CN109678960A - A kind of MSLN specificity fluorescent probe and its application - Google Patents
A kind of MSLN specificity fluorescent probe and its application Download PDFInfo
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- CN109678960A CN109678960A CN201910038339.3A CN201910038339A CN109678960A CN 109678960 A CN109678960 A CN 109678960A CN 201910038339 A CN201910038339 A CN 201910038339A CN 109678960 A CN109678960 A CN 109678960A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
- A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0058—Antibodies
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Veterinary Medicine (AREA)
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- Animal Behavior & Ethology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Materials Engineering (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of MSLN specificity fluorescent probe and its applications to be connected to the anti-MSLN single-chain antibody (Anti-MSLN-ICG) of fluorescent dye ICG-Sulfo-Osu including the single-chain antibody of anti-MSLN.Wherein, fluorescent dye indocyanine green ICG is connect by sulfo group Sulfo with the amido functional group-NH2 on single-chain antibody, realizes the fluorescent marker of single-chain antibody.MSLN specific single-chain antibody fluorescence probe provided by the invention, specific good, high sensitivity, no cytotoxicity are easy to remove, and immunogenicity is smaller in serum, are not easy to cause specific reaction in vivo since the molecular weight of single-chain antibody is smaller；In addition, MSLN specificity fluorescent probe of the invention also has the advantages that interference is small, penetration power is strong, it is suitable for clinical application.
Description
Technical field
Present invention design is related to optical molecular imaging field, and in particular to fluorescence probe field, in particular to a kind of MSLN
Specificity fluorescent probe and its application.
Background technique
Oophoroma is the malignant tumour of female reproductive system, and the death rate occupy the first place of female gynecological tumour.Due to its hair
Sick position is hidden, and diagnostic means lack, and 5 years survival rates of ovarian cancer patients are only 20%.It is early diagnosed, and is to be related to patient
One of an important factor for prognosis.
Mesothelin (mesothelin, MSLN) is a kind of tumour expressed on the mesothelial cell of pleura, peritonaeum and pericardium
Differentiation antigen becomes the reason of diagnosing tumor, treatment due to the characteristic of its differential expression in tumor tissues and normal tissue
Think target.About in 70% oophoroma, mesothelin is in high expression status.
Optical imagery is widely used in tumor research field with advantages such as its Non-Invasive, real-time, resolution ratio height, can be to swollen
Tumor is early diagnosed, and reflects tumour anatomical structure and metabolic condition.Indocyanine green (IndoCyanine Green, ICG) is
Be usually used in the non-specific fluorescence probe of medical diagnosis, be mainly used at present cardiac function measurement, retinal vessel radiography and
Liver function measurement.But ICG can with the nonspecific combination of albumin in blood plasma, therefore significantly limit its
Effect in clinical diagnosis.
Summary of the invention
In order to solve the problems in the existing technology, the present invention provides a kind of MSLN specificity fluorescent probe and its answers
With, solve the problems, such as in the prior art fluorescence probe in the diagnosing and treating of oophoroma using limited.
The technical scheme is that
The present invention provides the fluorescence probes of the anti-mesothelin of specificity (MSLN) albumen, including the list of anti-MSLN
Chain antibody is connected to the anti-MSLN single-chain antibody of fluorescent dye ICG-Sulfo-Osu.Wherein, fluorescent dye indocyanine green ICG is logical
It crosses sulfo group Sulfo to connect with the amido functional group-NH2 on single-chain antibody, realizes the fluorescent marker of single-chain antibody.
In another preferred embodiment of the present invention, the anti-MSLN single-chain antibody is as shown in SEQ ID NO.1
Nucleic acid sequence encoding.
In another preferred embodiment of the present invention, the affinity coefficient of the MSLN specificity fluorescent probe is
100nM。
The present invention also provides use of the MSLN specificity fluorescent probe in the reagent of preparation diagnosis and treatment oophoroma
On the way.
The present invention also provides a kind of pharmaceutical compositions, it includes the MSLN specificity fluorescent probe and pharmaceutically may be used
The carrier of receiving.
The present invention also provides a kind of kits, and it includes the MSLN specificity fluorescent probes.
Term as used herein " kit " includes MSLN specificity fluorescent probe and operation instructions of the invention.It should
Kit can further include at least one other reagent.Kit generally includes a label, and reagent is illustrated in brief
The purpose purposes of box content.
The invention has the benefit that MSLN specificity fluorescent probe specificity provided by the invention is good, and high sensitivity, nothing
Cytotoxicity is easy to remove in serum, and immunogenicity is smaller since the molecular weight of anti-MSLN single-chain antibody is smaller,
It is not easy to cause specific reaction in vivo；In addition, MSLN specificity fluorescent probe of the invention also has, interference is small, penetration power is strong
The advantages of, the diagnosing and treating application suitable for clinical oophoroma.
Detailed description of the invention
Fig. 1 is the SDS-PAGE result after each expression condition bacterial strain ultrasonication；
Wherein, the screening of Figure 1A -1, A-2 are inducing temperature when being 25 DEG C expression condition；Figure 1B -1, B-2 are inducing temperature
The screening of expression condition when being 37 DEG C, it is 25 DEG C that duct 1-3, which is inductive condition respectively, in Figure 1A -1, induces 1h, IPTG concentration is
Albumen (Before) before the induction of 0.5mM, total protein (Total) after induction, soluble protein (Soluble) after induction；Duct 4-6
Be respectively inductive condition be 25 DEG C, induce 1h, IPTG concentration be 1mM induction before albumen (Before), total protein after induction
(Total), soluble protein (Soluble) after induction；It is 25 DEG C that duct 7-9, which is inductive condition respectively, in Figure 1A -2, induces 2h,
Albumen (Before) before the induction that IPTG concentration is 0.5mM, total protein (Total) after induction, soluble protein after induction
(Soluble)；Duct 10-12 is that inductive condition is 25 DEG C respectively, induces 2h, albumen before the induction that IPTG concentration is 1mM
(Before), total protein (Total) after induction, soluble protein (Soluble) after induction；Duct 1-3 is lured respectively in Figure 1B -1
Conducting bar part is 37 DEG C, induces 1h, and IPTG concentration is albumen (Before) before the induction of 0.5mM, total protein (Total) after induction,
Soluble protein (Soluble) after induction；Duct 4-6 is that inductive condition is 37 DEG C respectively, induces 1h, and IPTG concentration is luring for 1mM
Leading albumen (Before), total protein (Total) after induction, soluble protein (Soluble) after induction；Duct 7-9 in Figure 1B -2
Be respectively inductive condition be 37 DEG C, induce 2h, IPTG concentration be 0.5mM induction before albumen (Before), total protein after induction
(Total), soluble protein (Soluble) after induction；Duct 10-12 is that inductive condition is 37 DEG C respectively, induces 2h, IPTG concentration
For albumen (Before) before the induction of 1mM, total protein (Total) after induction, soluble protein (Soluble) after induction；
Fig. 2 is in buffer；
Wherein, Fig. 2A -1 is the SDS-PAGE result of not urea-containing elution albumen and Fig. 2A -2 is without urea
Elution albumen His-tag Western-blot result.Fig. 2 B-1 is the elution albumen of the urea containing 8M
SDS-PAGE result and for the urea containing 8M elution albumen His-tag Western-blot result.Swimming lane point
Dui Ying not are as follows: M:loading marker；1:before；2:total；3:soluble；4:Flow-through；16:Wash
1；7:Wash 2；8:Elution 1；9:Elution 2；10:Elution 3；11:Elution 4；
Fig. 3 is verifying SDS-PAGE and Western-blot result after protein renaturation；
Wherein, Fig. 3 A is the eluent of the urea containing 8M, the SDS-PAGE result of protein renaturation after dialysed overnight.Fig. 3 B be containing
The eluent of 8M urea, the Western-blot result of the detection HIS-tag of protein renaturation after dialysed overnight.Swimming lane respectively corresponds
Are as follows: M:loading marker；1: the eluent after renaturation；
Fig. 4 is the result that ELISA detects anti-MSLN single-chain antibody affinity；
Fig. 5 is the result that CELL ELISA detects anti-MSLN single-chain antibody affinity；
Fig. 6 is that Anti-MSLN-ICG mouse tumor targets result.
Specific embodiment
Technical solution of the present invention is described below in conjunction with specific embodiment, described embodiment is only this
Invention a part of the embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art exist
Every other embodiment obtained under the premise of creative work is not made, shall fall within the protection scope of the present invention.
The expression of the anti-MSLN single-chain antibody of embodiment 1 is screened with expression condition
Its sequence of the nucleic acid of anti-MSLN single-chain antibody is encoded as shown in SEQ ID NO.1.Plasmid is converted into bacillus coli DH 5
α competent cell.After the monoclonal colonies grown on picking plate, plasmid is extracted.
Above-mentioned plasmid is converted into e. coli bl21 D3, bed board is simultaneously incubated overnight.Picking single colonie, with 5ml LB culture medium
It is resuspended, is placed in 37 DEG C of overnight incubations.In the ratio of 1:100 by above-mentioned 1ml media transfer to the LB culture medium equipped with 100ml
In flask, it is placed in 37 DEG C of shaking tables and cultivates 2 hours.IPTG induction is added in the ratio of 1:1000 and (IPTG final concentration is calculated
It is used as induction group for 0.5mM or 1mM), setting does not add the control group of IPTG, 37 DEG C or 25 DEG C cultures.Under the conditions of 4 DEG C with
The revolving speed of 9000rpm is centrifuged 5min, collects thallus.It washed once with 40ml PBS buffer solution, and matched thallus with 2ml buffer
At suspension.The container of thallus suspension is placed in ice bath, using ultrasonic disruption, power 30% is set, is crushed time 1s, interval
Time 1s runs 1min, and the control group bacterium solution after ultrasound, which is used as, induces preceding total protein (Before), the induction group bacterium solution after ultrasound
As total protein after induction (Total).10min is centrifuged with 15000rpm under the conditions of 4 DEG C, collects supernatant (Soluble) and thallus.
10 μ l Before, Total and Soluble is taken to carry out SDS-PAGE, the result is shown in Figure 1 respectively, it can be seen that have inside Total big
The inducible protein of amount exists, and without corresponding albumen in Soluble, it was demonstrated that inducible protein exists with inclusion bodies.Most
Selected 37 DEG C of 0.5mM IPTG concentration induction 2h carries out inclusion body purification and renaturation as inductive condition eventually.
The purifying and renaturation of the expression albumen of embodiment 2
Respectively using urea and not urea-containing buffer resuspension thallus is contained, it is (pre- that Ni+ column is added according to the ratio of 1:0.4
First magnetic bead is cleaned with PBS buffer solution), it overturns under the conditions of 4 DEG C and combines 1h.3min is centrifuged with 15000rpm in centrifuge, takes it
Supernatant 20ul is as Flow-through.Include with 3ml or the cleaning solution not comprising urea washs magnetic bead 2 times.Include with 2ml
Or the eluent of the 8M urea not comprising urea washs 4 times, and destination protein is eluted.Protein purification result is shown in Fig. 2.It will
Eluent after 4 washings merges.Under the conditions of 4 DEG C, above-mentioned eluent is placed in 5LPBS buffer the 48h that dialyses.It will be different
The inclusion body protein of condition renaturation carries out the Western-blot detection of SDS-PAGE and HIS-tag, as a result sees Fig. 3, it is seen that multiple
Purity of protein is higher after surname, almost without impurity presence.Measuring protein concentration is 1 μM.
Embodiment 3ELISA detects the affinity of anti-MSLN single-chain antibody
MSLN albumen is diluted to 0.25mg/ μ l, 100 μ l are added in every hole, and 4 DEG C of refrigerators are incubated overnight.Take out 96 within second day
Orifice plate carefully sops up the liquid in ELISA Plate with sample loading gun, again pats several times ELISA Plate against blotting paper after blotting only, pats
After observe ELISA Plate, liquid is not evident that in hole until looking at.It is washed twice with 0.1%PBS, each 10min, every hole
350ul.It is closed with 1%BSA, every hole 150ul, room temperature shakes 1h slowly.Every hole be added suitable concentration (0.001nM, 0.01nM,
0.1nM, 1nM, 10nM, 100nM, 1 μM) anti-MSLN single-chain antibody, be incubated for 1h under room temperature.It is washed again with 0.1%PBST
Twice, each 10min, every hole 350ul.Anti-his-HRP is added, is incubated at room temperature 1h.0.1%PBST is washed twice again, every time
10min, every hole 350ul.100 μ l TMB liquid are added in every hole, are incubated under the conditions of 37 DEG C and are no more than 30min.It is whole that 100 μ l are added in every hole
Only liquid detects OD value using Bioteck colour comparatour.Fig. 4 shows the affinity testing result of anti-MSLN single-chain antibody, it is seen that anti-
MSLN single-chain antibody has high affinity, and affinity costant reaches 100nM.
Embodiment 4CELL ELISA detects the affinity of anti-MSLN single-chain antibody
It is 96 orifice plate bed boards, every 50 μ l of hole with 2% gelatin.It is incubated for 30 to 60 minutes at 37 DEG C.It pats and discards gelatin liquid.
96 orifice plates are added in lung cancer cell line A549, every hole cell number is about 5 × 104.37 DEG C of overnight incubations.Next day takes out, and is washed with PBS
It washs cell 3 times.The anti-MSLN that suitable concentration (0.001nM, 0.01nM, 0.1nM, 1nM, 10nM, 100nM, 1 μM) is added in every hole is mono-
Chain antibody is incubated for 1h under the conditions of 4 DEG C.It is washed cell 3 times with PBS again.Anti-his-HRP is added, is incubated for 1h under the conditions of 4 DEG C.
It is washed cell 3 times with PBS again.100 μ l TMB liquid are added in every hole, are incubated under the conditions of 37 DEG C and are no more than 30min.Every hole is added
100 μ l terminate liquids detect OD value using bioteck colour comparatour.Fig. 5 shows the affinity detection knot of anti-MSLN single-chain antibody
Fruit, it is seen that anti-MSLN single-chain antibody has high affinity, and affinity costant reaches 41nM.
The combination of embodiment 5 fluorescent dye ICG and anti-MSLN single-chain antibody
Fluorescent dye indocyanine green ICG-Sulfo-Osu passes through the amido functional group-on sulfo group sulfo and single-chain antibody
NH2 connection.
Specific step is as follows:
The ratio of 1mM single-chain antibody, Mixed LB films G- are corresponded to according to every 10mM fluorescent dye indocyanine green ICG-Sulfo-Osu
Sulfo-Osu and anti-MSLN, wherein ICG-Sulfo-Osu is diluted to final concentration 1mM by DMSO, and anti-MSLN concentration is 1 μ
M.Slowly concussion mixes 2 hours mixed liquor at room temperature.It is placed in 4 DEG C of dialysed overnights in 5LPBS buffer.Obtain anti-MSLN-
ICG。
The foundation of 6 animal model of embodiment
With 2 × 105Ovarian cancer cell line A2780 subcutaneous injection be subcutaneously used to establish ovary in Immune deficient mice B-NDG
Cancer solid tumor mouse model.The mouse injected is continued to raise in SPF grades of animal houses, about 10 days or so strong point 2cm or so
Solid tumor.
The application zoopery detection 78C-PEG-ICG specificity of embodiment 7
The mouse of long tumor is divided into control group and two groups of Anti-MSLN-ICG, respectively by the PBS after being dialyzed overnight
Anti-MSLN-ICG after buffer and dialysis is entered in Mice Body by tail intravenous, keeps antibody finally dense in Mice Body
Degree is 1 μM.After for 24 hours, mouse tumor position is detected using small animal living body imager.After for 24 hours, target can be obviously observed
To the fluorescence (Fig. 6) in tumour.Prove the animal model for the identification MSLN that constructed fluorescence probe can be specific.
Invention applies specific embodiment, principle and implementation of the present invention are described, above embodiments
Illustrate to be merely used to help understand method and its central idea of the invention.It should be pointed out that for the ordinary skill people of this field
Member for, without departing from the principle of the present invention, can with several improvements and modifications are made to the present invention, these improve and
Modification also falls into the protection of the claims in the present invention.
<110>Medical University Of Tianjin
<120>a kind of MSLN specificity fluorescent probe and its application
<130>
<160> 2
<170>
<210> 1
<211> 723
<212> DNA
<213>artificial sequence
<400> 1
atgcaggtac aactgcagca gtctgggcct gagctggaga agcctggcgc ttcagtgaag 60
atatcctgca aggcttctgg ttactcattc actggctaca ccatgaactg ggtgaagcag 120
agccatggaa agagccttga gtggattgga cttattactc cttacaatgg tgcttctagc 180
tacaaccaga agttcagggg caaggccaca ttaactgtag acaagtcatc cagcacagcc 240
tacatggacc tcctcagtct gacatctgaa gactctgcag tctatttctg tgcaaggggg 300
ggttacgacg ggaggggttt tgactactgg ggccaaggga ccacggtcac cgtctcctca 360
ggtgtaggcg gttcaggcgg cggtggctct ggcggtggcg gatcggacat cgagctcact 420
cagtctccag caatcatgtc tgcatctcca ggggagaagg tcaccatgac ctgcagtgcc 480
agctcaagtg taagttacat gcactggtac cagcagaagt caggcacctc ccccaaaaga 540
tggatttatg acacatccaa actggcttct ggagtcccag gtcgcttcag tggcagtggg 600
tctggaaact cttactctct cacaatcagc agcgtggagg ctgaagatga tgcaacttat 660
tactgccagc agtggagtgg ttaccctctc acgttcggtg ctgggacaaa gttggaaata 720
aaa 723
Claims (5)
1. a kind of MSLN specificity fluorescent probe, including anti-MSLN single-chain antibody, fluorescent dye ICG connection single-chain antibody Anti-
MSLN-ICG, wherein fluorescent dye ICG-Sulfo-Osu passes through the amido functional group-NH2 on sulfo group Sulfo and single-chain antibody
Connection, realizes the fluorescent marker of single-chain antibody.
2. MSLN specificity fluorescent probe according to claim 1, the nucleic acid sequence such as SEQ of the anti-MSLN single-chain antibody
Shown in ID NO.1.
3. use of the MSLN specificity fluorescent probe according to claim 1 in the reagent of preparation diagnosis and treatment oophoroma
On the way.
4. a kind of pharmaceutical composition, it includes MSLN specificity fluorescent probes described in claim 1 and pharmaceutically acceptable
Carrier.
5. a kind of kit, it includes MSLN specificity fluorescent probes described in claim 1.
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ID=66193305
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116019939A (en) * | 2022-11-24 | 2023-04-28 | 华中科技大学同济医学院附属协和医院 | Molecular probe targeting MSLN and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116019939A (en) * | 2022-11-24 | 2023-04-28 | 华中科技大学同济医学院附属协和医院 | Molecular probe targeting MSLN and application thereof |
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Application publication date: 20190426 |