CN104892766A - Preparation of high-penetrability nano antibody fusion protein and application of high-penetrability nano antibody fusion protein in tumor resistance - Google Patents
Preparation of high-penetrability nano antibody fusion protein and application of high-penetrability nano antibody fusion protein in tumor resistance Download PDFInfo
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Abstract
The invention relates to preparation of a high-penetrability nano antibody fusion protein and an application of the high-penetrability nano antibody fusion protein in tumor resistance. The high-penetrability nano antibody fusion protein is formed by connecting a targeted human epidermal growth factor receptor (EGFR) nano antibody with a tumor penetrating peptide iRGD through a connecting peptide linker, namely GGGGSGGGGSGGGGS. The defects that a past existing fusion protein is only effective to a small part of tumors and cannot obtain good penetrability at tumor parts are overcome. The single-domain antibody encoding an anti-human EGFR antigen is connected with a nucleotide sequence of the tumor penetrating peptide iRGD, a fusion gene fragment encoding anti-EGFR-iRGD is obtained and then is inserted into a prokaryotic expression plasmid vector; a host cell capable of expressing the fusion protein is involved; the fusion protein can be efficiently expressed in escherichia coli, is applied in tumor resistance, and makes a drug accumulated in the local tumor; and at the same time, the fusion protein makes the drug obtain good penetrability in the local tumor by a NRP-1 way, and makes more drug entering tumor cells to play a role.
Description
Technical field
The invention belongs to biological with new medical technology field, particularly the preparation of high-penetrability nano antibody fusion rotein and the application in antitumor thereof.
Background technology
Before the present invention makes, the research of the targeted therapy of tumour be improve oncotherapy effect so far, reduce medicine toxic side effects the most effectively, one of the measure having practical application potentiality most.The structure of fusion rotein (recombinant protein) and the research and development being expressed as tumor-targeting drug provide method.But this kind of medicine only plays a role to sub-fraction (usual < 20%) tumour, curative effect of medication related biomarker is checked by individuation, could determine that minority is suitable for the patient of medication, and the prerequisite that medicine plays desirable anti-tumour effect is not only manage to allow drug accumulation in tumor by local, the more important thing is and medicine will be made to obtain good penetrance at tumor by local, make it enter tumour cell more and play a role.
Summary of the invention
Object of the present invention is just to overcome above-mentioned defect, the fusion rotein preparation of research high-penetrability nano antibody and the application in antitumor thereof.
Technical scheme of the present invention is as follows:
High-penetrability nano antibody fusion rotein, its technical characteristics is: this albumen is by targeted human EGFR nano antibody and tumour penetrating peptide iRGD via connection peptides linker, and namely GGGGSGGGGSGGGGS connects to form.
Another technical scheme of the present invention is:
The preparation method of high-penetrability nano antibody fusion rotein, its technical characteristics is that it comprises the following steps: on the carrier pSJF2 preserving ant i-EGFR gene fragment, carry out polymerase chain reaction (PCR) amplification obtain HIS-ant i-EGFR-linker-iRGD gene fragment by designing three primers, and be cloned on expression vector pET-28a, construction of expression vector pET-28a-ant i-EGFR-iRGD, and through sequence verification, be transformed into e. coli bl21 DE3 and expressed bacterium, through isopropyl-β-D-thiogalactoside(IPTG) induction expression of recombinant proteins, by nickel ion affinity chromatograph column purification recombinant protein, polyacrylamide gel electrophoresis pure 95% can be obtained, the fusion rotein of 15mg/L bacterial cultures,
Upstream primer P1:
5’-CATGCCATGGGCCATCACCATCACCATCACCAGGTAAAGCTGGAG-3’
45bp
Downstream primer P2:
5’-CGATCCGCCACCGCCAGAGCCACCTCCGCCTGAACCGCCTCCACCGTTCAGATCTTCTTCGCTGATC-3’
67bp
Downstream primer P3:
5’-CCCAAGCTTCTAGCAGTCCGGACCTTTGTCACCACGGCACGATCCGCCACCGCCAGAGCCACCTCCGCCTGAACCGCCTCCACC-3’
84bp
(1) recombinant human ant i-EGFR-iRGD gene fragment amplification:
Select upstream primer P1 and downstream primer P2, to preserve the pSJF2 carrier recombinant plasmid of ant i-EGFR gene fragment for template, polymerase chain reaction (PCR) amplifies Nco I-HIS-ant i-EGFR-linker, using this as template, carry out PCR reaction with upstream primer P1 and downstream primer P3 again, obtain Nco I-HIS-ant i-EGFR-linker-iRGD-Hind III;
(2) structure of ant i-EGFR-iRGD expression vector pET28a-ant i-EGFR-iRGD:
The ant i-EGFR-iRGD goal gene PCR primer purifying obtained in step (1) is reclaimed, and it is connected with carrier pET28a, build ant i-EGFR-iRGD expression vector pET28a-ant i-EGFR-iRGD, by being transformed into bacillus coli DH 5 alpha, carry out the bacterial strain that resistance screening selects successful connection, and by bacterium liquid PCR means, expression vector is identified, select and obtain positive colony and extract plasmid according to the little extraction reagent kit operation instruction of plasmid and send sequence verification;
(3) expression vector pET28a-ant i-EGFR-iRGD is converted into expression strain BL21DE3:
Getting the 100 μ g/ μ L expression vector pET28a-ant i-EGFR-iRGD0.1 μ L-5 μ L confirmed is transformed in expression strain BL21DE3 competent cell prepared by calcium chloride, be applied on the LB flat board of specific resistance, select glycerine in positive colony cryogenic refrigerator and preserve this bacterial classification;
(4) to recombinate the abduction delivering of ant i-EGFR-iRGD and purifying:
3) restructuring ant i-EGFR-iRGD isopropyl-β-D-thiogalactoside(IPTG) abduction delivering:
To restructuring ant i-EGFR-iRGD 1mM isopropyl-β-D-thiogalactoside(IPTG) abduction delivering, in induction after 4 hours, polyacrylamide gel whole protein electrophoresis;
4) ant i-EGFR-iRGD is recombinated through AKTA Purifier900FPLC system, nickel ion affinity chromatograph column purification:
Collect the BL21DE3 thalline of 1000mL after isopropyl-β-D-thiogalactoside(IPTG) abduction delivering, with the resuspended thalline of PBS, 5mM imidazoles, 350W power carries out ultrasonication, 12000rmin-1 collected after centrifugation supernatant liquor is in AKTA Purifier900FPLC system purifying, polyacrylamide gel electrophoresis is carried out finally by the albumen of purifying, detect albumen, measure purity;
(5) AXIMA MALDI-LNR-TOF verifies that the molecular weight of purifying protein is 18KD.
The another technical scheme of the present invention is:
High-penetrability nano antibody fusion rotein is as the application of antitumor drug, and its technical characteristics is that high-penetrability nano antibody fusion rotein is that activeconstituents is used for the treatment of tumour.
Described tumour refers to cancer of the stomach and stomach metastatic carcinoma especially.
Describedly be suitable for per rectum, in nose, lung, intravaginal, outside, oral or parenteral, comprise subcutaneous, implant, intravenously and intramuscular administration.
The invention has the beneficial effects as follows: dual specific target (EGFR and the integrin alpha of fusion rotein
vβ
3/ β
5) subject range of this fusion rotein can be improved, make drug accumulation in tumor by local, fusion rotein is by NRP-1 approach simultaneously, makes medicine obtain good penetrance at tumor by local, makes it enter tumour cell more and play a role.
Accompanying drawing explanation
Fig. 1---the Nco I-HIS-ant i-EGFR-linker gene fragment agarose electrophoresis figure that P1 P2 PCR obtains:
Swimming lane 1:DL2000marker; Swimming lane 2:PCR result (489bp).
The Nco I-HIS-ant i-EGFR-linker-iRGD-Hind III gene fragment agarose gel electrophoresis figure that Fig. 2---P1P3PCR obtains:
Swimming lane 1:DL2000marker; Swimming lane 2:PCR result (528bp).
Fig. 3---destination gene expression carrier pET28a-ant i-EGFR-iRGD DH5 α thalline PCR electrophorogram:
Swimming lane 1:DL2000marker; Swimming lane 2: positive control (with Fig. 2 swimming lane 2); Swimming lane 3:1 bacterium PCR result; Swimming lane 4:2 bacterium PCR result; Swimming lane 5:3 bacterium PCR result; Swimming lane 6:4 bacterium PCR result; Swimming lane 7:5 bacterium PCR result; Swimming lane 8:6 bacterium PCR result.
Fig. 4---the sequencing result schematic diagram in expression vector pET28a-ant i-EGFR-iRGD between Nco I and Hind III.
Fig. 5---BL21 DE3-pET28a-ant i-EGFR-iRGD solubility expression of protein and purifying:
Swimming lane 1: do not induce; Swimming lane 2: induction; Swimming lane 3: precipitation; Swimming lane 4: supernatant; Swimming lane 5: purifying protein (asterisk goes out); Swimming lane 6:Marker.
Fig. 6---affinity chromatography purifying target protein collection of illustrative plates (flow velocity: 5MI/min; A liquid: 1*PBS PH7.4; B liquid: 1*PBS PH7.4,1M imidazoles, arrow instruction target protein).
Fig. 7---AXIMA MALDI-LNR-TOF detected result schematic diagram.
Fig. 8---stomach cancer cell line EGFR expression schematic diagram.
Fig. 9---immunofluorescence analysis schematic diagram.
A ant i-EGFR group FITC fluorescence; B ant i-EGFR-iRGD group FITC fluorescence; C EGFR monoclonal antibody group FITC fluorescence; D ant i-EGFR group Hochest dyes; E ant i-EGFR-iRGD group Hochest dyes; F EGFR monoclonal antibody group Hochest dyes; G ant i-EGFR group FITC and Hochest merges; H ant i-EGFR-iRGD group FITC and Hochest merges; I EGFR monoclonal antibody group FITC and Hochest merges.
Figure 10---ant i-EGFR-iRGD fusion rotein is to the In-vitro Inhibitory Effect of BGC823 stomach cancer cell:
For Cetuximab X-coordinate 0.5 μM, 1 μM, 2 μMs, 4 μMs, 8 μMs, 16 μMs refer to 7.5 μ g/mL, 15 μ g/mL, 30 μ g/mL, 60 μ g/mL, 120 μ g/mL, 240 μ g/mL respectively.
Figure 11---the mouse Anticancer effect in vivo of ant i-EGFR-iRGD fusion rotein.
Embodiment
The technology of the present invention thinking is: the coding single domain antibody of anti-human EGFR antigen and the nucleotide sequence of tumour penetrating peptide iRGD are coupled together, and obtains the fusion gene fragment of coding ant i-EGFR-iRGD, and is inserted into prokaryotic expression plasmid vector.Invention also relate to can the host cell of expressed fusion protein, and this fusion rotein can at E. coli, and the application in antitumor.
Describe in detail below in conjunction with embodiment, but do not limit the present invention.
The expression and purification process with the fusion rotein of antitumor action is as follows:
1, recombinant human ant i-EGFR-iRGD gene fragment amplification:
Select upstream primer P1 and downstream primer P2, to preserve the pSJF2 carrier recombinant plasmid of ant i-EGFR gene fragment for template, PCR reaction amplifies Nco I-HIS-ant i-EGFR-linker; Using this as template, then carry out PCR reaction with upstream primer P1 and downstream primer P3, obtain Nco I-HIS-ant i-EGFR-linker-iRGD-Hind III; Reaction conditions is as follows:
95℃5min
(95℃30s；55℃30s；72℃1min)*30；
72℃7min；
4℃hold
50 μ L reaction systems are as follows:
ddH2037.5μL
Masterplate 1 μ L
Upstream primer 1 μ L
Downstream primer 1 μ L
Taq enzyme 0.5 μ L
Magnesium ion 3 μ L
dNTP1μL
KCI MgCl2-Buffer5μL
First time PCR primer Nco I-HIS-ant i-EGFR-linker gene fragment size is 489bp, and as shown in Figure 1: through 1% agarose gel electrophoresis, visible clip size is consistent with expection; Reclaim test kit (AxyPrep) with agarose gel DNA fragment and carry out the masterplate of this fragment recovery as second time PCR, take P1P3 as primer, reaction conditions, system are with first time PCR; Restructuring Nco I-HIS-ant i-EGFR-linker-iRGD-Hind III gene fragment size is 528bp, and as shown in Figure 2: through 1% agarose gel electrophoresis, visible clip size is consistent with expection.
2, the structure of ant i-EGFR-iRGD expression vector pET28a-ant i-EGFR-iRGD:
1) the ant i-EGFR-iRGD goal gene PGR product agarose gel DNA fragment obtained in step 1 is reclaimed test kit (AxyPrep) and carry out this fragment recovery.Restriction enzyme site according to upstream primer P1, downstream primer P3 design carries out double digestion respectively to the PCR primer of purifying and pET28a.
Endonuclease reaction system following (two kinds of enzymes are purchased from NEB company):
16 μ L PCR primer/vector plasmid pET28a
1μL Nco I
1μL Hind III
2μL buffer
React 37 DEG C of 3-4h
The DNA fragmentation that obtains is cut to enzyme and linear plasmid carries out gel-purified recovery.
2) ligation system:
17 μ L mix (carrier: goal gene=1: 3-4)
2μL ligation buffer
1 μ L T4 ligase enzyme (T4 ligase enzyme is purchased from TAKARA company)
React 16 DEG C of 12h
Adopt ligation to be connected the linear pET28a after goal gene ant i-EGFR-iRGD cuts with enzyme again, build ant i-EGFR-iRGD expression vector pET28a-ant i-EGFR-iRGD.
3) competence intestinal bacteria preparation:
(be seeded in the 3ml LB substratum containing 50ug/ml in DH5 α, 220rpm37 DEG C of incubated overnight from glycerol stock.Amplifying by 1: 100 next day is inoculated in containing in 50 μ g/ml LB substratum, and 37 DEG C of 220rpm joltings are 0.6-0.8 (about 3 hours) to OD600.This LB pipe places 30min on ice, get 1mL bacterium liquid 4 DEG C of centrifugal 3min of 12000g, abandon supernatant, add the calcium chloride (high-temperature sterilization) of 700 μ L precoolings, resuspended rearmounted 30min on ice, abandon supernatant after 4 DEG C of centrifugal 3min of 12000g, then add the calcium chloride (high-temperature sterilization) of 200 μ L precoolings, recipient cell prepares.Next directly transform.
4) method for transformation:
In competence bacterium, add 20 μ L connect product, mixing; 30min on ice; 42 DEG C, 90s thermal shock; Place 3-5min on ice; Add the LB of 700 μ L37 DEG C preheatings; 37 DEG C of 220rpm shake bacterium 1h, coated plate 37 DEG C of overnight incubation.
Product conversion after connecting is entered competence bacillus coli DH 5 alpha.
5) with the bacterial strain of that resistance screening successful connection of card, and by bacterium liquid PCR means, expression vector is identified.
Be cloned in LB pipe from LB plate rifle head random choose 6, add that microbiotic of card respectively, 37 DEG C of 220rpm shake bacterium and spend the night.
Set up PCR reaction system:
95℃5min
(95℃30s；55℃30s；72℃1min)*30；
72℃7min；
4℃hold
50 μ L reaction systems are as follows:
ddH2037.5μL
Masterplate (bacterium liquid) 1 μ L
Upstream primer P1 1 μ L
Downstream primer P3 1 μ L
Taq enzyme 0.5 μ L
Magnesium ion 3 μ L
dNTP1μL
KCI MgCl2-Buffer5μL
PCR primer 1% agarose gel electrophoresis, positive control is the product Nco I-HIS-ant i-EGFR-linker-iRGD-Hind III of second time PCR in 1, as shown in Figure 3: No. 2 bacterium liquid, No. 5 bacterium liquid are positive.Extract plasmid with No. 5 positive bacteria liquid according to the little extraction reagent kit of plasmid (axygen) operation instruction by No. 2 and send sequence verification (Jin Sirui company).The result of the nucleotide sequence of sequenced genes and the aminoacid sequence of derivation and design is analyzed.Show that expression No. 2 carrier pET28a-ant i-EGFR-iRGD successfully construct through contrast, sequencing result as shown in Figure 4.
3, expression vector pET28a-ant i-EGFR-iRGD is converted into expression strain BL21DE3:
Get the 100 μ g/ μ L expression vector pET28a-ant i-EGFR-iRGD0.1 μ L-5 μ L confirmed to be transformed in expression strain BL21DE3 competent cell prepared by calcium chloride, be applied on the LB flat board of that resistance of particular card.Select glycerine in positive colony cryogenic refrigerator and preserve this bacterial classification.
4, to recombinate the abduction delivering of ant i-EGFR-iRGD and purifying:
1) restructuring ant i-EGFR-iRGD IPTG abduction delivering:
To restructuring ant i-EGFR-iRGD 1mMIPTG abduction delivering, induce after 4 hours, SDS-PAGE whole protein electrophoresis, clear specific protein band can be seen at about 20KD, between 17KD to 26KD Marker, inducing without IPTG and there is IPTG induction contrast display, ant i-EGFR-iRGD albumen has been expressed successfully.
Whole protein I PTG abduction delivering, experimental procedure is as follows:
A) get 3 μ L bacterium liquid (expression strain glycerine preserve bacterium) and add 3mLLB (blocking that resistance) substratum, 220rpm37 DEG C is spent the night and shakes bacterium;
B) get 3 μ L bacterium liquid, add 3mLLB (blocking that resistance) substratum, 220rpm37 DEG C is shaken bacterium;
When c) surveying OD600 to 0.5;
D) IPTG that concentration is 1mM is added;
E) 220rpm37 DEG C is shaken bacterium 4 hours;
F) take out bacterium liquid, 12000g1min is centrifugal removes supernatant;
G) 100 μ LddH are added
20 resuspended bacterium liquid, adds 100 μ L2*lcading buffer;
H) 100 DEG C are boiled 10 minutes
I) configure 15% polyacrylamide gel, carry out electrophoresis;
J) coomassie brilliant blue staining, Gel Doc XR gel imaging system imaging (Bio-Rad, US);
2) ant i-EGFR-iRGD is recombinated through AKTA Purifier900FPLC system, nickel ion affinity chromatograph column purification:
Collect the BL21DE3 bacterium liquid of 1000mL after IPTG abduction delivering, through the centrifugal 10min of 4200rmin-1, collect thalline.Precipitation is resuspended in PBS (PH7.4), 5mM imidazoles 80mL carries out ultrasonic degradation, and ultrasound condition is 350W, work 3s, intermittently 3s, total time are 45min.Ultrasonic degradation thing is in 4 DEG C, the centrifugal 20min of 12000rmin-1.Collect supernatant liquor and AKTA Purifier900FPLC system purifying.Nickel post loading after 5 times of column volume PBS (PH7.4), 5mmol IL-1 imidazoles balance, after PBS (pH7.4), 40mmolL-1 imidazoles are washed and are mixed, target protein PBS (pH7.4), 500mmolL-1 imidazoles wash-out.Purifying protein after wash-out is dialysis in PBS (pH7.4).Obtain ant i-EGFR-iRGD albumen and have 15mg/L bacterium liquid.
5, with SDS-PAGE method testing goal albumen:
Choose the expression strain BL21DE3-pET28a-ant i-EGFR-iRGD bacterium liquid 1mL of non-abduction delivering, add the bacterium liquid 1mL of IPTG abduction delivering after 4 hours and carry out the centrifugal 1min of 12000rmin-1, abandon supernatant, precipitate with 100 μ LddH
2the resuspended conduct of O " is not induced " and " induction "; Bacterium liquid 100 μ L after ultrasonic carries out the centrifugal 1min of 12000rmin-1, and supernatant moves to new EP pipe, precipitates with 100 μ LddH
2o is resuspended respectively as " supernatant " and " precipitation "; The protein 10 0 μ L obtained through AKTA purifying is as " purifying protein "; Carry out SDS-PAGE electrophoresis, coomassie brilliant blue staining after electrophoresis also decolours, and takes pictures.Through BAND SCAN software analysis, the ant i-EGFR-iRGD albumen that result display purifying obtains reaches 95% purity.
6,
biacore (biomo lecular lnteraction Analysis) biological disperser analytical method utilizes surface plasma resonance technology to detect the dissociating of albumen and Human epidermal growth factor receptor, affinity costant:
Fusion rotein detects the dissociating of albumen and Human epidermal growth factor receptor, affinity costant by surface plasma resonance technology (Biacore), and obtaining KD value is 7.09nM.
7, AXIMA MALDI-LNR-TOF verifies that the molecular weight of purifying protein is about 18KD.
8, the stomach cancer cell line binding ability of immunofluorescence checking albumen and high expression level EGFR:
First BGC823EGFR high expression level in WESTERN BLOT method validation stomach cancer cell line.By stomach cancer cell line BGC823 kind in 12 orifice plates, after adherent, with PBS cleaning twice; The fusion rotein of flag F ITC and EGFR monoclonal antibody (EGFR mab) are added cell, hatches 1 hour for 37 degree, PBS cleaning twice; Hochest contaminates core 15min, and fluorescent microscope is taken pictures; Control selection EGFR monoclonal antibody.Result display albumen ant i-EGFR-iRGD target stomach cancer BGC823 ability is better than EGFR monoclonal antibody.
9, on stomach cancer cell line BGC823, contrast the anti-tumour cell proliferative ability of ant i-EGFR, ant i-EGFR-iRGD and Cetuximab.
Cultivate stomach cancer cell line BGC823 cell to 90% to merge, reach in 96 orifice plates, 12h cultivated by 5000/ every hole CO2 incubator.With serum-free 1640 substratum by setting gradient concentration dilution ant i-EGFR (0.5 μM, 1 μM, 2 μMs, 4 μMs, 8 μMs, 16 μMs), ant i-EGFR-iRGD (0.5 μM, 1 μM, 2 μMs, 4 μMs, 8 μMs, 16 μMs), Cetuximab (7.5 μ g/mL, 15 μ g/mL, 30 μ g/mL, 60 μ g/mL, 120 μ g/mL, 240 μ g/mL).After cell attachment, clean substratum in 96 orifice plates, add the albumen after 100 μ L dilutions according to the every hole of concentration gradient, often organize concentration and 3 parallel holes are set.Continue to cultivate 48h.Every hole adds 10 μ L0.5mg/ml MTT working fluids, continues to cultivate 4h.Substratum is abandoned in suction, and every hole adds 100 μ L DMSO room temperature concussion 15min, dissolving crystallized body.Measure light absorption value in 570nm place, reference wavelength 630nm, with OD
570-OD
630as net result.Using sample measurements divided by measure blank as growth of tumour cell survival ratio numerical value.Stronger inhibition (* < 0.05 is had than the propagation of nano antibody ant i-EGFR to BGC823 cell at multiple concentration point (0.5 μM, 2 μMs, 4 μMs, 8 μMs, 16 μMs) fusion rotein ant i-EGFR-iRGD, * < 0.01), in addition compared with Cetuximab (cetuximab), concentration 4 μMs, 8 μMs, 16 μMs time, ant i-EGFR-iRGD has the ability (# < 0.05, ## < 0.01) of stronger inhibition tumor cell propagation.
10, ant i-EGFR-iRGD suppresses the comparative studies of the growth of mouse tumor effectively.
Select 24 well-grown nude mices, often only people BGC823 cell 5*10^5 is injected in right oxter, 0.1mL.Treat that tumor bearing nude mice knurl volume increases to 100mm
3(about one week), is divided into 4 groups at random by it.Abdominal injection PBS (control group) 100 μ L, albumen ant i-EGFR0.4 μM/kg*100 μ L, ant i-EGFR-iRGD0.4 μM/kg*100 μ L of purifying, Cetuximab 6mg/kg*100 μ L respectively, is administered once (i.e. d0, d3, d6, d9, d12) and measures gross tumor volume (V=1/2 major diameter * minor axis * minor axis), Mouse Weight to nude mice for every three days.Within 15 day, put to death mouse.Use spss software analysis data.When the 12nd day, control group gross tumor volume is 1603 ± 572mm
3, ant i-EGFR group gross tumor volume is 1269 ± 480mm
3, inhibiting rate is 21%, ant i-EGFR-iRGD group gross tumor volume is 581 ± 229mm
3, inhibiting rate is 64%, and Cetuximab group gross tumor volume is 1098 ± 301mm
3, inhibiting rate is 31.5%; The growth of the remarkable Tumor suppression of high-penetrability nano antibody fusion rotein ant i-EGFR-iRGD group energy compared with control group PBS; And high-penetrability nano antibody fusion rotein ant i-EGFR-iRGD group also can the growth (* < 0.05, * * < 0.01) of Tumor suppression significantly compared with nano antibody ant i-EGFR.Experimental session, each group Bearing Mice Life sign is normal, and this heavy sensation of the whole body of mouse there is no and obviously alleviates, and major organs tissue morphology is good.
Claims (5)
1. high-penetrability nano antibody fusion rotein, is characterized in that: this albumen is by targeted human EGFR nano antibody and tumour penetrating peptide iRGD via connection peptides linker, and namely GGGGSGGGGSGGGGS connects to form.
2. the preparation method of high-penetrability nano antibody fusion rotein, it is characterized in that it comprises the following steps: on the carrier pSJF2 preserving ant i-EGFR gene fragment, carry out polymerase chain reaction (PCR) amplification obtain HIS-ant i-EGFR-linker-iRGD gene fragment by designing three primers, and be cloned on expression vector pET-28a, construction of expression vector pET-28a-ant i-EGFR-iRGD, and through sequence verification, be transformed into e. coli bl21 DE3 and expressed bacterium, through isopropyl-β-D-thiogalactoside(IPTG) induced fusion protein expression, by nickel ion affinity chromatograph column purification fusion rotein, polyacrylamide gel electrophoresis pure 95% can be obtained, the fusion rotein of 15mg/L bacterial cultures,
Upstream primer P1:
5’-CATGCCATGGGCCATCACCATCACCATCACCAGGTAAAGCTGGAG-3’
45bp
Downstream primer P2:
5’-CGATCCGCCACCGCCAGAGCCACCTCCGCCTGAACCGCCTCCACCGTTCAGATCTTCTTCGCTGATC-3’
67bp
Downstream primer P3:
5’-CCCAAGCTTCTAGCAGTCCGGACCTTTGTCACCACGGCACGATCCGCCACCGCCAGAGCCACCTCCGCCTGAACCGCCTCCACC-3’
84bp
(1) recombinant human ant i-EGFR-iRGD gene fragment amplification:
Select upstream primer P1 and downstream primer P2, to preserve the pSJF2 carrier recombinant plasmid of ant i-EGFR gene fragment for template, polymerase chain reaction (PCR) amplification goes out Nco I-HIS-ant i-EGFR-linker, using this as template, carry out polymerase chain reaction with upstream primer P1 and downstream primer P3 again, obtain Nco I-HIS-ant i-EGFR-linker-iRGD-Hind III;
(2) structure of ant i-EGFR-iRGD expression vector pET28a-ant i-EGFR-iRGD:
The ant i-EGFR-iRGD goal gene polymerase chain reaction product purification obtained in step (1) is reclaimed, and it is connected with carrier pET28a, build ant i-EGFR-iRGD expression vector pET28a-ant i-EGFR-iRGD, by being transformed into bacillus coli DH 5 alpha, carry out the bacterial strain that resistance screening selects successful connection, and by bacterium liquid polymerase chain reaction means, expression vector is identified, select and obtain positive colony and extract plasmid according to the little extraction reagent kit operation instruction of plasmid and send sequence verification;
(3) expression vector pET28a-ant i-EGFR-iRGD is converted into expression strain BL21DE3:
Getting the 100 μ g/ μ L expression vector pET28a-ant i-EGFR-iRGD0.1 μ L-5 μ L confirmed is transformed in expression strain BL21DE3 competent cell prepared by calcium chloride, be applied on the LB flat board of specific resistance, select glycerine in positive colony cryogenic refrigerator and preserve this bacterial classification;
(4) to recombinate the abduction delivering of ant i-EGFR-iRGD and purifying:
1) restructuring ant i-EGFR-iRGD isopropyl-β-D-thiogalactoside(IPTG) abduction delivering:
To restructuring ant i-EGFR-iRGD 1mM isopropyl-β-D-thiogalactoside(IPTG) abduction delivering, in induction after 4 hours, polyacrylamide gel whole protein electrophoresis;
2) ant i-EGFR-iRGD is recombinated through AKTA Purifier900FPLC system, nickel ion affinity chromatograph column purification:
Collect the BL21DE3 thalline of 1000mL after isopropyl-β-D-thiogalactoside(IPTG) abduction delivering, with the resuspended thalline of PBS, 5mM imidazoles, 350W power carries out ultrasonication, 12000rmin-1 collected after centrifugation supernatant liquor is in AKTA Purifier900FPLC system purifying, polyacrylamide gel electrophoresis is carried out finally by the albumen of purifying, detect albumen, measure purity;
(5) AXIMA MALDI-LNR-TOF verifies that the molecular weight of purifying protein is 18KD.
3. high-penetrability nano antibody fusion rotein is as the application of antitumor drug, it is characterized in that high-penetrability nano antibody fusion rotein is that activeconstituents is used for the treatment of tumour.
4. high-penetrability nano antibody fusion rotein according to claim 3 is as the application of antitumor drug, it is characterized in that described tumour refers to cancer of the stomach and stomach metastatic carcinoma especially.
5. high-penetrability nano antibody fusion rotein according to claim 3 is as the application of antitumor drug, it is characterized in that containing the liquid preparation as the high-penetrability nano antibody fusion rotein of activeconstituents, be suitable for per rectum, in nose, lung, intravaginal, outside, oral or parenteral, comprise subcutaneous, implant, intravenously and intramuscular administration.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN106265480A (en) * | 2016-09-03 | 2017-01-04 | 山西纳安生物科技有限公司 | Nano antibody vaginal drug delivery systems and preparation method and application |
| CN107739410A (en) * | 2017-10-18 | 2018-02-27 | 南京鼓楼医院 | CD3 single-chain antibody iRGD fusion proteins, preparation and its application as antineoplastic |
| CN108187064A (en) * | 2017-11-28 | 2018-06-22 | 南京大学 | The preparation method and purposes of double targent fused protein adriamycin couplets of one type elastin laminin-anti-EGFR nano antibodies-iRGD |
| CN108424924A (en) * | 2018-02-13 | 2018-08-21 | 天津大学 | Fusion protein HFBI-RGD genes and albumen |
| CN112457405A (en) * | 2018-09-27 | 2021-03-09 | 暨南大学 | Anti-human EGFR nano antibody and application |
| CN112646034A (en) * | 2020-12-25 | 2021-04-13 | 暨南大学 | Affinity maturation binding protein of EGFR (epidermal growth factor receptor) and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106075433A (en) * | 2016-06-17 | 2016-11-09 | 山西纳安生物科技有限公司 | Many targeted nanos antibody polymerization chain junctor that a kind of probiotic bacteria that can be used for treatment of cancer is expressed |
| CN106265480A (en) * | 2016-09-03 | 2017-01-04 | 山西纳安生物科技有限公司 | Nano antibody vaginal drug delivery systems and preparation method and application |
| CN107739410A (en) * | 2017-10-18 | 2018-02-27 | 南京鼓楼医院 | CD3 single-chain antibody iRGD fusion proteins, preparation and its application as antineoplastic |
| CN108187064B (en) * | 2017-11-28 | 2021-05-28 | 南京大学 | Preparation method and application of elastin-like protein-anti-EGFR nano antibody-iRGD double-targeting fusion protein adriamycin conjugate |
| CN108187064A (en) * | 2017-11-28 | 2018-06-22 | 南京大学 | The preparation method and purposes of double targent fused protein adriamycin couplets of one type elastin laminin-anti-EGFR nano antibodies-iRGD |
| CN108424924A (en) * | 2018-02-13 | 2018-08-21 | 天津大学 | Fusion protein HFBI-RGD genes and albumen |
| CN112457405A (en) * | 2018-09-27 | 2021-03-09 | 暨南大学 | Anti-human EGFR nano antibody and application |
| CN112457404A (en) * | 2018-09-27 | 2021-03-09 | 暨南大学 | Anti-human EGFR nano antibody and application |
| CN112521504A (en) * | 2018-09-27 | 2021-03-19 | 暨南大学 | Anti-human EGFR nano antibody and application thereof |
| CN112457405B (en) * | 2018-09-27 | 2022-08-12 | 暨南大学 | Anti-human EGFR nano antibody and application |
| CN112457404B (en) * | 2018-09-27 | 2022-08-12 | 暨南大学 | Anti-human EGFR nano antibody and application |
| CN112521504B (en) * | 2018-09-27 | 2022-08-16 | 暨南大学 | Anti-human EGFR nano antibody and application thereof |
| WO2022121928A1 (en) * | 2020-12-09 | 2022-06-16 | 江苏先声药业有限公司 | Anti-egfr nanobody and use thereof |
| CN112646034A (en) * | 2020-12-25 | 2021-04-13 | 暨南大学 | Affinity maturation binding protein of EGFR (epidermal growth factor receptor) and application thereof |
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