CN104987381A - Recombinant positive charge polypeptide interferon and application in anti-tumor and antiviral treatments - Google Patents
Recombinant positive charge polypeptide interferon and application in anti-tumor and antiviral treatments Download PDFInfo
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Abstract
The invention discloses recombinant positive charge polypeptide interferon and application in anti-tumor and antiviral treatments. According to the recombinant positive charge polypeptide interferon and the application in the anti-tumor and antiviral treatments, by means of the mode of a cDNA library, a polypeptide fragment and resistance protein in the same reading frame are cloned to vector plasmids; after antibiotic screening is conducted, an ATG-polypeptide-kalamycin fusion protein library in the same open reading frame is obtained, the library is cloned to a eukaryotic expression system through enzyme digestion to be connected with an interferon 3' end to form interferon-poolypeptide library plasmids, the library plasmids are packaged to be viruses to infect tumor cells with reporter genes, the tumor cells are steadily transferred in advance, the tumor cell of which the fluorescence is strongest as reported is selected through a flow cytometry, peptide sequences of the cell in DNA identification is extracted, the polypeptide fragment which possibly enhances the interferon effect is screened out, and verification is conducted on the anti-tumor effect by adopting a luciferase experiment and an MTT experiment to verify that the recombinant positive charge polypeptide interferon can enhance the anti-tumor effect.
Description
Technical field
The present invention adopts cDNA library method, filters out the polypeptide fragment that can strengthen Interferon, rabbit antitumor action, the application of restructuring positive charge polypeptide Interferon, rabbit in antitumor and antiviral therapy.
Background technology
Interferon, rabbit is that nineteen fifty-seven British scientist Isaacs and Lindenmann finds when studying virus interference, it is a kind of cytokine being produced, had antivirus action by virus infection induction, can suppress virus replication, be the highly active glycoprotein of a class.Recent study finds that Interferon, rabbit is except antiviral activity, also there is extensive regulatory function, as regulation and control cellular replication, hyperplasia and body immune system function, play a role in the pathological states such as malignant tumour, Immunological diseases, vascular proliferative disease and fibrotic disease.But Interferon, rabbit is extremely low body burden, again without suitable manufacture, thus limit the value of its clinical application.Up to date, biotechnology is advanced by leaps and bounds, and Interferon, rabbit is just able to volume production and is widely used in clinical.Mainly through inhibition tumor cell hyperplasia, promote apoptosis of tumor cells, suppress the transfer of oncogene expression, immunity moderation, Antineoplastic angiogenesis, Tumor suppression, the mechanism such as, induced tumor tumor cell differentiation collaborative with other cancer therapy drugs is antitumor.Interferon, rabbit is mainly divided into San great Lei ﹕ IFN-α at present, IFN-β and IFN-γ, because IFN-α and IFN-β all has many similarities in physicochemical property, antigenicity and acceptor etc., and play similar biological effect, therefore IFN-α and IFN-β is collectively referred to as interferon type Ⅰ in the past, IFN-γ is then classified as interferon type Ⅱ.
CDNA library specifically to organize or cell mRNA is template, after the complementary DNA (cDNA) that reverse transcription is formed is connected with suitable carrier (conventional phage or plasmid vector), transformation receptor bacterium forms recombinant DNA and clones group, and the cDNA clone collection that contain the whole mRNA information of cell is like this called the cDNA library of this tissue or cell.CDNA library reflects in certain tissue or cell specifically, and at the encoding gene of the protein that the specific etap expresses, therefore cDNA library has tissue or cell-specific.CDNA library is more much smaller than genome dna library, can than the gene being easier to therefrom screening and cloning and obtaining cell specific expression.Concerning eukaryotic cell, the gene obtained from genome dna library from obtain from cDNA library different, contained by genome dna library is genomic gene with intron and exon, and what obtain from cDNA library is through montage, the cDNA eliminating intron.Eukaryotic gene group DNA ten points is huge, and its complexity is about 100 times of protein and mRNA, simultaneously containing a large amount of tumor-necrosis factor glycoproteinss.Adopt the method for electrophoretic separation and hybridization, be all difficult to directly be separated to goal gene.Therefore be material Direct Cloning goal gene from chromosomal DNA more difficult relative to the cDNA clone from mRNA.Higher organism about has 10 usually
5kind different gene, but in the individual cells or individuality in certain hour stage, all only have the gene of about 15% to be expressed, produces about 15000 kinds of different mRNA molecules.Visible, the cDNA clone by mRNA, its complexity is than directly simply too much from genomic clone.The Function Identification of cDNA library genomic expression status and expressing gene in research certain class specific cells concrete conveniently has special advantage, thus make it in the research of the biological phenomenas such as ontogeny, cytodifferentiation, cell cycle regulating, cell aging and dead regulation and control, have using value more widely, be the gene library the most often used in research work.
In sum, Interferon, rabbit is applied to clinical antitumor drug as a kind of maturation, there is broad-spectrum anti-tumor, immunity moderation, the effects such as Tumor suppression vascularization, but, clinical practice proves existing Interferon, rabbit only to part entity tumour and leukemia treating sensitivity, offer limited effectiveness compared with a line antitumor drug, is used as adjunct antineoplastic and immunoregulation druge time more, its clinical application is still extensive not.For improving the antineoplastic effect of Interferon, rabbit, the mode that we are screened by cDNA library, Interferon, rabbit and polypeptide fragment are merged, forms recombinant polypeptide-Interferon, rabbit, filtered out the Interferon, rabbit-polypeptides in combination of Tumor suppression growth more effective in simple Interferon, rabbit by experiment in vitro.
Summary of the invention
The present invention is by the mode of cDNA library, vector plasmid is cloned into by polypeptide fragment and resistance protein (kalamycin) same reading frame, after antibiotic-screening, obtain the ATG-polypeptide-kalamycin fusion protein libraries in same open reading frame, this library enzyme cutting clone is held to eukaryotic expression system and with Interferon, rabbit 3 ' and connects into Interferon, rabbit-polypeptide libraries plasmid, this Library plasmid is packaged into viral postoperative infection and surely turns tumour cell with reporter gene in advance, the strongest tumour cell of reporter fluorescence is gone out by selected by flow cytometry apoptosis, extraction DNA identifies the peptide sequence in this cell, filter out the polypeptide fragment that may strengthen Interferon, rabbit effect, and adopt luciferase assay and MTT experiment to verify its antitumous effect, confirm that restructuring positive charge polypeptide Interferon, rabbit can strengthen antitumous effect.
Accompanying drawing explanation
Fig. 1 is Original Sequence:IFN-BamHI-Human plactnta somatomedin (Placenta growth factor, PGF) gene order.
Fig. 2 is ifn response element-luciferase experiment contrast figure mono-.
Fig. 3 is ifn response element-luciferase experiment contrast figure bis-.
Fig. 4 is MTT experimental control figure mono-.
Fig. 5 is MTT experimental control figure bis-.
Embodiment
One, polypeptide fragment cloning vector (Polypeptides Cloning Vector) is built
ATG and BamH/EcoRV restriction enzyme site is inserted after kalamycin promotor, and delete the ATG start methionine sequence of kalamycin, guarantee that polypeptide inserts after BamHI and EcoRV and kalamycin (open reading frame) in same open reading frame, otherwise bacterium can not survive in containing the substratum of kalamycin.
Two, IFN α/γ-polypeptide recombinant gene expression vector (Recombination-IFN-Expression Vector) is built
IFN α/γ is cloned in the multi-functional restriction enzyme site of Lentiviral, and holds at Interferon, rabbit 3 ' and build BamHI/EcoRV restriction enzyme site, to facilitate the insertion of polypeptide fragment, and guarantee polypeptide insert after and Interferon, rabbit in same open reading frame.
Three, build IFN and react original paper-green fluorescent protein reporter plasmid (Lenti-ISRE-GFP-Puro-Reporter)
Buy Cignal Lenti ISRE Reporter (luc) Kit:CLS-008L.Transformation CLS-008L plasmid, replaces with green fluorescent protein-T2A-tetracycline fusion gene, called after Lenti-ISRE-GFP-Puro-Reporter by luciferase gene.
Four, set up Lenti-ISRE-GFP-Puro-Reporter and surely turn tumor cell line (IFN responding element promoter-GFP-Puro-Reporter lentiviral stable clones)
Lenti-ISRE-GFP-Puro-Reporter, PSPAX2 and PMD2G corotation 293T cell strain is packaged into lentiviral particle, collects viral supernatants, infected tumor's cell, and adopt tetracycline to screen 1 week, obtain Interferon, rabbit reporting system and surely turn tumour cell.
Five, set up cDNA library and filter out target polypeptides:
1. from Normal human fibroblast, extract total serum IgE, and separation and purification mRNA.
2. mRNA reverse transcription is become cDNA, and then cDNA is synthesized dsDNA.
3. by ultrasonic wave, dsDNA is broken into little fragment
4. adopt PAGE glue separation and purification 50-100bp cDNA frag-ment libraries (Short double-strand cDNA fragments, DCF)
5. the DCF of purifying is cloned into the polypeptide fragment cloning vector built in advance and obtains plasmid library.
6. the plasmid library electricity built is forwarded in competent cell.
7. 37 DEG C of overnight incubation (bacterium wherein, only having ATG-DCF-kalamycin to express in same open reading frame could survive) in the LB-Agar culture dish containing kalamycin.
8. the LB nutrient solution of the bacterium on Agar culture dish is eluted in the Erlenmeyer flask of 500ml, in LB nutrient solution, adds kalamycin, 37 DEG C, 300rpm, overnight incubation.
9. bacterium is centrifugal and extract plasmid.
10. cut DCF library with BamHI/EcoRV enzyme, and cloned into IFN α/γ-polypeptide recombinant gene expression vector, make Interferon, rabbit-DCF in same open reading frame.
11. by the plasmid library electrotransfection that builds in competent cell.
12. in the LB-Agar culture dish containing Ampicillin Trihydrate 37 DEG C of overnight incubation.
13. is centrifugal and extract plasmid by bacterium.
The Library plasmid extracted and PsPAX2, PMD2G plasmid are proceeded to 293T cell by 14. together, are packaged into lentiviral particle.
15. collect viral supernatants and surely turn tumor cell line (IFN responding element promoter-GFP-Puro-Reporter lentiviral stable clones) with Lenti-ISRE-GFP-Puro-Reporter
16. go out the brightest tumour cell of fluorescence with selected by flow cytometry apoptosis
17. extract DNA, the peptide sequence increased in this mono-clonal, and are cloned into IFN α/γ-polypeptide recombinant gene expression vector.
18. repeat 10-17 step, 3 times
The DNA clone that the 4th is extracted is entered pJet plasmid, picking mono-clonal by 19., order-checking
20. by sequencing sequence and human gene group DNA's comparison, obtains peptide sequence i.e. our target fragment
Six, DCF library sequencing sequence
1, after order-checking, we obtain deriving from heterogeneic Transcriptional fragments, filter out 5 clones wherein having homology high further, by UCSC genome Blat, we find, these 5 polypeptide fragments are all from placenta growth factor (placenta growth factor, PGF), and these 5 fragments share 60bp sequence.As shown in Figure 1, be Original Sequence:IFN-BamHI-Human plactnta somatomedin (Placenta growth factor, PGF) gene order; Interferon-DCF1 ~ 5 are respectively 5 mono-clonals screened.
2, after the nucleotide sequence of 5 of above-mentioned screening polypeptide being translated into aminoacid sequence, called after placenta growth factor polypeptide (Placenta Growth Factor Polipeptides) 1 ~ 5 respectively, in table 1.
| Placenta growth factor peptide fragment | Placenta growth factor peptide amino acid sequence |
| PGFP1 | KMKPERRRPKGRGKRRREKQRPTDCHLCGDAVPRR |
| PGFP2 | KPERRRPKGRGKRRREKQRPTDCHLCGDAVPRR |
| PGFP3 | KPERRRPKGRGKRRREKQRPTD |
| PGFP4 | KERRRPKGRGKRRREKQRPTDC |
| PGFP5 | KMKPERRRPKGRGKRRREKQRPTDCHLCG |
Table 1: the aminoacid sequence of placenta growth factor polypeptide 1 ~ 5
Seven, recombinant placenta growth factor polypeptide-interferon alpha/γ 1 ~ 5 expression vector establishment
PGFP1 ~ 5 are connected to 3 ' the end composition fusion rotein of IFN α/γ with fragment round pcr, and be cloned into eukaryotic expression vector, obtain following 10 expression plasmids respectively, in table 2, the Nucleotide of 10 recombinant placenta growth factor polypeptide-Interferon, rabbit and aminoacid sequence are shown in attached 1 and attached 2:
Table 2: the title after interferon alpha, interferon-gamma, placenta growth factor polypeptide 1 ~ 5 recombination fusion protein build and english abbreviation
| Recombinant protein title | English abbreviation |
| Recombinant placenta growth factor polypeptide-Alfacon-1 | IFNα-PGFP1 |
| Recombinant placenta growth factor polypeptide-interferon α-2 | IFNα-PGFP2 |
| Recombinant placenta growth factor polypeptide-interferon alpha-3 | IFNα-PGFP3 |
| Recombinant placenta growth factor polypeptide-interferon alpha-4 | IFNα-PGFP4 |
| Recombinant placenta growth factor polypeptide-interferon alpha-5 | IFNα-PGFP5 |
| Recombinant placenta growth factor polypeptide-interferon-gamma-1 | IFNγ-PGFP1 |
| Recombinant placenta growth factor polypeptide-interferon-gamma-2 | IFNγ-PGFP2 |
| Recombinant placenta growth factor polypeptide-interferon-gamma-3 | IFNγ-PGFP3 |
| Recombinant placenta growth factor polypeptide-interferon-gamma-4 | IFNγ-PGFP4 |
| Recombinant placenta growth factor polypeptide-interferon-gamma-5 | IFNγ-PGFP5 |
Eight, ifn response element-luciferase reporter assay
By CLS-008L plasmid respectively with recombinant placenta growth factor polypeptide-Interferon, rabbit (see table 2) corotation in tumour cell, cultivate after 48h and adopt Promega Moduluas
tMluciferase reading measured by luciferase survey meter, and compares.As shown in Figure 2, Blank is blank group; No-ISRE is simple tumour cell group; ISRE+Vector is ifn response element-luciferase reporter plasmid+control plasmid group; ISRE+IFN α is ifn response original paper-luciferase reporter plasmid+Interferon, rabbit a group; All the other five groups all containing reporter plasmid and recombinant placenta growth factor polypeptide-interferon alpha, wherein ISRE+ control plasmid group luciferase activity well below containing Interferon, rabbit become grouping, p<0.0001; And the luciferase activity that recombinant placenta growth factor polypeptide-interferon alpha is respectively organized is higher than simple Interferon, rabbit group, p<0.05, but no difference of science of statistics between each group of recombinant interferon.
As shown in Figure 3, Blank is blank group; No-ISRE is simple tumour cell group; ISRE+Vector is ifn response element-luciferase reporter plasmid+control plasmid group; ISRE+IFN γ is ifn response original paper-luciferase reporter plasmid+interferon-gamma group; All the other five groups all containing reporter plasmid and recombinant placenta growth factor polypeptide-interferon-gamma, wherein ISRE+ control plasmid group luciferase activity well below containing Interferon, rabbit become grouping, p<0.01; And the luciferase activity that recombinant placenta growth factor polypeptide-interferon-gamma is respectively organized is higher than simple Interferon, rabbit group, p<0.05, but no difference of science of statistics between each group of recombinant interferon.
Nine, MTT test
1. collect logarithmic phase cell, adjustment concentration of cell suspension, about bed board makes cell density to 1000 to be measured/hole, every porocyte suspension final volume is 100ul (96 hole flat underside), parallel laid 4 96 level land, hole plates.
2. arrange during bed board and mark hole of returning to zero, control wells, medicine feeding hole.Zeroing hole only adds nutrient solution, does not add cell.Control wells and medicine feeding hole all add cell and nutrient solution.
3. 5%CO
2, hatch 1-7 days for 37 DEG C.
4. after bed board the 1st, 3,5,7 day, get a plate cell respectively.Every hole adds 20ulMTT solution (5mg/ml, i.e. 0.5%MTT), 5%CO
2, cultivate 4h for 37 DEG C.
5. stop after 4h cultivating, carefully suck nutrient solution in hole.
6. every hole adds 150ul dimethyl sulfoxide (DMSO), puts low-speed oscillation 10min on shaking table, crystallisate is fully dissolved.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument OD490nm place.
As shown in Figure 4, non treat: untreated tumour cell; PGreen-control: the control plasmid containing green fluorescent protein-tetracycline; IFN γ: Interferon, rabbit group; IFN γ+PGFP1 ~ 5: recombinant interferon-polypeptide group.
As shown in Figure 5, non treat: untreated tumour cell; PGreen-control: the control plasmid containing green fluorescent protein-tetracycline; IFN γ: Interferon, rabbit group; IFN γ+PGFP1 ~ 5: recombinant interferon-polypeptide group.
Claims (5)
1. a restructuring positive charge polypeptide Interferon, rabbit, is obtained by following steps:
One, polypeptide fragment cloning vector is built
ATG and BamH/EcoRV restriction enzyme site is inserted after kalamycin promotor, and delete the ATG start methionine sequence of kalamycin, guarantee that polypeptide inserts after BamHI and EcoRV and kalamycin (open reading frame) in same open reading frame, otherwise bacterium can not survive in containing the substratum of kalamycin;
Two, IFN α/γ-polypeptide recombinant gene expression vector is built
IFN α/γ is cloned in the multi-functional restriction enzyme site of Lentiviral, and holds at Interferon, rabbit 3 ' and build BamHI/EcoRV restriction enzyme site, to facilitate the insertion of polypeptide fragment, and guarantee polypeptide insert after and Interferon, rabbit in same open reading frame;
Three, build IFN and react original paper-green fluorescent protein reporter plasmid
Buy Cignal Lenti ISRE Reporter (luc) Kit:CLS-008L; Transformation CLS-008L plasmid, replaces with green fluorescent protein-T2A-tetracycline fusion gene, called after Lenti-ISRE-GFP-Puro-Reporter by luciferase gene;
Four, set up Lenti-ISRE-GFP-Puro-Reporter and surely turn tumor cell line
Lenti-ISRE-GFP-Puro-Reporter, PSPAX2 and PMD2G corotation 293T cell strain is packaged into lentiviral particle, collects viral supernatants, infected tumor's cell, and adopt tetracycline to screen 1 week, obtain Interferon, rabbit reporting system and surely turn tumour cell;
Five, set up cDNA library and filter out target polypeptides
1), from Normal human fibroblast total serum IgE is extracted, and separation and purification mRNA;
2), by mRNA reverse transcription become cDNA, and then cDNA is synthesized dsDNA;
3), by ultrasonic wave, dsDNA is broken into little fragment;
4) PAGE glue separation and purification 50-100bp cDNA frag-ment libraries, is adopted;
5), the DCF of purifying is cloned into the polypeptide fragment cloning vector built in advance and obtains plasmid library;
6), the plasmid library electricity built is forwarded in competent cell;
7), 37 DEG C of overnight incubation in the LB-Agar culture dish containing kalamycin; Wherein, the bacterium only having ATG-DCF-kalamycin to express in same open reading frame could survive;
8), the LB nutrient solution of the bacterium on Agar culture dish is eluted in the Erlenmeyer flask of 500ml, adds kalamycin in LB nutrient solution, 37 DEG C, 300rpm, overnight incubation;
9), that bacterium is centrifugal and extract plasmid;
10), with BamHI/EcoRV enzyme cut DCF library, and cloned into IFN α/γ-polypeptide recombinant gene expression vector, make Interferon, rabbit-DCF in same open reading frame;
11), by the plasmid library electrotransfection that builds in competent cell;
12), 37 DEG C of overnight incubation in the LB-Agar culture dish containing Ampicillin Trihydrate;
13), that bacterium is centrifugal and extract plasmid;
14), by the Library plasmid extracted and PsPAX2, PMD2G plasmid proceed to 293T cell together, be packaged into lentiviral particle;
15), collect viral supernatants and surely turn tumor cell line with Lenti-ISRE-GFP-Puro-Reporter;
16), the brightest tumour cell of fluorescence is gone out with selected by flow cytometry apoptosis;
17), extract DNA, the peptide sequence increased in this mono-clonal, and be cloned into IFN α/γ-polypeptide recombinant gene expression vector;
18), the 10th is repeated) ~ 17) step 3 time;
19), by the DNA clone that the 4th is extracted pJet plasmid is entered, picking mono-clonal, order-checking;
20), by sequencing sequence and human gene group DNA's comparison, peptide sequence i.e. our target patch is obtained.
Six, DCF library sequencing sequence
1, after order-checking, we obtain deriving from heterogeneic Transcriptional fragments, filter out 5 clones wherein having homology high further, by UCSC genome Blat, we find, these 5 polypeptide fragments are all from placenta growth factor (placenta growth factor, PGF), and these 5 fragments share 60bp sequence.
2, after the nucleotide sequence of 5 of above-mentioned screening polypeptide being translated into aminoacid sequence, called after placenta growth factor polypeptide 1 ~ 5 (Placenta Growth Factor Polipeptides) respectively, in table 1;
Table 1: the aminoacid sequence of placenta growth factor polypeptide 1 ~ 5
Seven, recombinant placenta growth factor polypeptide-interferon alpha/γ 1 ~ 5 expression vector establishment
PGFP1 ~ 5 are connected to 3 ' the end composition fusion rotein of IFN α/γ with fragment round pcr, and are cloned into eukaryotic expression vector, obtain following 10 expression plasmids respectively, in table 2,
Table 2: the title after interferon alpha, interferon-gamma, placenta growth factor polypeptide 1 ~ 5 recombination fusion protein build and english abbreviation
Table 2: the title after interferon alpha, interferon-gamma, placenta growth factor polypeptide 1 ~ 5 recombination fusion protein build and english abbreviation
Eight, ifn response element-luciferase reporter assay
By CLS-008L plasmid respectively with recombinant placenta growth factor polypeptide-Interferon, rabbit (see table 2) corotation in tumour cell, cultivate after 48h and adopt Promega Moduluas
tMluciferase reading measured by luciferase survey meter, and compares.
2. to recombinate described in a claim 1 application of positive charge polypeptide Interferon, rabbit in antineoplaston and antiviral therapy.
3. the application of restructuring positive charge polypeptide Interferon, rabbit according to claim 2 in antineoplaston, is characterized in that: described tumour comprises all entity class tumours or leukemia.
4. the application of restructuring positive charge polypeptide Interferon, rabbit in antineoplaston according to Claims 2 or 3, first adopts cDNA library method, filters out the polypeptide fragment that can strengthen Interferon, rabbit antitumor action.
5. recombinate described in claim 2 Nucleotide of positive charge polypeptide Interferon, rabbit and aminoacid sequence is shown in Nucleotide and aminoacid sequence.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111157434A (en) * | 2020-01-06 | 2020-05-15 | 四川大学华西医院 | Method for detecting luciferase-labeled cells using flow cytometry |
| CN113009156A (en) * | 2021-03-22 | 2021-06-22 | 华南农业大学 | Method for detecting dog IFN-alpha biological activity by using green fluorescent protein reporter gene |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1076729A (en) * | 1993-01-18 | 1993-09-29 | 中国预防医学科学院病毒学研究所 | Antineoplastic enkephalin-interferon fusion protein and method for making thereof |
| WO2007017637A1 (en) * | 2005-08-06 | 2007-02-15 | Riotech Pharmaceuticals Ltd | Method for producing recombinant interferon alpha-8 |
| CN102775502A (en) * | 2012-08-16 | 2012-11-14 | 天津禹王生物医药科技有限公司 | Alpha-interferon fusion protein |
| CN104583240A (en) * | 2012-08-13 | 2015-04-29 | Jw可瑞基因株式会社 | Interferon-[alpha] fusion protein in which cytoplasmic transduction peptide and polyethylene glycol are bonded to one another |
-
2015
- 2015-06-11 CN CN201510320365.7A patent/CN104987381B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1076729A (en) * | 1993-01-18 | 1993-09-29 | 中国预防医学科学院病毒学研究所 | Antineoplastic enkephalin-interferon fusion protein and method for making thereof |
| WO2007017637A1 (en) * | 2005-08-06 | 2007-02-15 | Riotech Pharmaceuticals Ltd | Method for producing recombinant interferon alpha-8 |
| CN104583240A (en) * | 2012-08-13 | 2015-04-29 | Jw可瑞基因株式会社 | Interferon-[alpha] fusion protein in which cytoplasmic transduction peptide and polyethylene glycol are bonded to one another |
| CN102775502A (en) * | 2012-08-16 | 2012-11-14 | 天津禹王生物医药科技有限公司 | Alpha-interferon fusion protein |
Non-Patent Citations (2)
| Title |
|---|
| 谷仲平,等: "重组人干扰素α2a和GFE-1多肽融合基因的克隆、表达及活性测定", 《现代肿瘤医学》 * |
| 郁子扬，等: "大鼠转化生长因子βⅡ型受体胞外区与γ干扰素融合蛋白真核表达载体的构建", 《浙江大学学报(医学版)》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111157434A (en) * | 2020-01-06 | 2020-05-15 | 四川大学华西医院 | Method for detecting luciferase-labeled cells using flow cytometry |
| CN113009156A (en) * | 2021-03-22 | 2021-06-22 | 华南农业大学 | Method for detecting dog IFN-alpha biological activity by using green fluorescent protein reporter gene |
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| CN104987381B (en) | 2018-11-27 |
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