CN104987381B - Recombinate positive charge polypeptide interferon and the application in antitumor and antiviral therapy - Google Patents
Recombinate positive charge polypeptide interferon and the application in antitumor and antiviral therapy Download PDFInfo
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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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/56—IFN-alpha
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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
- C07K14/475—Growth factors; Growth regulators
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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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/57—IFN-gamma
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses recombination positive charge polypeptide interferon and the applications in antitumor and antiviral therapy,The present invention is by way of cDNA library,Vector plasmid will be cloned into polypeptide fragment and the same reading frame of resistance protein,After antibiotic-screening,Obtain ATG- polypeptide-kalamycin fusion protein libraries in same open reading frame,Interferon-polypeptide libraries plasmid is connected by this library enzyme cutting clone to eukaryotic expression system and with the end of interferon 3 ',This Library plasmid is packaged into viral postoperative infection and surely turns the tumour cell with reporter gene in advance,Go out the strongest tumour cell of reporter fluorescence by selected by flow cytometry apoptosis,It extracts DNA and identifies the polypeptide sequence in the cell,Filter out the polypeptide fragment that may enhance interferon effect,And its antitumous effect is verified using luciferase assay and MTT experiment,Confirm that recombination positive charge polypeptide interferon can enhance antitumous effect.
Description
Technical field
The present invention uses cDNA library method, filters out the polypeptide fragment that can enhance interferon antitumor action, recombination is just
Application of the charged polypeptide interferon in antitumor and antiviral therapy.
Background technique
Interferon is that nineteen fifty-seven British scientist Isaacs and Lindenmann has found when studying virus interference,
Be it is a kind of generated by virus infection induction, the cell factor with antivirus action, be able to suppress virus replication, be a kind of high
Active glycoprotein.Recent study finds interferon in addition to antiviral activity, also has extensive regulatory function, such as adjusts and control
Cellular replication, hyperplasia and body immune system function processed, in malignant tumour, immunological diseases, vascular proliferative disease and fibrosis
It plays a role in the pathological states such as disease.But interferon is extremely low in body burden, and without manufacture appropriate, thus limits
The value of its clinical application.Up to date, biotechnology is advanced by leaps and bounds, and interferon is just able to volume production and is widely used in and faces
Bed.Mainly pass through and inhibits tumor cell proliferation, promote apoptosis of tumor cells, inhibit oncogene expression, adjusting to be immunized, antitumor blood
Pipe generates, inhibits metastases, cooperates with other anticancer drugs, induces the mechanism such as tumor cell differentiation antitumor.Interference
Element is broadly divided into three big class ﹕ IFN-α, IFN-β and IFN-γ at present, due to IFN-α and IFN-β physicochemical property, antigenicity and
There are many similarities for receptor etc., and play similar biological effect, therefore previous IFN-α and IFN-β are collectively referred to as I
Type interferon, and IFN-γ is then classified as interferon type Ⅱ.
CDNA library is the complementary DNA (cDNA) that reverse transcription is formed specifically to organize or cell mRNA is template and suitable
When carrier (common bacteriophage or plasmid vector) be ligated and transformed into recipient bacterium and form recombinant DNA clone group, in this way include thin
The cDNA clone collection of born of the same parents' whole mRNA information is collectively referred to as the cDNA library of the tissue or cell.CDNA library specifically reflects certain
In tissue or cell, in the encoding gene of the protein of expression of specific stage of development, therefore cDNA library has tissue or cell
Specificity.CDNA library is more much smaller than genome dna library, can be easier therefrom screening and cloning and obtain cell specific expression
Gene.For eukaryocyte, the gene obtained from genome dna library and the difference obtained from cDNA library, genome
It is the genomic gene with introne and exon contained by DNA library, and what is obtained from cDNA library is to have been subjected to cut
Meet, eliminate the cDNA of introne.Eukaryotic gene group DNA is very huge, and complexity is the 100 of protein and mRNA
Times or so, contain a large amount of repetitive sequence simultaneously.Using the method for electrophoretic separation and hybridization, all it is difficult to be directly separated purpose base
Cause.Therefore relatively more tired relative to the cDNA clone from mRNA from chromosomal DNA be material Direct Cloning target gene
It is difficult.Higher organism normally about has 105The different gene of kind, but in the individual cells or individual in stage certain time, all only
There is 15% or so gene to be expressed, generates about 15000 kinds of different mRNA molecules.As it can be seen that by cDNA grams mRNA
It is grand, complexity than directly from genomic clone it is much simpler.CDNA library base in studying certain specific class specific cells
Because the expression status of group and the Function Identification of expressing gene conveniently have special advantage, to make it in ontogeny, cell
There is more wide application value in the researchs of biological phenomenas such as differentiation, cell cycle regulating, cell ageing and dead regulation,
It is the most-often used gene library arrived in research work.
In conclusion interferon has broad-spectrum anti-tumor, adjusts as a kind of mature anti-tumor drug for being applied to clinic
The effects of section is immune, inhibits Tumor angiogenesis, however, clinical practice proves existing interferon only to part entity tumour and white
The treatment of blood disease is sensitive, the offer limited effectiveness compared with a line anti-tumor drug, is used as adjunct antineoplastic and immune tune when more
Drug is saved, clinical application is still not enough extensively.To improve the antitumor effect of interferon, the side that we are screened by cDNA library
Formula merges interferon with polypeptide fragment, forms recombinant polypeptide-interferon, is filtered out by experiment in vitro than simple interferon
More effective interferon-the polypeptides in combination for inhibiting tumour growth.
Summary of the invention
The present invention is by way of cDNA library, by polypeptide fragment and resistance protein (kalamycin) same reading frame grams
It is grand to obtain ATG- polypeptide-kalamycin fusion egg in same open reading frame after antibiotic-screening to vector plasmid
Text of an annotated book library connects into interferon-polypeptide libraries plasmid by this library enzyme cutting clone to eukaryotic expression system and with the end of interferon 3 ',
This Library plasmid is packaged into viral postoperative infection and surely turns the tumour cell with reporter gene in advance, passes through selected by flow cytometry apoptosis
The strongest tumour cell of reporter fluorescence out extracts DNA and identifies the polypeptide sequence in the cell, and interferon effect may be enhanced by filtering out
The polypeptide fragment of fruit, and its antitumous effect is verified using luciferase assay and MTT experiment, it was demonstrated that recombination positive charge
Polypeptide interferon can enhance antitumous effect.
Detailed description of the invention
Fig. 1 is Original Sequence：IFN-BamHI- Human plactnta growth factor (Placenta growth
Factor, PGF) gene order.
Fig. 2 is ifn response element-luciferase experiment contrast figure one.
Fig. 3 is ifn response element-luciferase experiment contrast figure two.
Fig. 4 is MTT experimental control figure one.
Fig. 5 is MTT experimental control figure two.
Specific embodiment
One, polypeptide fragment cloning vector (Polypeptides Cloning Vector) is constructed
It is inserted into ATG and BamH/EcoRV restriction enzyme site after kalamycin promoter, and deletes the ATG starting of kalamycin
Methionine sequence, it is ensured that polypeptide is inserted into after BamHI and EcoRV and kalamycin (open in same open reading frame
Reading frame), otherwise, bacterium will cannot survive in the culture medium containing kalamycin.
Two, IFN α/γ-polypeptide recombinant gene expression vector (Recombination-IFN-Expression is constructed
Vector)
IFN α/γ is cloned into the multifunctional enzyme enzyme site of Lentiviral, and holds and constructs in interferon 3 '
BamHI/EcoRV restriction enzyme site to facilitate the insertion of polypeptide fragment, and is read with interferon in same opening after ensuring polypeptide insertion
In frame.
Three, building IFN reacts original part-green fluorescent protein reporter plasmid (Lenti-ISRE-GFP-Puro-Reporter)
Buy Cignal Lenti ISRE Reporter (luc) Kit:CLS-008L.CLS-008L plasmid is transformed, it will be glimmering
Light element enzyme gene replaces with green fluorescent protein-T2A- puromycin fusion, is named as Lenti-ISRE-GFP-Puro-
Reporter。
Four, it establishes Lenti-ISRE-GFP-Puro-Reporter and surely turns 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 are packaged into slow disease
Malicious particle is collected viral supernatants, infected tumor's cell, and is screened 1 week using puromycin, obtains interferon reporting system and surely turns
Tumour cell.
Five, it establishes cDNA library and filters out target polypeptides：
1. extracting total serum IgE from Normal human fibroblast, and isolate and purify mRNA.
2. then cDNA is synthesized dsDNA again at cDNA by mRNA reverse transcription.
3. dsDNA to be broken into small segment with ultrasonic wave
4. isolating and purifying 50-100bp cDNA frag-ment libraries (Short double-strand cDNA using PAGE glue
fragments,DCF)
5. the DCF of purifying is cloned into the polypeptide fragment cloning vector built in advance obtains plasmid library.
6. the plasmid library electricity built is gone in competent cell.
7. (wherein, only ATG-DCF- OK a karaoke club is mould for 37 DEG C of overnight incubations in the LB-Agar culture dish containing kalamycin
The bacterium that element is expressed in same open reading frame could survive).
8. the bacterium on Agar culture dish is eluted in the conical flask of 500ml with LB culture solution, it is added in LB culture solution
Kalamycin, 37 DEG C, 300rpm, overnight incubation.
9. bacterium is centrifuged and extracts plasmid.
10. using the library BamHI/EcoRV digestion DCF, and cloned into IFN α/γ-polypeptide recombinant gene expression vector,
Make interferon-DCF in same open reading frame.
11. by the plasmid library electrotransfection built into competent cell.
12. 37 DEG C of overnight incubations in the LB-Agar culture dish containing ampicillin.
13. bacterium is centrifuged and extracts plasmid.
14. by the Library plasmid and PsPAX2 of extraction, PMD2G plasmid is transferred to 293T cell together, is packaged into slow virus
Grain.
15. collecting viral supernatants and surely turning tumor cell line (IFN with Lenti-ISRE-GFP-Puro-Reporter
responding element promoter-GFP-Puro-Reporter lentiviral stable clones)
16. going out the most bright tumour cell of fluorescence with selected by flow cytometry apoptosis
17. extracting DNA, the polypeptide sequence in the monoclonal is expanded, and be cloned into IFN α/γ-polypeptide recombination
Expression vector.
18. 10-17 step is repeated, 3 times
19. the DNA clone that the 4th is extracted is sequenced into pJet plasmid, picking monoclonal
20. sequencing sequence and human gene group DNA are compared, polypeptide sequence i.e. our target fragment is obtained
Six, the library DCF sequencing sequence
1, after being sequenced, we obtain the Transcriptional fragments from different genes, and further screening wherein has homology pole
5 high clones, pass through UCSC genome Blat, it has been found that this 5 polypeptide fragments are all from placenta growth factor
(placenta growth factor, PGF), 5 segments share 60bp sequence.As shown in Figure 1, being Original
Sequence：IFN-BamHI- Human plactnta growth factor (Placenta growth factor, PGF) gene order；
Interferon-DCF1~5 are respectively 5 monoclonals screened.
2, after the nucleotide sequence of 5 polypeptides of above-mentioned screening being translated into amino acid sequence, it is raw to be respectively designated as placenta
Growth factor polypeptide (Placenta Growth Factor Polipeptides) 1~5, is shown in Table 1.
| Placenta growth factor peptide fragment | Placenta growth factor peptide amino acid sequence |
| PGFP1 | KMKPERRRPKGRGKRRREKQRPTDCHLCGDAVPRR |
| PGFP2 | KPERRRPKGRGKRRREKQRPTDCHLCGDAVPRR |
| PGFP3 | KPERRRPKGRGKRRREKQRPTD |
| PGFP4 | ERRRPKGRGKRRREKQRPTDC |
| PGFP5 | KMKPERRRPKGRGKRRREKQRPTDCHLCG |
Table 1：The amino acid sequence of placenta growth factor polypeptide 1~5
Seven, recombinant placenta growth factor polypeptide -1~5 expression vector establishment of interferon-' alpha '/γ
PGFP1~5 are connected to the end IFN α/γ 3 ' composition fusion protein with segment with round pcr, and are cloned into
Eukaryotic expression vector obtains following 10 expression plasmids respectively, is shown in Table 2,10 recombinant placenta growth factor polypeptide-interference
The nucleotide and amino acid sequence of element are shown in attached 1 and attached 2：
Table 2：Title and English after interferon-' alpha ', interferon gamma, the building of 1~5 recombination fusion protein of placenta growth factor polypeptide
Text abbreviation
Eight, ifn response element-luciferase reporter assay
By CLS-008L plasmid respectively with recombinant placenta growth factor polypeptide-interferon (being shown in Table 2) corotation to tumour cell
In, Promega Moduluas is used after cultivating 48hTMLuciferase measuring instrument measures luciferase reading, and compares.Such as Fig. 2
Shown, Blank is blank control group；No-ISRE is simple tumour cell group；ISRE+Vector is ifn response element-firefly
Light element enzyme reporter plasmid+control plasmid group；ISRE+IFN α is ifn response original part-luciferase reporter plasmid+interferon a
Group；Remaining five groups contain reporter plasmid and recombinant placenta growth factor polypeptide-interferon-' alpha ', wherein ISRE+ control plasmid group firefly
Light element enzymatic activity well below containing interferon at grouping, p<0.0001；And recombinant placenta growth factor polypeptide-interferon-' alpha ' is each
The luciferase activity of group is higher than simple interferon group, p<0.05, however no difference of science of statistics between recombinant interferon each group.
As shown in figure 3, Blank is blank control group；No-ISRE is simple tumour cell group；ISRE+Vector is interference
Plain response element-luciferase reporter plasmid+control plasmid group；ISRE+IFN γ is ifn response original part-luciferase report
Accuse plasmid+interferon gamma group；Remaining five groups contain reporter plasmid and recombinant placenta growth factor polypeptide-interferon gamma, wherein
ISRE+ control plasmid group luciferase activity well below containing interferon at grouping, p<0.01；And recombinant placenta growth factor
Polypeptide-interferon gamma each group luciferase activity is higher than simple interferon group, p<0.05, however between recombinant interferon each group
No difference of science of statistics.
Nine, MTT is tested
1. collecting logarithmic phase cell, concentration of cell suspension is adjusted, bed board makes cell density to be measured to 1000 or so/hole,
Every hole cell suspension final volume is 100ul (96 hole flat underside), 4 96 hole level land plates of parallel laid.
2. being arranged and having marked zeroing hole, control wells, medicine feeding hole when bed board.Culture solution is only added in zeroing hole, is added without thin
Born of the same parents.Cell and culture solution is added in control wells and medicine feeding hole.
3.5%CO2, 37 DEG C are incubated for 1-7 days.
4. taking a plate cell respectively the 1st after bed board, 3,5,7 days.Every hole be added 20ulMTT solution (5mg/ml, i.e.,
0.5%MTT), 5%CO2, 37 DEG C of culture 4h.
Culture is terminated after 5.4h, carefully sucks culture solution in hole.
6. 150ul dimethyl sulfoxide is added in every hole, low-speed oscillation 10min on shaking table is set, dissolves crystal sufficiently.In enzyme
Join the light absorption value in each hole of measurement at immune detector OD490nm.
As shown in figure 4, non treat:Untreated tumour cell；pGreen-control:Contain green fluorescence
Albumen-puromycin control plasmid；IFNγ：Interferon group；IFN γ+PGFP1~5：Recombinant interferon-polypeptide group.
As shown in figure 5, non treat:Untreated tumour cell；pGreen-control:Contain green fluorescence
Albumen-puromycin control plasmid；IFNγ：Interferon group；IFN γ+PGFP1~5：Recombinant interferon-polypeptide group.
Claims (3)
1. a kind of recombination positive charge polypeptide interferon is made by following steps：
One, polypeptide fragment cloning vector is constructed
It is inserted into ATG and BamH/EcoRV restriction enzyme site after kalamycin promoter, and deletes the ATG starting egg ammonia of kalamycin
Acid sequence, it is ensured that polypeptide is inserted into after BamHI and EcoRV and kalamycin (open reading in same open reading frame
Frame), otherwise, bacterium will cannot survive in the culture medium containing kalamycin；
Two, IFN α/γ-polypeptide recombinant gene expression vector is constructed
IFN α/γ is cloned into the multifunctional enzyme enzyme site of Lentiviral, and holds building BamHI/ in interferon 3 '
EcoRV restriction enzyme site, to facilitate the insertion of polypeptide fragment, and ensure polypeptide insertion after and interferon in same open reading frame
It is interior；
Three, building IFN reacts original part-green fluorescent protein reporter plasmid
Buy Cignal Lenti ISRE Reporter (luc) Kit:CLS-008L；CLS-008L plasmid is transformed, by fluorescein
Enzyme gene replaces with green fluorescent protein-T2A- puromycin fusion, is named as Lenti-ISRE-GFP-Puro-
Reporter；
Four, it establishes Lenti-ISRE-GFP-Puro-Reporter and surely turns tumor cell line
Lenti-ISRE-GFP-Puro-Reporter, PSPAX2 and PMD2G corotation 293T cell strain are packaged into slow virus
Grain is collected viral supernatants, infected tumor's cell, and is screened 1 week using puromycin, obtains interferon reporting system and surely turns tumour
Cell；
Five, it establishes cDNA library and filters out target polypeptides
1) total serum IgE, is extracted from Normal human fibroblast, and isolates and purifies mRNA；
2), then cDNA is synthesized into dsDNA again at cDNA by mRNA reverse transcription；
3) dsDNA, is broken into small segment with ultrasonic wave；
4) 50-100bp dsDNA frag-ment libraries (Short double-strand cDNA, is isolated and purified using PAGE glue
fragments,DCF)；
5), the DCF of purifying is cloned into the polypeptide fragment cloning vector built in advance obtain plasmid library；
6), the plasmid library electricity built is gone in competent cell；
7), 37 DEG C of overnight incubations in the LB-Agar culture dish containing kalamycin；Wherein, only ATG-DCF- kalamycin
The bacterium expressed in same open reading frame could survive；
8) bacterium on Agar culture dish is eluted in the conical flask of 500ml with LB culture solution, OK a karaoke club is added in LB culture solution
Mycin, 37 DEG C, 300rpm, overnight incubation；
9), bacterium is centrifuged and extracts plasmid；
10) it, with the library BamHI/EcoRV digestion DCF, and is cloned into IFN α/γ-polypeptide recombinant gene expression vector, is made
Interferon-DCF is in same open reading frame；
11), by the plasmid library electrotransfection built into competent cell；
12), 37 DEG C of overnight incubations in the LB-Agar culture dish containing ampicillin；
13), bacterium is centrifuged and extracts plasmid；
14), by the Library plasmid of extraction and PsPAX2, PMD2G plasmid is transferred to 293T cell together, is packaged into lentiviral particle；
15) viral supernatants, are collected and surely turn tumor cell line with Lenti-ISRE-GFP-Puro-Reporter；
16), go out the most bright tumour cell of fluorescence with selected by flow cytometry apoptosis；
17) DNA, is extracted, expands the polypeptide sequence in the monoclonal, and be cloned into IFN α/γ-polypeptide recombinant gene expression
Carrier；
18) the, is repeated 10)~17) step 3 time；
19), the DNA clone for extracting the 4th is sequenced into pJet plasmid, picking monoclonal；
20), sequencing sequence and human gene group DNA are compared, obtain polypeptide sequence i.e. our target patch.
Six, the library DCF sequencing sequence
1, after being sequenced, we obtain the Transcriptional fragments from different genes, and further screening wherein has high 5 of homology
A clone passes through UCSC genome Blat, it has been found that this 5 polypeptide fragments are all from placenta growth factor (placenta
Growth factor, PGF), which shares 60bp sequence.
2, after the nucleotide sequence of 5 polypeptides of above-mentioned screening being translated into amino acid sequence, it is respectively designated as placenta growth factor
Sub- polypeptide 1~5 (Placenta Growth Factor Polipeptides), is shown in Table 1；
Table 1：The amino acid sequence of placenta growth factor polypeptide 1~5
Seven, recombinant placenta growth factor polypeptide -1~5 expression vector establishment of interferon-' alpha '/γ
PGFP1~5 are connected to the end IFN α/γ 3 ' composition fusion protein with segment with round pcr, and are cloned into eukaryon
Fibrocyte expression vector obtains following 10 expression plasmids respectively, is shown in Table 2,
Table 2：Title and English contracting after interferon-' alpha ', interferon gamma, the building of 1~5 recombination fusion protein of placenta growth factor polypeptide
It writes
Eight, ifn response element-luciferase reporter assay
By CLS-008L plasmid respectively with recombinant placenta growth factor polypeptide-interferon (being shown in Table 2) corotation into tumour cell, training
Promega Moduluas is used after supporting 48hTMLuciferase measuring instrument measures luciferase reading, and compares.
2. a kind of recombination positive charge polypeptide interferon described in claim 1 is preparing antineoplaston and antiviral therapy drug
In application.
3. recombination positive charge polypeptide interferon according to claim 2 is preparing antineoplaston and antiviral therapy drug
In application, it is characterised in that：The tumour includes all entity class tumours or leukaemia.
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| CN113009156B (en) * | 2021-03-22 | 2022-08-12 | 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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| WO2007017637A1 (en) * | 2005-08-06 | 2007-02-15 | Riotech Pharmaceuticals Ltd | Method for producing recombinant interferon alpha-8 |
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Inventor after: Wang Hong Inventor after: Yin Hongmei Inventor after: Guo Rui Inventor after: Chen Naifei Inventor after: Cui Jiuwei Inventor after: Hu Jifan Inventor before: Hu Jifan Inventor before: Guo Rui Inventor before: Wang Hong Inventor before: Yin Hongmei |
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