, and eluent is in charge of collection, and the HIP/PAP albumen of purifying is carried out SDS-
PAGE is analyzed.
Purification result is shown in Fig. 2, and it is purpose albumen at 16kD that molecular weight, which is about, and 1,2,3,4 swimming lanes are respectively 4,2,1,0.5 μ g
The HIP/PAP albumen of purifying, through gray scale scanning software analysis, purity of protein is 94.51% after purification.
7th, the determination of activity of HIP/PAP albumen
The separation and culture of Rat Primary Hepatocytes：Buy 6 week old SD rats, liver separated after execution, be transferred to containing
In the L15 culture mediums of antibiotic, with 70uM nylon membrane filtrations after liver grinding, and 2min is centrifuged with 600rpm.Cell is trained with L15
Base cleaning is supported, and cell viability measure is carried out with trypan blue.Separated primary hepatocyte is transferred to 10%FCS culture mediums, really
It is per 30,000, hole to protect cell number, is added to 96 orifice plates and carries out adhere-wall culture.Serum free medium is changed to after when 3-4 is small, is carried out
Suspend culture, and changes liquid daily.
It is apoptosis-induced：(tumour is bad with 20ng/mL Act D (actinomycin D)+80ng/ml TNF-α for the liver cell of culture
Necrosis factor α) processing 24h, establishes model of cell apoptosis.
Anti-apoptotic activities measures：To compare the HIP/ of the HIP/PAP albumen of the method for the present invention preparation and Bacillus coli expression
The activity difference of PAP albumen, buys the HIP/PAP albumen (being purchased from ProSpec companies) of the Bacillus coli expression of commercialization.To show area
Not, the HIP/PAP albumen of Bacillus coli expression is expressed as HIP/PAP-E, and HIP/PAP prepared by the method for the present invention (is implemented
The HIP/PAP albumen that example 5 obtains) it is expressed as HIP/PAP-C.Respectively with the HIP/PAP-C of various concentrations and HIP/PAP-E (0,
250th, 500,1000,2000,4000ng/ml) processing apoptotic cell model 18 it is small when, each processing sets 6 parallel repeating holes.It
Afterwards using 3/7 kit measurement HIP/PAP-C and HIP/PAP-E anti-apoptotics of Caspase Glo activity, determine respective
IC50。
Testing result is shown in Fig. 3, it can be seen from the figure that the IC of HIP/PAP-C prepared by the method for the present invention50For 62.2ng/
Ml, and the IC of the HIP/PAP-E of Bacillus coli expression50For 153.3ng/ml, HIP/PAP activity prepared by the method for the present invention is significantly
Higher than the HIP/PAP (P ＜ 0.05) of normal intestinal bacteria expression.
The present invention establishes a kind of preparation method of high activity HIP/PAP albumen, and mammalian cell expression system can
The correct of protein is instructed to fold and glycosylation modified, thus expression product is in molecular structure, physicochemical property and biological function
Convenient closest natural HIP/PAP albumen.The expression of exocytosis type is the current state-of-the-art expression suitable for industrialized production
Mode, extracellular expression can intracellular accumulation forms inclusion body again to avoid albumen, makes the downstream purification of destination protein simpler
Just, easy to large-scale industrial production.The advantage of the invention is that preparation method is easy to operate, it is not necessary to expression product into
Row renaturation, and expression quantity is high, is easy to purify using serum free medium and the expression of exocytosis form, it is only necessary to using once
Ion-exchange chromatogram purification, product purity reaches more than 94%, and because using serum free medium, product is immunized
Originality is low, and obtained HIP/PAP albumen has significantly higher biology compared with the HIP/PAP albumen of prokaryotic expression
Activity.
The better embodiment of this patent is explained in detail above, but this patent is not limited to above-mentioned embodiment party
Formula, can also be on the premise of this patent objective not be departed from the knowledge that one skilled in the relevant art possesses
Make a variety of changes.
Sequence table
<110>Guangzhou Co., Ltd of An Chen new drug researches institute
<120>A kind of preparation method of high activity HIP/PAP albumen
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 150
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Met Glu Glu Pro Gln Arg Glu Leu Pro Ser Ala Arg Ile Arg Cys Pro
1 5 10 15
Lys Gly Ser Lys Ala Tyr Gly Ser His Cys Tyr Ala Leu Phe Leu Ser
20 25 30
Pro Lys Ser Trp Thr Asp Ala Asp Leu Ala Cys Gln Lys Arg Pro Ser
35 40 45
Gly Asn Leu Val Ser Val Leu Ser Gly Ala Glu Gly Ser Phe Val Ser
50 55 60
Ser Leu Val Lys Ser Ile Gly Asn Ser Tyr Ser Tyr Val Trp Ile Gly
65 70 75 80
Leu His Asp Pro Thr Gln Gly Thr Glu Pro Asn Gly Glu Gly Trp Glu
85 90 95
Trp Ser Ser Ser Asp Val Met Asn Tyr Phe Ala Trp Glu Arg Asn Pro
100 105 110
Ser Thr Ile Ser Ser Pro Gly His Cys Ala Ser Leu Ser Arg Ser Thr
115 120 125
Ala Phe Leu Arg Trp Lys Asp Tyr Asn Cys Asn Val Arg Leu Pro Tyr
130 135 140
Val Cys Lys Phe Thr Asp
145 150
<210> 2
<211> 531
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gaattcccgc cgccaccatg ggttggtcct gtatcatcct gtttctggtc gccactgcca 60
ctggggtcca ctccgaagag cctcagcgtg aactgccctc agcaaggatc cggtgcccaa 120
agggaagcaa ggcctacggc tctcactgtt atgctctgtt cctgtcccct aagagctgga 180
ccgacgccga tctggcttgc cagaagaggc catctggcaa cctggtgtct gtgctgtccg 240
gagctgaggg ctccttcgtg tcctccctgg tgaagagcat cggcaacagc tactcttacg 300
tgtggatcgg cctgcacgac ccaacccagg gaacagagcc taatggcgag ggctgggagt 360
ggtcttccag cgatgtgatg aactacttcg cttgggagag aaatccatcc accatctctt 420
ccccaggaca ttgtgcctcc ctgagccgct ctacagcttt tctgaggtgg aaggactata 480
actgtaatgt gcggctgccc tatgtctgta aattcactga ctgataagct t 531
Claims (6)
1. a kind of preparation method of high activity HIP/PAP albumen, it is characterised in that comprise the following steps：
(1) gene order of purpose of design albumen：According to amino acid sequence (the SEQ ID NO of HIP/PAP albumen：And lactation 1)
The codon preference of animal, the nucleotide sequence of coding HIP/PAP albumen is obtained by codon optimization, and is designed for accordingly
Express the genetic fragment F of HIP/PAP albumenHIP/PAP(SEQ ID NO：2)；
(2) synthesis is used for the genetic fragment F for expressing HIP/PAP albumenHIP/PAP；
(3) construction of expression vector：The genetic fragment FHIP/PAP for being used to express HIP/PAP albumen of synthesis is utilized into I Hes of EcoR
After III double digestions of Hind, the pTT5 plasmids of same double digestion, construction of expression vector pTT5-HIP/PAP are inserted into；
(4) transfect：By expression vector pTT5-HIP/PAP transfection mammalian cells, culture supernatant is obtained；
(5) purifying of destination protein：Expression supernatant is obtained after centrifugation, is purified using ion-exchange chromatography；
(6) determination of activity of destination protein：Using kit, the anti-apoptotic activities of testing goal albumen.
2. the preparation method of high activity HIP/PAP albumen according to claim 1, it is characterised in that described in step (1)
Genetic fragment FHIP/PAPIncluding EcoRI sites-Kozak sequences-signal peptide-HIP/PAP (SEQ ID NO：2)-terminator codon-
HindIII sites.
3. the preparation method of the high activity HIP/PAP albumen according to claim 1 or 2, it is characterised in that step is fed described in (4)
Newborn zooblast is Chinese hamster ovary celI, preferably CHO 3E7 cells.
4. the preparation method for the high activity HIP/PAP albumen stated according to claim 3, it is characterised in that step transfects described in (4)
Including transient transfection and stable transfection.
5. the preparation method for the high activity HIP/PAP albumen stated according to claim 4, it is characterised in that ion described in step (5)
Exchange chromatography uses ion-exchange chromatography chromatography Q Sepharose.
6. the preparation method for the high activity HIP/PAP albumen stated according to claim 5, it is characterised in that reagent described in step (6)
Box is 3/7 kits of Caspase Glo.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114702551A (en) * | 2018-09-18 | 2022-07-05 | 广州领晟医疗科技有限公司 | Polypeptide for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1835764A (en) * | 2003-06-18 | 2006-09-20 | 国立健康与医学研究所 | Hip/pap polypeptide composition for use in liver regeneration and for the prevention of liver failure |
| US20100172922A1 (en) * | 2007-02-12 | 2010-07-08 | The Board Of Regents Of The University Of Texas System | Expression and Purification of HIP/PAP and Uses Therefor |
| CN102559759A (en) * | 2011-12-19 | 2012-07-11 | 西安交通大学医学院第一附属医院 | HIP/PAP recombinant adenovirus and application thereof in resisting ulcerative colitis |
| CN102625707A (en) * | 2009-06-11 | 2012-08-01 | 阿尔法科特创新公司 | Novel applications of HIP/PAP or derivatives thereof |
| CN103343134A (en) * | 2013-07-05 | 2013-10-09 | 江苏普罗赛生物技术有限公司 | EGF-Fc fusion protein and High-expression production method of mammal expression system of EGF-Fc fusion protein |
| WO2014013032A1 (en) * | 2012-07-19 | 2014-01-23 | Alfact Innovation | Hip/pap protein and derivatives thereof for use in treating cancer |
| CN105658280A (en) * | 2013-04-10 | 2016-06-08 | 艾尔法特创新公司 | Composition including the hip/pap protein or one of the derivatives thereof for treating insulin resistance |
-
2017
- 2017-12-16 CN CN201711355675.8A patent/CN107936111B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1835764A (en) * | 2003-06-18 | 2006-09-20 | 国立健康与医学研究所 | Hip/pap polypeptide composition for use in liver regeneration and for the prevention of liver failure |
| US20100172922A1 (en) * | 2007-02-12 | 2010-07-08 | The Board Of Regents Of The University Of Texas System | Expression and Purification of HIP/PAP and Uses Therefor |
| CN102625707A (en) * | 2009-06-11 | 2012-08-01 | 阿尔法科特创新公司 | Novel applications of HIP/PAP or derivatives thereof |
| CN102559759A (en) * | 2011-12-19 | 2012-07-11 | 西安交通大学医学院第一附属医院 | HIP/PAP recombinant adenovirus and application thereof in resisting ulcerative colitis |
| WO2014013032A1 (en) * | 2012-07-19 | 2014-01-23 | Alfact Innovation | Hip/pap protein and derivatives thereof for use in treating cancer |
| CN105658280A (en) * | 2013-04-10 | 2016-06-08 | 艾尔法特创新公司 | Composition including the hip/pap protein or one of the derivatives thereof for treating insulin resistance |
| CN103343134A (en) * | 2013-07-05 | 2013-10-09 | 江苏普罗赛生物技术有限公司 | EGF-Fc fusion protein and High-expression production method of mammal expression system of EGF-Fc fusion protein |
Non-Patent Citations (4)
| Title |
|---|
| SAKIKO MATSUMOTO 等: "Expression Analysis of the Regenerating Gene (Reg) Family Members Reg-III b and Reg-III c in the Mouse During Development", 《THE JOURNAL OF COMPARATIVE NEUROLOGY》 * |
| YUJIRO NITTA 等: "Urinary levels of Hepatocarcinoma-intestinepancreas/Pancreatitis-associated protein as a diagnostic biomarker in patients with bladder cancer", 《BMC UROLOGY》 * |
| 庄强: "构建新型的高效哺乳动物细胞表达系统及其在CHO细胞中的应用", 《中国学位论文全文数据库》 * |
| 郑惠惠 等: "ＣＨＯ细胞表达系统研究进展", 《生物技术进展》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114702551A (en) * | 2018-09-18 | 2022-07-05 | 广州领晟医疗科技有限公司 | Polypeptide for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis and application thereof |
| CN114702551B (en) * | 2018-09-18 | 2023-09-12 | 广州领晟医疗科技有限公司 | Polypeptide for promoting hepatocyte proliferation and/or inhibiting hepatocyte apoptosis and application thereof |
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