CN104592395A - Preparation method and application of VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) single-chain antibody and MICA fusion protein - Google Patents
Preparation method and application of VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) single-chain antibody and MICA fusion protein Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering antibodies and particularly discloses a preparation method and an application of a fusion antibody for linking a single-chain antibody (AK404R) of anti-human vascular endothelial growth factor receptor 2 (VEGFR2) and MICA. The invention discloses the construction of fusion antibody expression plasmid and transfection of the recombinant plasmid. The invention also provides a method for expressing and purifying the fusion antibody. The fusion antibody is obtained by carrying out secreting expression and affinity chromatography purification on eukaryotic cells. The fusion antibody AK404R-MICA can be specifically bound to human vascular endothelial growth factor receptor 2 (VEGFR2) so that the proliferation, migration, cell invasion and tubule formation of human umbilical vein endothelial cells (HUVEC) can be significantly inhibited. The fusion antibody AK404R-MICA can also significantly inhibit the growth of tumor cells K562 and can be used in the preparation of diagnostic and therapeutic agents.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of newly can with the fusion antibody (AK404R-MICA) be connected with MICA of the high-affinity of human vascular endothelial growth factor receptor 3 body 2 specific combination, the propagation of endotheliocyte, migration, cell invasion and tubule can be suppressed to be formed, the growth of all right significantly inhibition tumor cell K562 is a kind of genetically engineered fusion antibody with anti-tumor activity.
Background technology
Tumour cell can be infiltrated lymphatic vessel and blood vessel and be invaded in normal tissue by blood circulation, makes cancer become the disease of potential threat life.A necessary requirement of the transfer of tumour cell is exactly the new life of blood vessel.But, one of key of the formation of new vessel is exactly vascular endothelial growth factor (VEGF) family and associated receptor, it by with vascular endothelial growth factor receptor (VEGFR) in conjunction with signal path in activating cells, stimulate vascular endothelial cell proliferation, migration, promote the generation of new vessel, have close relationship with the morbidity of the multiple kinds of tumor of body and the transfer of tumour.VEGFR-2, is also called KDR (Kinase Insert Domain Receptor), has higher avidity with VEGF in human body, and it plays an important role in the biologic activity such as mediation VEGF stimulating endothelial cell propagation and vascular permeability.。KDR belongs to tyrosine kinase receptor (Receptorprotein-tyrosine kinase), 2nd ~ 3 district and VEGF in conjunction with relevant, 2 with its being separated with VEGF of 4 district's major effects, 1 district's primary responsibility regulates the combination of acceptor and part, the site suppressing to be combined with VEGF may be there is in 4th ~ 7 district, wherein 3rd district (i.e. KDR3, containing 97 amino acid) combination contribution to VEGF is the most outstanding.
MHC work chain molecule A (Major Histocompatibility Complex class I-relatedchain molecules A, MICA), it is a kind of transmembrane glycoprotein of being encoded by mhc gene of high glycosylation, play an important role in immunosurveillance, MICA is the part of NK cell (NK) cell activation receptors NKG2D, research shows, the tumour cell of expressing MICA is generally comparatively responsive to NK cell killing.Natural killer cell surface receptor 2D (NKG2D), is a member in C type lectin superfamily, is not only expressed in all NK cells, and is expressed in the Macrophage Surface of CD8+T cell, activation.The crosslinked cytotoxic activity that can trigger NK cell of NKG2D and its part, in cancer immunosurveillance, play very important effect, on target cell, the expression level of NKG2D part almost determines the power of body NK cellullar immunologic response.
The single-chain antibody AK404R that it is antigen that this laboratory utilizes display technique of bacteriophage to obtain with KDR Bao Wai 3 district before, but consider the relatively little molecular weight (30KDa) of single-chain antibody and lack Fc fragment, our a construction expression fusion antibody AK404R-MICA goes the validity strengthening its oncotherapy.
RhuMAb-VEGF (Bevacizumab, trade(brand)name Avastin) is most commonly used to the antibody drug for VEGF-VEGFR2 signal path in the market.Being the Humanized monoclonal antibodies of restructuring, within 2004, obtaining the approval of FDA, is that first, the U.S. gets the Green Light the medicine of Tumor suppression vasculogenesis of listing.Confirm that IgGl antibody capable and human vascular endothelial growth factor (VEGF) combine and block its biological activity by examination of the inside with outside system.And Arastin contain human antibody structural area and can in conjunction with the complementary determining region of the mouse source monoclonal antibody of VEGF.
Summary of the invention
Goal of the invention
The invention provides a kind of fusion antibody AK404R-MICA with potential medical science and pharmacy value.The feature of fusion antibody of the present invention is that the single-chain antibody of VEGFR2 is connected with MICA, can suppress the propagation of Leukemia K562 cell in vitro.
Technical scheme
The single-chain antibody of VEGFR2 is connected a preparation for fusion antibody with MICA, it is characterized in that:
Single-chain antibody AK404R and MICA that it is antigen that the fusion antibody that the single-chain antibody of VEGFR2 is connected with MICA comprises with KDR Bao Wai 3 district, is connected by a Linker (G4S).
A nucleic acid for separation, is characterized in that the antibody that this nucleic acid encoding is above-mentioned.
A kind of expression vector, containing above-mentioned nucleic acid.
A kind of recombinant host cell, containing above-mentioned expression vector.
A kind of pichia spp secreting, expressing method, for the fusion antibody of secreting, expressing.
The fusion antibody of above-mentioned any one or the application of fusion antibody fragment, be optionally combined with KDR3 or NKG2D combines.
The fusion antibody fragment of above-mentioned any one, is selected from single-chain antibody, Fab, scFv, double antibody and three antibody.
The fusion antibody of above-mentioned any one or the conjugate of fusion antibody fragment.
Described conjugate, containing antitumor drug, target integral part or reporter moiety.
Invention further illustrates:
Fusion antibody gene order total length 1596 Nucleotide in the present invention, wherein single-chain antibody 735 Nucleotide, MICA828 Nucleotide, by a Linker (G
4s) connect.
Expression vector containing the present inventor's vascular endothelial growth factor receptor single-chain antibody gene and Host Strains, expression vector and the host cell of the expression vector of MICA gene and Host Strains and fusion antibody all belong to protection scope of the present invention.Increase the primer pair of any fragment of fusion antibody gene of the present invention also within protection scope of the present invention.
Another object of the present invention be to provide a kind of can the method for the above-mentioned fusion antibody AK404R-MICA of expression and purification.
The present invention is by a G
4s connects single-chain antibody and MICA and imports to that screening fermentation in pichia spp obtains can the fusion antibody of specific binding KDR3 and NKG2D.Cultivate after 5 days, low-temperature centrifugation removes cell debris, supernatant is crossed nickel affinity chromatography post and carries out separation and purification; Western Blot identifies the AK404R-MICA that separation and purification obtains; The keying action of elisa assay fusion antibody and KDR3 and NKG2D; Utilize the avidity of surface plasma body resonant vibration analysis fusioning protein and KDR3 and NKG2D; Inspection fusion rotein is to the restraining effect of Leukemia K562 cell.
The fusion antibody AK404R-MICA that the present invention obtains has high specific with Leukemia K562 cell and Human umbilical vein endothelial cells HUVEC and is combined, extracorporeal suppression tumor cell growth experiment result shows, the fusion antibody that the present invention obtains can the growth of obvious inhibition tumor cell.The present invention is that treatment and diagnosis provide the therapy based on antibody with the disease that the expression of people's interior cutaneous vessel growth factor receptors, particularly overexpression are feature, and relative disease includes but not limited to autoimmune disease and cancer.
Accompanying drawing explanation
Fig. 1.Figure 1A is the structure of expression plasmid.L1:linker1, L2:linker2, restriction site: EcoR1 and XbaI.Figure 1B is the product figure after the PCR of MICA and AKA404R.Fig. 1 C is the product figure of AK404R-MICA after PCR.
Fig. 2.Fig. 2 A shows SDS-PAGE protein electrophorese figure, the fusion antibody describing fermentation expression carries out the result of separation and purification by nickel post, swimming lane 1 ~ 9 different concns imidazole buffer washing nickel post result, swimming lane 2 ~ 9 be target protein (70KD) Fig. 2 B display obtained containing 100mM imidazole buffer eluting nickel post be the schematic diagram of fusion rotein; Fig. 2 C shows the MICA after the purified renaturation of SDS-PAGE, and swimming lane 1 is irreducibility MICA, and swimming lane 2 is reductibility MICA.Fig. 2 D shows the NKG2D after the purified renaturation of SDS-PAGE, and swimming lane 1 is irreducibility NKG2D, and swimming lane 2 is reductibility NKG2D.Fig. 2 E display utilizes the combination of WesternBlot fusion antibody and restructuring KDR3.Fig. 2 F display utilizes the combination of Western Blot fusion antibody and restructuring NKG2D.
Fig. 3.What Fig. 3 A showed is the affinity constant waiting surface plasma resonance to analyze fusion antibody and recombinate between NKG2D, Ka=ka=6.77 × 10
9/ MS, k
d=267.4/S and K
d=3.95 × 10
-8m.What Fig. 3 B showed is the affinity constant waiting surface plasma resonance to analyze fusion antibody and recombinate between KDR3, Ka=4.26 × 10
6/ MS, kd=261/S and K
d=6.13 × 10
-7m.What Fig. 3 C showed is fusion antibody and the ELISA binding activities of restructuring NKG2D.What Fig. 3 D showed is fusion antibody and the ELISA binding activities of restructuring KDR3.
Fig. 4.The combination of Fig. 4 A, D Flow cytometry fusion antibody, AK404R and HUVEC cell, combination rate is respectively 62.7% and 67.0%, and control group MICA and PBS is respectively 2.1% and 1.5%.The combination of figure B, D Flow cytometry fusion antibody, MICA and U937, combination rate is respectively 69.7% and 58.0%, and control group A K404R and PBS and U937 combines without significance.Figure C, D Flow cytometry fusion antibody, AK404R, MICA and PBS all with HEK293 (HEKC) cell without remarkable combination.
Fig. 5.Fig. 5 A shows that the restraining effect of fusion antibody to HUVEC cell presents dosage and time-dependent manner.What Fig. 5 B showed is the Inhibit proliferaton effect of fusion antibody to HUVEC cell, the half inhibiting rate IC of 48 hours
50: ~ 333.186nM, HEK293 are as negative cells.
Fig. 6.It is Transwell Matrigel that Fig. 6 A shows, fusion antibody effectively can suppress the invasion and attack of endotheliocyte.Fig. 6 B quantitative analysis Transwell Matrigel, fusion antibody can with the invasion and attack of dose-dependent inhibition endotheliocyte.
Fig. 7.Fig. 7 A shows the cellulotoxic experiment of K562, and the ratio along with effector cell/target cell raises and raises.Fig. 7 B shows the cellulotoxic experiment of MDA-MB-435, and the ratio along with effector cell/target cell raises and raises.The B16F1 cellulotoxic experiment that Fig. 7 C shows, the ratio along with effector cell/target cell raises and raises.Fig. 7 D shows the cellulotoxic experiment of 3T3-L1 (control).
Lift by non-limiting example below and describe the present invention in detail.To those skilled in the art, without departing from the premise in the spirit of the present invention to any apparent change that it does, particularly to the equivalent replacement of some parts, all form infringement of patent right of the present invention, corresponding legal obligation will be born.
Embodiment
The per-cent related in embodiment, wherein fixating reagent is percent weight in volume, and liquid reagent is volume percent.
The structure of embodiment 1AK404R-MICA plasmid and transfection
The single-chain antibody gene of human vessel endothelium growth factor resisting acceptor 2 obtains with pHEN2-AKA404R (laboratory preservation) amplification, devises pair of primers accordingly, upstream primer P1:5 '-
gGAATTCgAGGTGCAGCTGGTG-3 ' and downstream primer P2:5 '-GGTTCTGAACCACCACCACCACCTAGGACGGTCAG-3 ', the pET28a-MICA amplification of MICA gene obtains, upstream primer P3:5 '-TAGGTGGTGGTGGTGGTTCAGAACCTCACAGCCTG-3 ' and downstream primer: 5 '-GC
tCTAGAgCTCAATGATGATGATGATGATGCTTGCCGCTTGGGAC3 '.The base pair of lower end line is the restriction enzyme site of EcoR1 and Xba1.C-terminal and the MICA of single-chain antibody use one to link thing (linker) G
4s (G-glycine, S-Serine) is merged by overlap PCR, uses upstream primer P1 and downstream primer P4.The goal gene (AK404R-MICA) increased and carrier pPICZ α carry out being connected with restriction enzyme EcoR1 with Xba1 and are transferred to DH5 α.Picking is out and check order in containing the LB flat board of 25 μ g/ml bleomycins for positive colony.
The recombinant vectors pPICZ α-AK404R-MICA obtained uses electroporation method to carry out electricity and proceeds to pichia spp (X-33), positive colony screens at yeast extract powder peptone glucose culture plate (YPD) containing 100 μ g/ml bleomycins, and schematic diagram and result are as shown in Figure 1.
The expression and purification of embodiment 2 fusion antibody
The clone using SDS-PAGE and western blot to pick out high expression level from 50 positive colonies is fermented.High-expression clone in 10mlYPD liquid nutrient medium (containing 100 μ g/ml bleomycins) 30 DEG C, 220rpm incubated overnight to OD
600: 2-4, is inoculated into BMGY (2% peptone, 1% yeast extract, 100mM potassium phosphate buffer (pH6.0), 1.34%YNB, 4 × 10 of 200ml with 1: 1000
-5vitamin H, 1% glycerine) 30 DEG C, 220rpm incubated overnight is to OD
600: the centrifugal 5min of 1-2,3000g uses BMMY (2% peptone, 1% yeast extract, 100mM potassium phosphate buffer (pH6.0), 1.34%YNB, 4 × 10 of 200ml after removing supernatant
-5vitamin H) and the methyl alcohol 30 DEG C, the 220rpm that add 0.5% cultivate, added the methanol fermentation 4 days of 0.5% every 24 hours.6th day, the fusion antibody nickel post that collection supernatant obtains secreting carried out purifying.After purifying as shown in Figure 2 A.
Embodiment 3 fusion antibody Western Blot identify
The KDR3 that purifying obtains and NKG2D and AK404R (as control) carries out sex change SDS-PAGE electrophoresis, and resolving gel concentration is 15%; 4 DEG C, 20mA constant current transfer printing 1.5h, is transferred to pvdf membrane (purchased from Millipore) by albumen; Transfer printing terminates, by film 37 DEG C of closed 2h in 5%MTBS (TBS containing 5% skimmed milk); Dilute fusion antibody with 5%MTBS, 4 DEG C of night incubation, TBST (containing 0.05%Tween-20 in TBS) washs 3 times, each 10min; Carry out after 2h hatches with the antibody 37 DEG C of anti-MICA, with 5%MTBS by 1: 5000 dilution HRP-anti-Mouse bis-anti-(purchased from Millipore), hatch 1.5h for 37 DEG C, TBS washs 3 times, each 10min; With ECL solution exposure, result as Fig. 2 E, shown in 2F.
Embodiment 4 surface plasma body resonant vibration analyzes part and receptor interaction
Adopt BiacoreX100 (GE) kinetics to KDR3, NKG2D and fusion antibody to study, fusion antibody is sequestered in detection chip GM5 (GE Healthcare, BR-1000-12), KDR3 (250,125,62.5,31.5,15.6,7.8nM) and NKG2D (250,125,62.5,31.5,15.6,7.8nM) sample detection obtains and combines and dissociation curve, and BIA assessment software is analyzed, and calculates and obtains kinetic constant.Experimental result as Fig. 3 A, shown in 3B.
Embodiment 5 enzyme linked immunosorbent assay (ELISA)
Every hole adds 100 μ 1NKG2D or KDR3 (10000nm is dissolved in CBS), 4 DEG C of bag quilts that spend the night.After cleaning, every hole adds skimmed milk 37 DEG C of closed 2h of 200 μ l 5%.Every hole adds 250 μ l TPBS (PBS containing 0.05%Tween 20) trace concussion instrument cleaning 5min, repeat 3 times, with the fusion antibody (0.5 of different concns, 1, 2, 3.9, 7.8, 15.6, 31.3, 62.5, 125, , 250, 500, , 1000nM) room temperature bag is by 1.5h, TPBS and PBS respectively washes three times, add anti-MICAmonoclonal antibody incubated at room 1.5h again, with 5%MTBS by anti-(purchased from Millipore) the incubated at room 1.5h of 1: 5000 dilution HRP-anti-Mouse bis-, TBS washs 3 times, each 10min.Every hole adds 100 μ l nitrite ion (0.03%H
2o
20.1M NaAc buffer is dissolved in, pH 6.0 with 2mg/ml TMB, now with the current), hatch 15min for 37 DEG C.Every hole adds 50 μ l stop buffer (1M H
2sO4), microplate reader detects OD
450-OD
650nm light absorption value.Result as Fig. 3 C, shown in 3D.
The combination rate of embodiment 6 Flow cytometry fusion antibody and HUVEC, U937 and HEK293
Regulate HUVEC, U937 and HEK293 cell concn to 2 × 10
6individual/mL, respectively adds 250 μ L single cell suspensions in each 1.5mL EP pipe.Negative control group: first close with 5% skimmed milk, after 2%FBS-PBS washs 3 times, 1h is hatched in the fluorescence two anti-ice bath only adding 500 μ L FITC marks; Sample sets: first close with 5% skimmed milk, after 2%FBS-PBS washs 3 times, the fusion antibody ice bath of 2000nM hatches 1h, after 2%FBS-PBS washs 3 times, then 1h is hatched in the fluorescence two anti-ice bath adding 500 μ L FITC marks; After finally each group of sample 2%FBS-PBS being washed 3 times, with 500 μ L PBS solution re-suspended cells, BD FACS flow cytomery.Experimental result as Fig. 4 A, shown in 4B, 4C, 4D.
Embodiment 7 cell proliferation experiment
4x10
3individual HUVEC, U937 and HEK293 cell is inoculated at 37 DEG C on 96 orifice plates, 5%CO
2incubator in cultivate 24h, different concns gradient (0nM, 3.9nM, 7.8nM, 15.6nM, 31.2nM, 62.5nM, 125nM, 250nM, 500nM, fusion antibody 1000nM) adds cultivation 24,48 and 72 hours, and AK404R, MICA and PBS are as a control group.Every hole adds 11 μ l MTT, and 37 DEG C of incubators hatch 4h.Discard the substratum containing MTT, add DMSO, microplate reader (Thermo) detects absorbancy at 570nm and 630nm, analysis design mothod result, calculates IC
50value.Experimental result as Fig. 5 A, shown in 5B.
Embodiment 8Transwell Matrigel
1 × 10
4the fusion rotein (20nM, 100nM and 500nM) of individual HUVEC cell and different concns gradient with serum-free ECM substratum re-suspended cell and be laid on by 1: 2 dilution Matrigel matrix (being purchased from Millipore) glue on.AK404R (20nM, 100nM and 500nM), MICA (500nM) and PBS (500nM) are in contrast.In 37 DEG C, hatch 30min in 5%C02 incubator after, the ECM perfect medium of 600 μ L containing 5%FBS and 20ng/mLECGS is added in Zhong Mei hole, lower room, finally, transwell cell is transferred in the lower room containing nutrient solution, is then placed in 37 DEG C, 5%C02 incubator cultivation effect 12h.Fix 20 minutes poststainings with 4% paraformaldehyde 600 μ L/ hole of 4 DEG C of precoolings after cultivating to take pictures.Experimental result as Fig. 6 A, shown in 6B.
Embodiment 9 cellulotoxic experiment
CytoTox 96 Nonradioactive Cytotoxicity assay (being purchased from Promega), target cell is respectively K562, MDA-MB-435, B16F10 and 3T3-L1 (control group).White corpuscle is separated and obtains action effect cell from Healthy People.Effector cell and target cell are with 5: 1, and 30: 1 and 100: 1 co-cultivation also add fusion rotein, AK404R and MICA cultivates 5h at 37 DEG C.Draw 50 μ L supernatants and carry out LDH activity detection according to test kit instruction.Killing activity (%)=[(the total Spontaneous release of OD experimental group-OD)/(the total Spontaneous release of OD maximum release group-OD)] × 100%.Experimental result as shown in Figure 7.
Claims (8)
1. the single-chain antibody of VEGFR2 and a MICA fusion rotein, has following characteristics: single-chain antibody AK404R and MICA that to comprise with KDR Bao Wai 3 district be antigen, by means such as gene recombination, utilizes the connection of a Linker (G4S).
2. the nucleic acid be separated, containing the antibody that this nucleic acid encoding claim 1 Suo Shi is above-mentioned.
3. an expression vector, the nucleic acid containing claim 1.
4. a recombinant host cell, containing above-mentioned expression vector.
5. a pichia spp secreting, expressing method, is characterized in that: for secreting, expressing fusion antibody AK404R-MICA according to claim 1.
6. the fusion antibody of above-mentioned any one or the application of fusion antibody fragment, is optionally combined with KDR3 or fusion antibody that NKG2D combines.
7. the conjugate of claim 1 antibody.
8. the single-chain antibody of VEGFR2 according to claim 1 and the application of MICA fusion rotein in tumor.
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| CN109762785A (en) * | 2018-12-10 | 2019-05-17 | 吴江近岸蛋白质科技有限公司 | A kind of efficient application is in the method for the non-trophoderm in vitro culture of NK cells of human beings |
| CN110903402A (en) * | 2019-11-27 | 2020-03-24 | 中国药科大学 | Bispecific fusion protein and construction method and application thereof |
| CN111333732A (en) * | 2019-12-17 | 2020-06-26 | 中国药科大学 | Preparation and application of bispecific antibody targeting human BCMA and activating NK cells |
| CN114292337A (en) * | 2021-12-17 | 2022-04-08 | 中南大学 | Soluble NK-CAR fusion protein, preparation method and application in mediated immune cell targeted tumor cell killing medicine |
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| WO2017222556A3 (en) * | 2016-06-24 | 2018-04-19 | Avidbiotics Corp. | Insertable variable fragments of antibodies and modified a1-a2 domains of nkg2d ligands |
| CN109762785A (en) * | 2018-12-10 | 2019-05-17 | 吴江近岸蛋白质科技有限公司 | A kind of efficient application is in the method for the non-trophoderm in vitro culture of NK cells of human beings |
| CN109762785B (en) * | 2018-12-10 | 2022-06-24 | 苏州近岸蛋白质科技股份有限公司 | Method for efficiently applying to in-vitro culture of human NK cell non-trophoblast |
| CN109678963A (en) * | 2018-12-18 | 2019-04-26 | 中国药科大学 | A kind of preparation and its application of the bispecific antibody for targeting CD24 and activating NK cell |
| CN110903402A (en) * | 2019-11-27 | 2020-03-24 | 中国药科大学 | Bispecific fusion protein and construction method and application thereof |
| CN110903402B (en) * | 2019-11-27 | 2023-02-28 | 中国药科大学 | Bispecific fusion protein and construction method and application thereof |
| CN111333732A (en) * | 2019-12-17 | 2020-06-26 | 中国药科大学 | Preparation and application of bispecific antibody targeting human BCMA and activating NK cells |
| CN111333732B (en) * | 2019-12-17 | 2023-02-28 | 中国药科大学 | Preparation and application of bispecific antibody targeting human BCMA and activating NK cells |
| WO2023274352A1 (en) * | 2021-07-02 | 2023-01-05 | Laekna Therapeutics Shanghai Co., Ltd. | Depletion of activated hepatic stellate cells (hscs) and uses thereof |
| CN114292337A (en) * | 2021-12-17 | 2022-04-08 | 中南大学 | Soluble NK-CAR fusion protein, preparation method and application in mediated immune cell targeted tumor cell killing medicine |
| WO2024055995A1 (en) * | 2022-09-14 | 2024-03-21 | 寻济生物科技（北京）有限公司 | Anti-vegfa fusion protein, and preparation method therefor and use thereof |
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