CN104628867A - Hepatocyte growth factor receptor key structural domain fusion protein and application thereof - Google Patents
Hepatocyte growth factor receptor key structural domain fusion protein and application thereof Download PDFInfo
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- CN104628867A CN104628867A CN201510036266.6A CN201510036266A CN104628867A CN 104628867 A CN104628867 A CN 104628867A CN 201510036266 A CN201510036266 A CN 201510036266A CN 104628867 A CN104628867 A CN 104628867A
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Abstract
The invention relates to a hepatocyte growth factor receptor key structural domain fusion protein and an application thereof. The fusion protein comprises an epidermal growth factor receptor strong signal peptide, a hepatocyte growth factor receptor key structural domain and a His tag. The fusion protein is used for the preparation of an antibody. The hepatocyte growth factor receptor key structural domain fusion protein is stably expressed by a lentiviral vector in 293T cells in a long-acting manner and can be specifically bound with the hepatocyte growth factor. The fusion protein has an important role in research and development on neutralizing antibody drugs for screening and blocking a C-Met/HGF signal transduction pathway to further inhibit tumor growth.
Description
Technical field
The invention belongs to fusion rotein field, particularly a kind of hepatocyte growth factor receptor key structure domain fusion protein and application thereof.
Background technology
C-Met is the only acceptor that pHGF (Hepatocyte growth factor, HGF) is known, is also the transmembrane receptor that a class has autophosphorylation activity, in the developing of Several Kinds of Malignancy, have vital role.Clinical study finds, the overexpression of C-Met is found in the higher lung cancer of China's sickness rate, liver cancer, large bowel cancer, mammary cancer, and is proportionate with malignancy of tumor degree, Metastasis and prognosis.C-Met mainly comprises the different structural domain of three functions: extracellular region, cross-film district and intracellular region.C-Met extracellular region (Extracellular domain, ED) comprise semaphorins axially albuminoid structural domain, one be rich in the PSI region of Cys and the IPT1-4 structural domain of four immunoglobulin (Ig)-plexin-transcription factor homolog structure compositions.Intracellular region then comprises a membrane-proximal region, a Tyrosylprotein kinase district and a C-end sequence.ED, as the identification of part recognition site and in conjunction with HGF, has vital role to a series of biological regulation effects of follow-up generation.Recent study also finds, the IPT3-4 structural domain in C-Met extracellular region is also the key structure territory that C-Met and HGF combines.HGF and ED combines and its conformation is changed, and many barss transduction pathway in phosphorylation activation born of the same parents promotes the propagation of tumour cell, migrates and vasculogenesis, finally causes malignant cell to invade healthy tissues, penetrates organs and tissues layer, finally diffuse to whole body.Once the HGF/C-Met signal path of abnormal activation is blocked in tumour cell, tumour cell just there will be cells proliferation slowed down, Tumor formation reduces, a series of change of degradation under invasive ability.The tyrosine kinase inhibitor cabozantinib blocking HGF/C-Met signal path has obtained the treatment of FDA approval for medullary thyroid carcinoma in late period in 2012.At present, the neutralizing antibody medicine of the anti-C-Met also not having FDA to ratify.
Neutrality antibody, because of features such as its high specificity, side effect are little, plays a significant role in kinds of tumors treatment.Neutrality antibody can not only be combined with corresponding antigens receptor-specific, can also effectively in and part to the effect of acceptor, there are the potentiality developing into therapeutic antibodies medicine, in human disease treatment, play keying action.As can be specificity be combined with human epidermal growth factor receptor 2 through neutralizing antibody class medicine Herceptin (trastuzumab) of FDA approval treatment mammary cancer, the signal transduction of block ligand mediation, Tumor suppression grows.The Cetuximab (Cetuximab) being used for the treatment of colorectal carcinoma and lung cancer through FDA approval also belongs to neutralizing antibody class medicine, can the combination of emulative suppression EGFR and part thereof, and then inhibition tumor cell grows, and induces its tune to die.
The neutralizing antibody of so-called C-Met, can be combined with C-Met exactly, and occupy the key structure territory that C-Met and HGF combines, both preventions combine, and blocks HGF/C-Met signal transduction, and then inhibition tumor cell proliferate.And the key structure territory that C-Met and HGF combines is its extracellular region ED, the particularly IPT3-4 structural domain of extracellular region.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of hepatocyte growth factor receptor key structure domain fusion protein and application thereof, this fusion rotein can specificly be combined with pHGF, in the neutralizing antibody medicament research and development that screening blocking-up C-Met/HGF signal transduction pathway and then Tumor suppression grow, have significant role.
A kind of hepatocyte growth factor receptor key structure domain fusion protein of the present invention, described fusion rotein is made up of the strong signal peptide of EGF-R ELISA, hepatocyte growth factor receptor key structure territory and His label; Wherein, the key structure territory of hepatocyte growth factor receptor is extracellular region ED (Extracellular domain) or immunoglobulin (Ig)-plexin-transcription factor homolog structural domain 3-4IPT34 (Immunoglobulin plexin transcription factor homology Domains); The nucleotide sequence of ED is as shown in SEQ ID NO:1 or the variant of this sequence, and the aminoacid sequence of ED is as shown in SEQ ID NO:2 or the variant of this sequence; The nucleotide sequence of IPT34 is as shown in 2152-2724 nucleotide sequence in above-mentioned sequence SEQ ID NO:1 or the variant of this sequence, and the aminoacid sequence of IPT34 is as shown in 718-908 aminoacid sequence in above-mentioned sequence SEQ ID NO:2 or the variant of this sequence.
The nucleotide sequence of the strong signal peptide of described EGF-R ELISA is as shown in SEQ ID NO:3, and aminoacid sequence is as shown in SEQ ID NO:4.
Described fusion rotein one has 940 amino acid, as shown in SEQ ID NO:5, wherein 1-26 amino acids is the strong signal peptide of EGF-R ELISA, and 27-934 amino acids is hepatocyte growth factor receptor ED albumen, and 935-940 amino acids is 6 × His label.
Described fusion rotein one has 223 amino acid, as shown in SEQ ID NO:6, wherein 1-26 amino acids is the strong signal peptide of EGF-R ELISA, and 27-217 amino acids is hepatocyte growth factor receptor IPT34 albumen, and 218-223 amino acids is 6 × His label.
Coding has the plasmid of the DNA of hepatocyte growth factor receptor key structure domain fusion protein.
Coding has the expression of hepatocyte growth factor receptor key structure domain fusion protein, is included in protokaryon and eukaryotic expression.
Described fusion rotein and pHGF specific binding.
The application of a kind of hepatocyte growth factor receptor key structure domain fusion protein of the present invention, described fusion rotein is used for antibody preparation.
C-Met extracellular region of the present invention (Extracellular domain, ED) comprise semaphorins axially albuminoid structural domain, one be rich in the PSI region of Cys and the IPT1-4 structural domain of four immunoglobulin (Ig)-plexin-transcription factor homolog structure compositions.The IPT-Fc of prior art 201110215692.8 is made up of IPT1-4, and the IPT34 that the present invention builds only includes IPT3 and IPT4.C-Met only has to be combined with HGF and just can be activated, and plays effect.At present, the key binding sites that c-Met extracellular region is combined with HGF is not clear, and IPT1-4 is considered to critical area, and the nearly step of IPT34 that the present invention builds reduces search coverage.
Compared with the fusion rotein of prior art 201110215692.8, the present invention has changed the signal peptide of c-MET itself, and used the strong signal peptide of EGF-R ELISA (EGFR), this signal peptide is repeatedly authenticated, the extracellular expression of fusion rotein can be realized, and do not change the three-dimensional structure of fusion rotein.
beneficial effect
(1) utilize genetic engineering technique to have expressed key structure territory ED and the IPT34 fusion rotein of C-Met, this region is the critical area that C-Met is combined with its part HGF, in its intracellular signaling process, play very important effect.
(2) for making fusion rotein high expression in 293T cell, select slow virus expression system, because lentiviral vectors can effectively integrate into host genome, mediation goal gene is stable, long-term expression, and the neutrality antibody blocking HGF/C-Met signal path for preparation C-Met monoclonal antibody and screening lays the foundation; Construction process is simple, and cost is low, has a good application prospect.
Accompanying drawing explanation
Fig. 1 is ED or the IPT34 district gene PCR product electrophorogram of agarose electrophoresis identifier C-Met, and wherein (a) is that (swimming lane 1 is 500bp Ladder to ED district gene electrophoresis result; Gene band 2700bp for the purpose of swimming lane 2); B () is that (swimming lane 1 is DL2000DNA Ladder to IPT34 district gene electrophoresis result; Gene band 670bp for the purpose of swimming lane 2);
Fig. 2 is lentiviral vectors double digestion rear electrophoresis figure, and wherein (a) is that (swimming lane 1 is 500-15000bp DNA Marker to p RRL-CMV-ED double digestion electrophorogram; Swimming lane 2 is p RRL-CMV-ED); B () is that (swimming lane 1 is DL2000DNA Ladder to p RRL-CMV-IPT34 double digestion electrophorogram; Swimming lane 2 is p RRL-CMV-IPT34);
Fig. 3 is the 293T cell that transfection slow virus plasmid observed by fluorescent microscope (× 100), wherein (a) and (b) egfp expression (a: common light microscopic that is basis of microscopic observation p RRL-CMV-ED group; B: fluorescence light microscopic); C egfp expression (c: common light microscopic that () and (d) is basis of microscopic observation p RRL-CMV-ED group; D: fluorescence light microscopic); E egfp expression (e: common light microscopic that () and (f) is basis of microscopic observation positive controls; F: fluorescence light microscopic);
Fig. 4 is hepatocyte growth factor receptor fusion protein S DS-PAGE Xylene Brilliant Cyanine G result in embodiment 3; Wherein (a) is C-Met-ED fusion rotein; B () is C-Met-IPT34 fusion rotein;
Fig. 5 is the Activity determination result of the hepatocyte growth factor receptor fusion rotein of 293T cell expressing in embodiment 4; Wherein (a) is C-Met-ED fusion rotein; B () is C-Met-IPT34 fusion rotein.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1
The structure of ED-His fusion rotein Lentiviral
With the pCR-Blunt-EGFR containing Human epidermal growth factor receptor full length sequence for template, primer P1 and P2 (synthesis of Suzhou Jin Weizhi company) is designed and synthesized according to EGFR gene signal peptide sequence, and add restriction enzyme site Age I at upstream primer, amplification obtains the signal peptide of 78bp.With the pCR-Blunt-C-Met containing people C-Met full-length gene for template, PCR primer P3 and P4 (synthesis of Suzhou Jin Weizhi company) is designed and synthesized according to the people C-Met gene extracellular domain sequence that GenBank logs in, downstream primer adds restriction enzyme site Sal I and His label, and amplification obtains the object fragment ED of about 2800bp.With these two PCR primer for template, with P1 and P4 for primer, carry out second time pcr amplification reaction, by Overlap extension PCR, two PCR primer are linked together.Wherein, primer sequence is as shown in the table:
Above-mentioned PCR reaction system is as follows:
| Reagent name | Volume (μ l) |
| Upstream primer | 2.5 |
| Downstream primer | 2.5 |
| 10×buffer | 10 |
| d NTP(10mM) | 1 |
| Template DNA | <100ng |
| NEB high-fidelity enzyme | 0.5 |
| dd H 2O | Add water to 50 μ l |
PCR reaction conditions: denaturation 98 DEG C, 3min; Sex change 98 DEG C of 15s, anneal 56 DEG C of 30s; Extend 72 DEG C, 1.5min, circulate 30 times; Extend 72 DEG C of 10min, reaction terminates.
After PCR reaction terminates, above-mentioned PCR primer identified by nucleic acid electrophoresis, reclaim test kit with rubber tapping and reclaim specific band, obtain the ED district gene of the people C-Met of purifying, detect its concentration by Eppendorf nucleic acid-protein determinator ,-20 save backup.
C-Met-ED PCR primer after purifying is connected into pCR-Blunt cloning vector by the flat end of T4 ligase enzyme, connect product conversion E. coli competent DH5 α, coat overnight incubation on the LB solid medium containing kantlex, picking individual colonies is overnight incubation on the LB liquid nutrient medium containing kantlex, preliminary evaluation positive colony is cut by bacterium liquid PCR and enzyme, positive colony called after Blunt-C-Met-ED, carries out order-checking qualification by Blunt-C-Met-ED recombinant plasmid.
The correct Blunt-C-Met-ED plasmid of order-checking and Lentiviral p RRL-CMV are used Age I and Sal I double digestion respectively, reclaim after kits through glue, the object fragment of enzyme being cut gained is connected with Lentiviral, connect product conversion E. coli competent DH5 α, coat overnight incubation on the LB solid medium containing penbritin, picking individual colonies is overnight incubation on the LB liquid nutrient medium containing penbritin, preliminary evaluation positive colony is cut by enzyme, positive colony called after p RRL-CMV-ED, pRRL-CMV-ED slow virus expression plasmid is carried out order-checking qualification, fragment constructed by confirmation is the encoding gene of C-Met-ED.
Embodiment 2
The structure of IPT34-His fusion rotein Lentiviral
With the pCR-Blunt-EGFR containing Human epidermal growth factor receptor full length sequence for template, design and synthesize primer P1 and P2 (synthesis of Suzhou Jin Weizhi company) according to EGFR gene signal peptide sequence.Take pCR-Blunt-C-Met as template, according to the sequences Design in people C-Met gene IPT34 district and synthetic primer P5 and P6 (synthesis of Suzhou Jin Weizhi company).Carry out first time pcr amplification reaction respectively, obtain two PCR primer.With these two PCR primer for template, with P1 and P6 for primer, carry out second time pcr amplification reaction, by Overlap extension PCR, two PCR primer are linked together.First amplified production is cloned in pCR-Blunt cloning vector, cuts afterwards, the step such as connection through enzyme, finally successfully obtains Lentiviral pRRL-CMV-IPT34, and passes through sequence verification.The concrete operation step of pcr amplification and follow-up lentiviral vectors construction step are with embodiment 1.Primer sequence is as follows:
Embodiment 3
Recombinant C-Met-ED and C-Met-IPT34 expressing fusion protein and purifying
Inoculation is in the 293T cell of logarithmic phase, when Growth of Cells to 50% ~ 60% merges, utilize calcium phosphate method by pRRL-CMV-C-Met-ED with containing control vector (p RRL-CMV-GFP) the cotransfection 293T cell of green fluorescent protein to express C-Met-ED albumen.Meanwhile, at 293T cells C-Met-IPT34 albumen, by pRRL-CMV-C-Met-IPT34 and p RRL-CMV-GFP cotransfection 293T cell.With p RRL-CMV-GFP transfection 293T cell for positive control, using the 293T cell of untransfected as blank.After cell cultures 48h, as shown in Figure 3, find that C-Met-ED group, C-Met-IPT34 group and positive controls cell all have egfp expression by fluorescence microscope, the negative control group cell then unstressed configuration expression of untransfected plasmid, illustrates slow-virus transfection success.
Collect the 293T cell culture fluid of above-mentioned C-Met-ED group and C-Met-IPT34 group respectively, 10000rpm is centrifugal, and 20min gets supernatant, by its membrane filtration through 0.45 μm.The nutrient solution filtered is mixed with PBS damping fluid equal-volume, carrys out purifying protein by Ni-NTAagarose purification column.After purifying, liquid can use BCA determination of protein concentration test kit test proteins content, detects the biology binding activities of albumen with purifying protein and pHGF binding tests, adopts SDS-PAGE its purity of electrophoresis detection and molecular weight.
Embodiment 4
The combination of fusion rotein and pHGF
With CBS, recombinant human HGF albumen is diluted to 2 μ g/ml, spends the night bag by 96 hole enzyme plates according to 4 DEG C, 100 μ L/ hole.After PBST washs 3 times, with 1%BSA 37 DEG C of closed 2h.Added in enzyme plate by fusion rotein prepared by different volumes embodiment 1,2 and mix with HGF, 37 DEG C of effect 2h, simultaneously using PBS as negative control, PBST washs 3 times.Using Goat anti human C-Met polyclonal antibody as primary antibodie, 37 DEG C of effects 1h, PBST wash 3 times.HRP marks rabbit anti goat igg antibody and anti-ly to detect as two, 37 DEG C of effect 1h.After PBST washing, every hole adds 100 μ l TMB nitrite ions, and 37 DEG C of lucifuge colour developing 10min, after termination reaction, microplate reader measures the absorbance at 450nm place.Detected result is as Fig. 5, and compared with negative control, the light absorption value of test group is all higher than control group, and result shows that fusion rotein prepared by embodiment 1,2 can effectively be combined with HGF, and increasing along with fusion rotein amount, increase with HGF combination rate.Confirm the success of C-Met-ED group, C-Met-IPT34 expressing fusion protein, and there is the ability with people HGF specific combination.
Claims (8)
1. a hepatocyte growth factor receptor key structure domain fusion protein, is characterized in that: described fusion rotein is made up of the strong signal peptide of EGF-R ELISA, hepatocyte growth factor receptor key structure territory and His label; Wherein, the key structure territory of hepatocyte growth factor receptor is extracellular region ED or immunoglobulin (Ig)-plexin-transcription factor homolog structural domain 3-4IPT34; The nucleotide sequence of ED is as shown in SEQ ID NO:1 or the variant of this sequence, and the aminoacid sequence of ED is as shown in SEQ ID NO:2 or the variant of this sequence; The nucleotide sequence of IPT34 is as shown in 2152-2724 nucleotide sequence in above-mentioned sequence SEQ ID NO:1 or the variant of this sequence, and the aminoacid sequence of IPT34 is as shown in 718-908 aminoacid sequence in above-mentioned sequence SEQ ID NO:2 or the variant of this sequence.
2. a kind of hepatocyte growth factor receptor key structure domain fusion protein according to claim 1, it is characterized in that: the nucleotide sequence of the strong signal peptide of described EGF-R ELISA is as shown in SEQ ID NO:3, and aminoacid sequence is as shown in SEQ ID NO:4.
3. a kind of hepatocyte growth factor receptor key structure domain fusion protein according to claim 1, it is characterized in that: described fusion rotein one has 940 amino acid, as shown in SEQ ID NO:5, wherein 1-26 amino acids is the strong signal peptide of EGF-R ELISA, 27-934 amino acids is hepatocyte growth factor receptor ED albumen, and 935-940 amino acids is 6 × His label.
4. a kind of hepatocyte growth factor receptor key structure domain fusion protein according to claim 1, it is characterized in that: described fusion rotein one has 223 amino acid, as shown in SEQ ID NO:6, wherein 1-26 amino acids is the strong signal peptide of EGF-R ELISA, 27-217 amino acids is hepatocyte growth factor receptor IPT34 albumen, and 218-223 amino acids is 6 × His label.
5. coding is just like the plasmid of the DNA of described hepatocyte growth factor receptor key structure domain fusion protein arbitrary in claim 1-4.
6. coding is just like the expression of described hepatocyte growth factor receptor key structure domain fusion protein arbitrary in claim 1-4, is included in protokaryon and eukaryotic expression.
7., according to described a kind of hepatocyte growth factor receptor key structure domain fusion protein arbitrary in claim 1-4, it is characterized in that: described fusion rotein and pHGF specific binding.
8. an application for hepatocyte growth factor receptor key structure domain fusion protein as claimed in claim 1, is characterized in that: described fusion rotein is used for antibody preparation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108611367A (en) * | 2018-04-24 | 2018-10-02 | 北京诺思兰德生物技术股份有限公司 | The gene therapy recombinant vector that one kind is mediated by plasmid vector |
| WO2022048521A1 (en) * | 2020-09-01 | 2022-03-10 | 荣昌生物制药(烟台)股份有限公司 | Anti-c-met antibody-drug conjugate and applications thereof |
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| CN101619361A (en) * | 2008-07-04 | 2010-01-06 | 梅思尔斯平移研究有限公司 | High affinity combined sites of hgfr and method for identifying its antagonist |
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| GENBANK: "Homo sapiens mRNA for met proto-oncogene，GenBank:X54559.1", 《GENBANK》 * |
| POOPAK FARNIA ET AL: "Cloning and expression of soluble vascular endothelial growth factors receptor-1(sFlt-1) fragments in CHO-K1", 《INT J CLIN EXP MED》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108611367A (en) * | 2018-04-24 | 2018-10-02 | 北京诺思兰德生物技术股份有限公司 | The gene therapy recombinant vector that one kind is mediated by plasmid vector |
| WO2022048521A1 (en) * | 2020-09-01 | 2022-03-10 | 荣昌生物制药(烟台)股份有限公司 | Anti-c-met antibody-drug conjugate and applications thereof |
| JP2023524678A (en) * | 2020-09-01 | 2023-06-13 | レメゲン シーオー．，エルティーディー． | Anti-c-Met antibody drug conjugate and its application |
| JP7510518B2 (en) | 2020-09-01 | 2024-07-03 | レメゲン シーオー．，エルティーディー． | Anti-c-Met antibody-drug conjugate and its application |
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