CN111410693A - CDK5 resistant nano antibody and application thereof - Google Patents
CDK5 resistant nano antibody and application thereof Download PDFInfo
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Abstract
The invention provides an anti-CDK 5 nano antibody and application thereof, belonging to the technical field of biological engineering, wherein the amino acid sequence of the anti-CDK 5 nano antibody is shown as SEQ ID No. 1. The anti-CDK 5 nano antibody provided by the invention has the effects of inhibiting growth and migration of melanoma cells and inhibiting growth of solid tumors of mice melanoma, and is possibly suitable for ideal medicines for treating or controlling growth of melanoma.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to an anti-CDK 5 nano antibody and application thereof.
Background
Melanoma is the most invasive cancer of skin caused by the canceration of melanocytes, and is cyclin-dependent kinase 5 (Cdk 5), one of the family members of the serine/threonine kinases. The role of Cdk5 is related to the maturation and movement of nerve cells, normal development of the central nervous system, and sensory pathways such as pain signaling mechanisms. Once Cdk5 is abnormal, various neurodegenerative diseases, such as alzheimer's syndrome, will result. The research of the prior art shows that CDK5 is abnormally and highly expressed in mouse melanoma (B16) cells, Cdk5 can promote the growth and proliferation of the melanoma cells, and the prepared Cdk5 nano antibody can be a tracing tool for researching the action mechanism of Cdk5 by using a molecular imaging technology on one hand and can be used as a small molecular drug for inhibiting the growth of melanoma on the other hand.
Disclosure of Invention
In view of the above, the present invention provides an anti-CDK 5 nanobody and an application thereof, and the anti-CDK 5 nanobody provided by the present invention can inhibit the growth of melanoma.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides an anti-CDK 5 nano antibody, wherein the amino acid sequence of the anti-CDK 5 nano antibody is shown as SEQ ID No. 1.
The invention also provides application of the anti-CDK 5 nano antibody in the technical scheme in preparation of a medicine for inhibiting proliferation of melanoma cells.
The invention also provides application of the anti-CDK 5 nano antibody in the technical scheme in preparation of a drug for inhibiting melanoma cell migration.
The invention also provides application of the anti-CDK 5 nano antibody in the technical scheme in preparation of a medicine for inhibiting melanin generation of melanoma cells.
The invention also provides application of the anti-CDK 5 nano antibody in the technical scheme in preparation of a drug for inhibiting growth of solid tumors of mice melanoma.
The anti-CDK 5 nano antibody provided by the invention has the effects of inhibiting growth and migration of melanoma cells and inhibiting growth of solid tumors of mice melanoma, and is possibly suitable for an ideal medicine for treating or controlling growth of the melanoma.
Drawings
FIG. 1 SDS-PAGE electrophoresis of purified expression products;
FIG. 2 detection of the specificity of the purified product against CDK5 nanobody with anti-His-tagged secondary antibody;
FIG. 3 anti-CDK 5 nanobody inhibits the proliferation of B16-F10 cells;
FIG. 4 anti-CDK 5 nanobody inhibits the production of melanin in B16-F10 cells;
FIG. 5 anti-CDK 5 nanobody inhibits migration of B16-F10 cells (dose effect of 100ng/ml is evident);
FIG. 6 anti-CDK 5 nanobody inhibits growth of mouse melanoma solid tumor;
Detailed Description
The invention provides an anti-CDK 5 nano antibody, wherein the amino acid sequence of the anti-CDK 5 nano antibody is shown as SEQ ID No.1, and the amino acid sequence is as follows:
ESGGGLVQPGESLRLSCKVSGGSTLDYYAIGWFRQAPGKEREAVSCISGSGEVTSWAESVEGRFTISRDNSKNMVYLQMNSLKSEDTGVYYCAGRLYGSHWCDQDFGLWGQGTQVTVSS。
in the invention, the anti-CDK 5 nano antibody is preferably obtained by screening from a melanoma nano antibody library disclosed in Chinese patent CN201910057953.4 by using a conventional screening method.
The invention also provides application of the anti-CDK 5 nano antibody in the technical scheme in preparation of a medicine for inhibiting proliferation of melanoma cells. In the invention, the drug takes anti-CDK 5 nanobody as the only active substance. The dosage form of the medicine is not particularly limited, and the medicine can be prepared from the medically acceptable medicine of the anti-CDK 5 nano antibody. The content of the anti-CDK 5 nanobody in the medicament is not particularly limited, and the content of an active substance in a conventional dosage form is adopted. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a corresponding preparation formulation.
The invention also provides application of the anti-CDK 5 nano antibody in the technical scheme in preparation of a drug for inhibiting melanoma cell migration. In the invention, the drug takes anti-CDK 5 nanobody as the only active substance. The dosage form of the medicine is not particularly limited, and the medicine can be prepared from the medically acceptable medicine of the anti-CDK 5 nano antibody. The content of the anti-CDK 5 nanobody in the medicament is not particularly limited, and the content of an active substance in a conventional dosage form is adopted. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a corresponding preparation formulation.
The invention also provides application of the anti-CDK 5 nano antibody in the technical scheme in preparation of a medicine for inhibiting melanin generation of melanoma cells. In the invention, the drug takes anti-CDK 5 nanobody as the only active substance. The dosage form of the medicine is not particularly limited, and the medicine can be prepared from the medically acceptable medicine of the anti-CDK 5 nano antibody. The content of the anti-CDK 5 nanobody in the medicament is not particularly limited, and the content of an active substance in a conventional dosage form is adopted. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a corresponding preparation formulation.
The invention also provides application of the anti-CDK 5 nano antibody in the technical scheme in preparation of a drug for inhibiting growth of solid tumors of mice melanoma. In the invention, the drug takes anti-CDK 5 nanobody as the only active substance. The dosage form of the medicine is not particularly limited, and the medicine can be prepared from the medically acceptable medicine of the anti-CDK 5 nano antibody. The content of the anti-CDK 5 nanobody in the medicament is not particularly limited, and the content of an active substance in a conventional dosage form is adopted. The preparation method of the medicine is not particularly limited, and the medicine can be prepared by adopting a conventional preparation method of a corresponding preparation formulation.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The main reagents used in the examples are as follows:
TG1 strain, KM13 helper phage, pCANTAN5E Vector, skimmed milk powder, SfiI, DreamTaq GreenPCRMix, HisTrap HP 5ml, 1640 culture medium, DMEM culture medium, L ipofectamine 2000 transfection reagent, imported fetal bovine serum, luciferase substrate, cell lysate, BSA, E L ISA coating solution, E L ISA stop solution, TMB monocomponent chromogenic solution, pMD19-T Simple Vector, PCR Master Mix, HRP-His mouse/rabbit monoclonal antibody, plasmid miniprep kit, reverse transcription kit, DH5 α competent cells, DNA extraction kit, DNA product purification kit, gel recovery kit and endotoxin-free plasmid extraction kit.
Example 1
Construction of an expression vector:
designing a primer according to a positive clone sequence screened from a melanoma nano antibody library disclosed in Chinese patent CN201910057953.4, and adding enzyme cutting sites of BamHI and HindIII at the 5 'end and the 3' end of the primer respectively, inoculating glycerol bacteria of positive expression bacteria into 5ml of 2 × YTAG culture medium for culture, extracting plasmids as template plasmids of prokaryotic expression by using a plasmid miniprep kit, amplifying VHH sequences of the positive cloning by using the designed primer through a PCR method, and connecting the VHH sequences into a pCold I prokaryotic expression vector through the enzyme cutting sites to construct a nano antibody prokaryotic expression recombinant plasmid (the product name is pCold I-5-VHH) so as to carry out CDK5 specific identification of the nano antibody.
The primers were as follows (5 '-3'):
SEQ ID No.2：F：gtgaggatccgagtctggaggrgcttggtggca (underlined BamH i);
SEQ ID No.3：R：tctgagtcgactgaggacagrgtcascstgtc (underlined Hind iii).
Coli expresses anti-CDK 5 nanobodies:
prokaryotic induction expression, namely transforming the constructed pCold I-CDK 5-VHH plasmid into DH5 α escherichia coli by a heat shock method (42 ℃ and 40S), cloning, extracting the plasmid by using a plasmid minim extraction kit, continuously transforming the extracted plasmid into B L (DE3) escherichia coli by the heat shock method, coating a plate, picking the monoclonal in L B culture medium at 37 ℃ and 200rpm for overnight shaking culture the next day, transferring a bacterial liquid into a new L B culture medium according to the proportion of 1:100, shaking the bacterial liquid at 37 ℃ until the OD value is 0.4, adding IPTG with the final concentration of 0.6mM, carrying out overnight induction at 15 ℃ and 80rpm, taking the same monoclonal liquid without IPTG induction as a negative control, taking B L (DE3) empty bacteria as a blank control, and detecting the escherichia coli (DH5 α) to express the anti-CDK 5 nano antibody product, wherein the result is shown in figure 1.
After the DH5 α expression product was purified by SDS-PAGE, a protein with a molecular weight of about 15KD was found in the total protein, which was in the molecular weight range of nanobody.
Purification and identification of expression product:
purification of the expression product: and (3) enabling the supernatant obtained after ultrasonic centrifugation to enter a nickel column through an AKTApure protein purifier and be combined with the nickel column, then eluting through imidazole with the concentration of 200mM to obtain the target protein of the single anti-CDK 5 antibody band, dialyzing the purified protein through a dialysis bag, and removing redundant salt to obtain the pure protein of the anti-CDK 5 nano antibody (SEQ ID No. 1).
And (3) identification after purification: the dialyzed protein was subjected to SDS-PAGE and Western Blotting for detection. The results are shown in FIG. 2.
Since the pColdI carrier is provided with the His label, a secondary antibody containing an anti-His label is used for detecting the specificity of the purified product, and the result shows that the purified product has an immune positive substance specifically combined with the anti-His secondary antibody, namely the anti-CDK 5 nano antibody.
Example 2
The purified anti-CDK 5 nanobody prepared in example 1 inhibited the growth of melanoma cells B16-F10 cells
Cell addition assay: recovering melanoma B16 cells frozen in liquid nitrogen tank at 37 deg.C under 5% CO2The cells were cultured in a cell culture box for subculture, and when the cells were grown to a density of 90%, the cells were added at a concentration of 100ng/ml for the anti-CDK 5 nanobody, and the cells were cultured in FBS-free DMEM medium, and the control group was tested by adding an equal volume of PBS to the FBS-free medium. After 12h, the medium containing the anti-CDK 5 nanobody was aspirated and the FBS-free medium was added again.
CCK8 proliferation assay: after the cell addition is stopped, the cell proliferation is detected by using a CCK-8 kit, and the specific experimental steps are as follows:
(1) digesting the cells with 0.25% trypsin digest, terminating the digestion, centrifuging and suspending the cells with medium, and counting the cells;
(2) adding the cell suspension into a 96-well enzyme label plate, wherein each well is provided withAdding 100 μ l of cell suspension containing 2000 cells/ml, each group is provided with 3 replicates, and placing 96-well enzyme label plate in CO2Incubating the cell culture box for 24 hours;
(3) adding 10ul of CCK-8 working solution into each of two groups of 96-well enzyme label plates, and incubating for 4 hours in a CO2 cell culture box
(4) Detecting the light absorption value of each group at the wavelength of 450nm by using a microplate reader;
(5) the proliferation of cells per well was calculated from the number of cells.
The results are shown in FIG. 3.
As can be seen from FIG. 3, after the anti-CDK 5 nanobody is added into B16-F10 cells, the antibody can effectively inhibit the proliferation of B16-F10 cells through CCK8 detection.
Example 3
Purified anti-CDK 5 nanobody inhibits synthesis of melanin in melanoma cells
After the cell addition experiment was performed as in example 2, the following experiment was performed:
and (3) measuring the total melanin content, namely discarding old culture medium 48h after the addition of the cells is stopped, washing the cells for 3 times by using PBS respectively, digesting by using 0.25% trypsin, after the digestion is stopped, extracting 10 mu l respectively to count the cells, centrifuging the rest cells at 4 ℃ and 1000rpm for 10min, discarding supernatant, preparing 0.2 mol/L NaOH solution, adding 400 mu l of the NaOH solution into each tube for resuspension, placing the tubes at 80 ℃ for heating and cracking for 10min, dividing cell lysate into three holes per group, adding 100 mu l of cell lysate into a 96-hole enzyme label plate per hole, measuring the light absorption value of the cell lysate at 475nm, and calculating the total melanin content of the cells in each group according to the number of the cells.
The content of eumelanin is determined by discarding old culture medium 48h after the addition of cells is terminated, washing the cells 3 times with PBS, digesting with 0.25% trypsin, counting the cells by extracting 10 mul after the digestion is terminated, centrifuging the remaining cells at 4 deg.C for 10min at 1000rpm, discarding the supernatant, adding 750 mul of 30% HI to each group, reacting at 80 deg.C for 2h in a metal bath, cooling to room temperature, adding 50% ethanol in equal amount, mixing, centrifuging at 2234Xg for 10min, discarding the supernatant, adding 500 mul of 0.2 mol/L NaOH to each group, reacting at 80 deg.C for 4h in a metal bath, centrifuging at 10700Xg for 10min, collecting the supernatant, repeating the steps for 3 wells, adding 100 mul per well to a 96-well enzyme plate, determining the absorbance at 350nm, and calculating the content of eumelanin in each group of cells according to the number of cells.
The method comprises the steps of discarding old culture medium 48h after the addition of cells is stopped, washing the cells for 3 times by PBS, digesting by 0.25% trypsin, extracting 10 mu l of the cells after the digestion is stopped, counting the cells, centrifuging the rest of the cells at 4 ℃ and 1000rpm for 10min, discarding supernatant, adding 500 mu l of PBS (0.1 mol/L) with the pH value of 10.5 into each hole, violently mixing the cells, centrifuging the cells at 10000rpm for 10min, taking supernatant, adding 350 mu l of chloroform, centrifuging the supernatant at 4000rpm for 10min, taking supernatant, adding 100 mu l of the supernatant into a 96 enzyme-labeled plate, repeating the steps for 3 times for each group, measuring the light absorption value at the wavelength of 400nm, and calculating the content of the brown melanin of each group of cells according to the number of the cells.
The results are shown in FIG. 4.
As can be seen from FIG. 4, after the anti-CDK 5 nano antibody is added into B16-F10 cells, the detection of ultraviolet spectrophotometry shows that the antibody can effectively inhibit the content of total melanin and eumelanin in the B16-F10 cells, but the influence on the content of the pheomelanin is not obvious.
Example 4
Purified anti-CDK 5 nanobody inhibits migration of melanoma cells
After the cell addition experiment was performed as in example 2, the following experiment was performed: after the termination of the cell addition, the cell surface was scratched with an autoclaved small gun head and washed 3 times with PBS, and the scratches were photographed under a microscope with FBS-free medium and recorded as 0 h. After 12h old medium was discarded and washed 3 times with PBS and then medium without FBS was added and the scratch change was photographed under a microscope as 12 h. The results are shown in FIG. 5.
As can be seen from FIG. 5, after the anti-CDK 5 nano antibody is added into B16-F10 cells, the antibody can generate effective inhibition effect on B16-F10 cell migration within 12h through scratch experiments, and especially the dosage effect of 100ng/ml is obvious.
Example 5
Purified anti-CDK 5 nanobody inhibits the growth of melanoma in a melanoma mouse model
B16 cell preparation: recovering B16 cells frozen in liquid nitrogen tank, spreading in 25t Coriolis bottle, culturing and passaging, removing culture medium when the cells grow to density of about 90%, washing with PBS for 3 times, blowing B16 with PBS, and counting cells to density of 1x106The injection is ready for each ml.
Constructing a melanoma mouse model: 20 healthy Kunming mice of 6-8 weeks old were used as experimental groups and injected subcutaneously into the axilla of mice at a concentration of 1X10 to give a 200. mu.l injection6The B16 cells per ml are injected 1-2 weeks later, and melanoma with the diameter of 1cm appears in the mouse armpit, namely the tumor is successfully established. Another 20 healthy Kunming mice of 6-8 weeks old were injected subcutaneously under the mouse axilla with 200. mu.l/mouse as a control group.
Effect of purified anti-CDK 5 antibody on melanoma growth: after the melanoma mice successfully made tumors, 10 mice in the experimental group and the control group respectively were injected with purified anti-CDK 5 nano antibody via tail vein of mice, and the mice were administered with 100 ug/g/day, and the body weight, the size of melanoma and the mental state of the mice were observed after administration. Also, 10 mice in each of the experimental group and the control group were injected with an equal volume of PBS buffer as a control group. The body weight of the mice, the size of the melanoma and the mental state of the mice were also observed after the administration. The results are shown in FIG. 6.
As can be seen from fig. 6, significant growth inhibition occurred after 7 consecutive injections of anti-CDK 5 nanobody every three days into melanoma solid, compared to the control group.
From the above examples, it can be seen that the anti-CDK 5 nanobody provided by the present invention has the effects of inhibiting the growth and migration of melanoma cells and inhibiting the growth of solid tumors of mouse melanoma, and may be suitable for ideal drugs for treating or controlling the growth of melanoma.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Shanxi university of agriculture
<120> anti-CDK 5 nano antibody and application thereof
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>119
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>1
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu Ser Leu Arg Leu Ser
1 5 10 15
Cys Lys Val Ser Gly Gly Ser Thr Leu Asp Tyr Tyr Ala Ile Gly Trp
20 25 30
Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val Ser Cys Ile Ser
35 40 45
Gly Ser Gly Glu Val Thr Ser Trp Ala Glu Ser Val Glu Gly Arg Phe
50 55 60
Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Val Tyr Leu Gln Met Asn
65 70 75 80
Ser Leu Lys Ser Glu Asp Thr Gly Val Tyr Tyr Cys Ala Gly Arg Leu
85 90 95
Tyr Gly Ser His Trp Cys Asp Gln Asp Phe Gly Leu Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210>2
<211>33
<212>DNA
<213> Artificial sequence (artificacial sequence)
<400>2
gtgaggatcc gagtctggag grrgcttggt gca 33
<210>3
<211>33
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
tctgagtcga ctgaggagac grtgacstsg gtc 33
Claims (5)
1. An anti-CDK 5 nanobody, which is characterized in that the amino acid sequence of the anti-CDK 5 nanobody is shown in SEQ ID No. 1.
2. Use of the anti-CDK 5 nanobody of claim 1 in the preparation of a medicament for inhibiting melanoma cell proliferation.
3. Use of the anti-CDK 5 nanobody of claim 1 in the preparation of a medicament for inhibiting melanoma cell migration.
4. Use of the anti-CDK 5 nanobody of claim 1 in the preparation of a medicament for inhibiting the production of melanin in melanoma cells.
5. Use of the anti-CDK 5 nanobody of claim 1 in the preparation of a medicament for inhibiting the growth of solid tumor in mouse melanoma.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111454355A (en) * | 2020-04-26 | 2020-07-28 | 山西农业大学 | SOX6 nano antibody and application thereof |
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| WO2010034863A1 (en) * | 2008-09-23 | 2010-04-01 | Consejo Superior De Investigaciones Científicas (Csic) | Use of kinase inhibitors for the preparation of pharmaceutical compositions for treatment of parkinson’s disease, pharmaceutical compositions and diagnostic procedure for parkinson’s disease |
| CN108635579A (en) * | 2018-06-06 | 2018-10-12 | 暨南大学 | Anti-human bFGF nano antibodies are preparing the application in treating melanoma drug |
| CN109678962A (en) * | 2019-01-22 | 2019-04-26 | 山西农业大学 | A kind of Cdk5 nano antibody and screening technique |
| CN110215516A (en) * | 2018-03-02 | 2019-09-10 | 南京大学 | It is a kind of to inhibit the treatment of CDK5 synergetic immunity in the application inhibited in breast cancer |
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2020
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| WO2010034863A1 (en) * | 2008-09-23 | 2010-04-01 | Consejo Superior De Investigaciones Científicas (Csic) | Use of kinase inhibitors for the preparation of pharmaceutical compositions for treatment of parkinson’s disease, pharmaceutical compositions and diagnostic procedure for parkinson’s disease |
| CN110215516A (en) * | 2018-03-02 | 2019-09-10 | 南京大学 | It is a kind of to inhibit the treatment of CDK5 synergetic immunity in the application inhibited in breast cancer |
| CN108635579A (en) * | 2018-06-06 | 2018-10-12 | 暨南大学 | Anti-human bFGF nano antibodies are preparing the application in treating melanoma drug |
| CN109678962A (en) * | 2019-01-22 | 2019-04-26 | 山西农业大学 | A kind of Cdk5 nano antibody and screening technique |
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| Title |
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| 姬凯元等: "黑色素瘤细胞单域抗体文库的构建及鉴定", 《中国生物化学与分子生物学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111454355A (en) * | 2020-04-26 | 2020-07-28 | 山西农业大学 | SOX6 nano antibody and application thereof |
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