CN106806907B - TEM1 specific nuclear magnetic probe and application thereof - Google Patents

Abstract

The invention provides a TEM1 specific nuclear magnetic probe, which comprises an anti-TEM 1 single-chain antibody and an aminated SPIO nano-particle which are connected through a cross-linking agent, wherein the aminated SPIO nano-particle is connected with the cross-linking agent through an amido bond, and the C-terminal of the anti-TEM 1 single-chain antibody is cysteine and is connected with the cross-linking agent through a thioether bond. The invention also provides a preparation method of the TEM1 specific nuclear magnetic probe, application of the TEM1 specific nuclear magnetic probe in preparing a reagent for diagnosing ovarian cancer and a kit containing the TEM1 specific nuclear magnetic probe. The TEM1 specific nuclear magnetic probe provided by the invention has good specificity, high sensitivity, no cytotoxicity, easy removal in serum, small immunogenicity and difficult initiation of specific reaction in vivo; in addition, the small molecular weight of the antibody makes it easy to enter the periphery of solid tumors, and the binding efficiency with receptors is high, so that sufficient image contrast can be obtained.

Description

TEM1 specific nuclear magnetic probe and application thereof

Technical Field

The invention relates to the field of molecular imaging, in particular to the field of nuclear magnetic probes, and particularly relates to a TEM1 specific nuclear magnetic probe and application thereof.

Background

Ovarian cancer is a malignant tumor of a female reproductive system, has high mortality rate and is the first of gynecological cancers, and an effective early diagnosis method is lacked at present due to the hidden onset and atypical clinical symptoms and signs. The ultrasonic examination is the most common method for detecting ovarian cancer, the accuracy is higher than that of CT, but the imaging influence factors are more, such as the body type, the thickness of the abdominal wall, the depth of the tumor position, ascites influence and the like of a patient, and the patient is often confused with the intestinal cavity, so that missed diagnosis is easy to occur. CT has better positioning diagnosis on ovarian tumor, but has higher accuracy in the aspect of diagnosing cystic teratoma, and has lower identification accuracy on other ovarian tumors than B-mode ultrasound. MRI (magnetic resonance imaging) is a non-invasive, non-radiative imaging method, but MRI is non-specific and suffers from the size of magnetic nanoparticles, making the results unstable.

It has been newly found that tumor endothelial cell markers (TEMs) exist in tumor tissues, which are expressed in normal tissues in small amounts or are not expressed, and can be good tumor markers. The study demonstrated that endosialin (TEM1/endosialin/CD248) is highly expressed in ovarian cancer tissues, and is significantly different from other organs in vivo.

The core of the technology is different kinds and sizes of molecular probes with cell targeting function, including proteins, antibodies, polypeptides, chemical small molecules, etc. The application of antibody targeting technology in the field of tumor detection is receiving more and more attention. However, because of its large molecular weight, the antibody is not easily cleared in serum, and the antibody, as an exogenous protein, has a large immunogenicity and is liable to cause a specific reaction in vivo; on the other hand, the large molecular weight of antibodies makes them less likely to enter the periphery of solid tumors, affecting their binding efficiency to receptors, and making it difficult to obtain sufficient image contrast.

Disclosure of Invention

In order to solve the above problems, the present invention provides a TEM 1-specific nuclear magnetic probe, which includes an anti-TEM 1 single-chain antibody and an aminated SPIO (superparamagnetic iron oxide) nanoparticle connected by a cross-linking agent, wherein the aminated SPIO nanoparticle is connected to the cross-linking agent by an amide bond, and the C-terminal of the anti-TEM 1 single-chain antibody is cysteine and is connected to the cross-linking agent by a thioether bond.

In a preferred embodiment of the invention, the amino acid sequence of the anti-TEM 1 single chain antibody is shown in SEQ id No. 2.

In another preferred embodiment of the invention, the anti-TEM 1 single chain antibody is encoded by the nucleic acid sequence shown in SEQ ID No. 1.

The nucleic acid encoding the anti-TEM 1 single-chain antibody of the present invention can be synthesized by a method known in the art, such as the solid-phase phosphoramidite method, etc., based on its sequence.

In another preferred embodiment of the present invention, the particle size of the aminated SPIO nanoparticles is 60-120nm, preferably 70-90 nm.

In another preferred embodiment of the invention, the lateral relaxation rate of the aminated SPIO nanoparticles is in the range of 70-90mM^-1S^-1。

In another preferred embodiment of the invention, the cross-linking agent is SMCC or sulfo-SMCC.

In another preferred embodiment of the present invention, said TEM 1-specific nuclear magnetic probe contains iron ions at a concentration of 0.15-2.2nM, preferably 0.5-2nM, more preferably 1-1.5 nM.

The invention also provides a preparation method of the TEM1 specific nuclear magnetic probe, which comprises the following steps:

(1) synthesizing an anti-TEM 1 single-chain antibody with cysteine at the C terminal, and then purifying and renaturing;

(2) synthesizing aminated SPIO nanoparticles;

(3) the anti-TEM 1 single chain antibody and the aminated SPIO nanoparticles were linked by a cross-linker.

The invention also provides application of the TEM1 specific nuclear magnetic probe in preparing a reagent for diagnosing ovarian cancer.

The invention also provides a kit comprising a TEM 1-specific nuclear magnetic probe.

The term "kit" as used herein includes the TEM 1-specific nuclear magnetic probe of the present invention and instructions for use. The kit can further comprise at least one additional reagent. The kit will generally include a label that briefly indicates the intended use of the contents of the kit.

The TEM1 specific nuclear magnetic probe provided by the invention has good specificity, high sensitivity and no cytotoxicity, and is easy to remove in serum and less in immunogenicity due to the small molecular weight of the anti-TEM 1 single-chain antibody, so that the specific reaction is not easy to initiate in vivo; in addition, the small molecular weight of the antibody makes it easy to enter the periphery of solid tumors, and the binding efficiency with receptors is high, so that sufficient image contrast can be obtained.

Drawings

FIG. 1 shows the results of clone verification of the constructed plasmid;

FIG. 2 shows the SDS-PAGE results after ultrasonication of each expression strain;

FIG. 3 shows SDS-PAGE results of different batches of renatured inclusion body proteins;

FIG. 4 shows the result of the affinity detection of the anti-TEM 1 single-chain antibody;

FIG. 5 is a DLS measurement of the particle size of aminated SPIO nanoparticles;

fig. 6 is a measurement of the transverse relaxation rate of aminated SPIO nanoparticles;

FIG. 7 is a standard curve for NP-linker-78C concentration determination using the Bradford protein assay kit;

FIG. 8 is the result of detection of the saturation curve of NP-linker-78C;

FIG. 9 shows the results of toxicity test of NP-linker-78C;

FIG. 10 shows the results of affinity assay of NP-linker-78C;

FIG. 11 shows the results of specific detection of NP-linker-78C;

FIG. 12 is a result of measuring the endocytosis capacity of NP-linker-78C;

FIG. 13 is a statistical analysis of the endocytosis assay of NP-linker-78C;

FIG. 14 is a method of fluorescence confocal measurement to verify that NP-linker-78C has endocytosis effect;

FIG. 15 shows the results of an MRI scan of NP-linker-78C binding specifically to antigen;

FIG. 16 is a result of statistical analysis of the ability of NP-linker-78C to specifically bind to an antigen.

Detailed Description

The technical solutions of the present invention will be described below with reference to specific embodiments, and the described embodiments are only a part of embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 vector construction and expression, purification and renaturation of anti-TEM 1 Single chain antibodies

The sequence of the nucleic acid for encoding the anti-TEM 1 single-chain antibody is shown in SEQ ID NO.1, the 3' end is a codon for encoding cysteine (Cys), and the codon is cloned on a PET302 vector by using restriction endonuclease Nsi I/Xho I to construct a complete plasmid. The plasmid was transformed into E.coli competent cells, plated and cultured overnight. After the colonies grow on the plate, the plasmid is extracted, PCR is carried out by using one primer on the exogenous gene and the other primer on the vector, the result of clone verification is shown in figure 1, and except that No.2 clone has no specific band, other clones have specific bands. Sequencing results also demonstrated successful vector construction.

Coli BL21D3 was transformed with the above plasmid, plated and cultured overnight. Single colonies were picked, resuspended in 5ml LB medium and incubated overnight at 37 ℃. 5ml of the above medium was transferred to a flask containing 500ml of LB medium at a ratio of 1:100, and cultured on a shaker at 37 ℃ for 2 hours. IPTG induction (final concentration of IPTG calculated to be 1mM) was added at a ratio of 1:1000 as an induction group, and a control group to which no IPTG was added was set and cultured at 37 ℃ for 2 hours. The cells were centrifuged at 4000rpm for 15 minutes at 4 ℃ to collect the cells. The cells were washed once with 40ml EQ buffer and suspended in 40ml EQ buffer containing 8M urea. And (3) placing a container of the thallus suspension in an ice bath, crushing by adopting ultrasonic waves, setting the power to be 40%, the crushing time to be 1 second, the intermittent time to be 2 seconds, operating for 6 minutes, taking the control group bacteria liquid after the ultrasonic waves as protein before induction, and taking the induction group bacteria liquid after the ultrasonic waves as total protein after induction. The cells were centrifuged at 4 ℃ for 15 minutes at maximum speed, and the supernatant (soluble protein after induction) and the cells were collected. And respectively taking 10 mu l of protein before induction, total protein after induction and soluble protein after induction to perform SDS-PAGE, wherein the result is shown in figure 2, and the result shows that a large amount of induced protein exists in the total protein after induction, but no corresponding induced protein exists in the soluble protein after induction, thereby proving that the induced protein exists in an inclusion body form. Thus inclusion body purification and renaturation were performed as follows.

The cells were resuspended in PBS, 3ml of a magnetic bead solution (beads were washed with 10ml of EQ buffer in advance) was added thereto, and binding was carried out at 4 ℃ for 2 hours. The beads were washed 2 times with 20ml of wash solution containing 8M urea. The target protein 78C was eluted by washing 2 times with 3ml of an eluent containing 8M urea. The eluates after 2 washes were combined in total 6ml and made up to 25ml with EQ buffer containing 8M urea. The above 25ml suspension was dialyzed overnight at 4 ℃ in 1L of PBS buffer. Different batches of renatured inclusion body protein (batches 1-12) were subjected to SDS-PAGE in amounts of 5. mu.l or 10. mu.l. As a result, as shown in FIG. 3, it can be seen that the protein was highly pure with almost no impurities present. The protein suspension was concentrated and centrifuged using a 50ml concentration tube from Millipore to concentrate the protein concentration to 1 mg/ml.

Example 2 ELISA detection of affinity of anti-TEM 1 Single chain antibodies

96 well plates were plated with 50. mu.l each using 2% gelatin. Incubate at 37 ℃ for 30 to 60 minutes. MS1-TEM1 cells (mouse islet endothelial cells transfected with TEM1 plasmid and capable of highly expressing TEM1 protein) were added to 96-well plates at about 10 cells per well⁴-10⁵. Incubated at 37 ℃ overnight. Cells were washed 3 times with PBS solution. anti-TEM 1 single-chain antibody was added to each well at an appropriate concentration (0.0001nM, 0.001nM, 0.01nM, 0.1nM, 1nM, 10nM, 100nM, 1mM, 10mM) and incubated at 4 ℃ for 1 hour. The cells were washed again 3 times with PBS. anti-His-HRP secondary antibody was added and incubated at 4 ℃ for 1 hour. The cells were washed again 3 times with PBS. Mu.l of TMB solution was added to each well and incubated at 37 ℃ for no more than 30 minutes. Mu.l of stop solution was added to each well, and OD was measured using a bioteck colorimeter. FIG. 4 shows the result of the affinity detection of the anti-TEM 1 single-chain antibody, and it can be seen that the anti-TEM 1 single-chain antibody has very high affinity and an affinity constant of 3.941 nM.

Example 3 Synthesis of SPIO nanoparticles

A250 ml flask was taken and a stirrer was placed inside the flask. 12.5g of dextran were added. 25ml of deionized water were added while a heatable stirring stand (ika RCT basic type heating magnetic stirrer) was prepared, and the stirrer speed was set to 4 stages. The temperature of the stirrer is adjusted to 120 ℃ andthe flask was then sealed with aluminum foil and placed in a glass container filled with dimethicone, and the stirrer was turned on for 1 hour (this step was also chosen overnight, with the stirrer temperature set at 75-80 ℃ to ensure that it did not exceed 100 ℃). After 1 hour, the heating was stopped and the mixture was allowed to cool naturally. The dextran solution was filtered through a 0.22 μm filter and then allowed to mix overnight. The overnight dextran solution was passed through nitrogen (N)₂) And (5) cleaning. The washing process was continued for 30 minutes, and after the first 20 minutes, the glass container was filled with crushed ice. (2 needles were inserted into the rubber cap of the flask, one of which was connected to nitrogen, the bottom of which was attached to the wall of the flask and submerged in the solution, and the other was connected to outside air to maintain the pressure in the flask balanced

Weigh 0.985g FeCl₃And 0.366g FeCl₂Put into 50ml centrifuge tubes, respectively, and 6.25ml deionized water was added. The solute is completely melted and mixed by rotary shaking. Followed by FeCl injection using a large syringe₃Solution and FeCl₂The solution was poured into the flask containing the dextran solution, ensuring that the syringe head entered the dextran solution at the time of pouring. (note: when weighing ferric chloride, the amount of ferric chloride minus the mass of 60mg of rare metal if additional rare metal is needed later.) the resulting mixed solution was placed on ice and purged again with nitrogen for 45 to 60 minutes. The Kds elution apparatus (model bole 422, usa) was started and 15ml of ammonium hydroxide solution was prepared in the syringe. The ammonium hydroxide solution will be added to the solution obtained in step 7, the addition process follows the following steps (due to instability of the nanoparticle solution, time concerns and timely rate adjustment): (1) the machine rate was adjusted to 1 drop every 3 to 4 minutes for 1 hour; (2) adjusting the machine speed to 2 times the previous step (1) for 30 minutes; (3) adjusting the machine speed to 3 times that of step (1) for 30 minutes; (4) adjusting the machine speed to 4 times that of step (1) for 30 minutes; (5) the machine rate was adjusted to 5ml per hour for 15 minutes; (6) the rate was adjusted to 4 to 5ml per hour and prepared 15ml ammonium hydroxide solution was added. After all, the flask with the solution was removed from the ice bath and stoppedAnd (5) nitrogen purification. The flask was placed in a dry bath at 90 ℃ for 1 hour. Stir at room temperature overnight.

The nanoparticle solution was centrifuged at 20000g for 30 minutes at 4 ℃. After Centrifugation, the supernatant was transferred to an ultrafiltration centrifuge tube (Amicon Ultra Centrifugation tubes) of 100kd MW. Centrifuged at 2000 rpm overnight at 4 ℃. After centrifugation, the nanoparticles will be stored in the upper solution of the ultrafiltration tube. All the solution in the upper part was collected as much as possible and placed in a 50ml Erlenmeyer flask. The method comprises the following steps of transferring residual nanoparticles by using a citrate buffer solution (the bottom of a filter is V-shaped, and the residual nanoparticles are easy to remain, sucking 5ml of citrate buffer solution (35 g of sodium chloride, 23.5g of dehydrated sodium citrate and 4L of deionized water) by using a liquid transfer machine to blow the residual nanoparticles, sucking out all the solution, filtering the nanoparticle solution, flushing the filtering system by using the citrate buffer solution firstly to ensure that waste liquid enters a waste liquid bottle, flushing the filtering system by using the citrate buffer solution firstly, wherein the pressure cannot exceed 20psi, adding the synthesized SPIO nanoparticles into the citrate buffer solution when the volume reaches 25ml, clarifying the waste liquid, finishing filtering, and flushing the filtering system by using 2L of the citrate buffer solution after the filtering is finished.

Example 4 SPIO nanoparticles plus amino tails

25ml of the SPIO nanoparticle solution prepared in example 3 was taken and added to a 500ml triangular flask, 25ml of 10M NaOH was added, and then the mixed liquid was uniformly stirred on a stirrer for 10 minutes. After 10 minutes, 50ml of epichlorohydrin were added, and the bottle was closed with a foil paper and then stirred on a stirrer overnight. The next day, the mixture was transferred to a 50ml centrifuge tube and centrifuged at 2000g for 10 minutes. After centrifugation, the uppermost epichlorohydrin layer was removed using a glass tube, and the lower SPIO nanoparticles were then transferred again to a new 500ml Erlenmeyer flask. The same volume of ammonium hydroxide was added to the same flask and then stirred overnight with an open mouth. The next day, the now aminated SPIO nanoparticles were continued to be stirred overnight at 35 ℃ while the bottle was capped with tinfoil. The aminated SPIO nanoparticles were filtered using 0.45 μm and 0.22 μm filter tubes, respectively. And after the ammonia gas is volatilized, filtering and purifying the aminated SPIO nano particles by using a filtering system again, wherein the volume of the finally obtained aminated SPIO nano particles needs to be reduced to 5ml at most.

Example 5 characterization of aminated SPIO nanoparticles

Taking 100 μ l of aminated SPIO nanoparticle solution, performing Dynamic Light Scattering (DLS) measurement by using a Malvern HPPS5001 instrument for 3 times, and taking an average value to analyze the particle size of the aminated SPIO nanoparticles. As can be seen from fig. 5, the particle size of the aminated SPIO nanoparticles was around 80 nm.

The transverse relaxation rate (R2) of the aminated SPIO nanoparticles was measured using a Bruker (usa) 1.4T minispec mq60 NMR analyzer. Nanocluster samples were first diluted to different concentrations in the range of 0.001-10mM in iron concentration and thermostatted to 310K. 0.3mL of the solution was placed in a test tube and its T2 relaxation time was measured using a standard Carr-Purcell-Meiboom-Gill pulse sequence. And performing linear fitting by taking the measured T2 time as an ordinate and the iron concentration as an abscissa, and obtaining the slope of a straight line as the transverse relaxation rate of the sample. From FIG. 6, it can be seen that the transverse relaxation rate of the aminated SPIO nanoparticles is 80.48mM^-1S^-1。

Example 6 linking of aminated SPIO nanoparticles to anti-TEM 1 Single chain antibodies

(1) Conjugation of aminated SPIO nanoparticles with crosslinker sulfo-SMCC

Mu.g of aminated SPIO nanoparticles were added to 100. mu.l of sulfo-SMCC, and diluted with water to a final concentration of sulfo-SMCC of 4.8mg/ml, at which time the mixture had an excess of sulfo-SMCC. The mixture was left to bind at room temperature for 30 minutes with slow shaking. The desalting column (Thermo) was washed 2 times with water in advance and centrifuged at 1000g for 2 minutes. Excess sulfo-SMCC was removed in a desalting column using binding buffer (PBS). And slowly injecting the mixed solution into a desalting column, centrifuging at the rotating speed of 1000g for 2 minutes, and collecting the solution to obtain a connector of the aminated SPIO nano-particles and the sulfo-SMCC, which is called NP-linker for short.

(2) Mu.g of anti-TEM 1 single chain antibody was added to the NP-linker solution and bound with gentle shaking at room temperature for 2 hours, or overnight at 4 ℃. The linker of NP-linker and anti-TEM 1 single-chain antibody is abbreviated as NP-linker-78C.

(3) Add 100. mu.l of freshly prepared cysteine solution at 20mM concentration and bind slowly with shaking at room temperature for 1 hour to block the remaining maleimide groups on the sulfo-SMCC.

(4) The magnetic column (Invitrogen) was washed 2 times with binding buffer in advance.

(5) NP-linker-78C was purified by magnetic column. Add binding buffer and NP-linker-78C to the magnetic column, centrifuge and purify, finally adjust the NP-linker-78C concentration to 40. mu.g/ml.

Example 7 detection of NP-linker-78C saturation Curve

NP-linker-78C prepared in example 6 was diluted in equal proportion, and a standard curve for detecting NP-linker-78C concentration was established using the Bradford protein assay kit with aminated SPIO nanoparticles as a blank (FIG. 7). Single-chain antibodies against TEM1 at different concentrations (72. mu.g, 200. mu.g, 400. mu.g, 800. mu.g, 1600. mu.g) were added to 500ng of the aminated SPIO nanoparticles, respectively, and the reaction was performed. After the reaction was completed, the protein concentration was measured using the Bradford protein assay kit. As can be seen from FIG. 8, the concentration of NP-linker-78C increased as the concentration of the anti-TEM 1 scFv in the reaction system increased, but the tendency of the NP-linker-78C concentration increased decreased when the concentration of scFv in the system reached 800. mu.g.

Example 8 toxicity test of NP-linker-78C

Logarithmic phase growth of MS1 (mouse islet endothelial cells), MS1-TEM1 (mouse islet endothelial cells transfected with TEM1 plasmid and capable of highly expressing TEM1 protein) cells at 2X 10⁴The density of individual/ml was seeded in 96-well plates. The plates were incubated in a cell incubator for 24 hours. Nanoparticles containing different concentrations of iron ions were added to the cells separately and incubated overnight for 24 hours. Mu.l of 5mg/ml MTT was added to each well and incubation was continued for an additional 4 hours. Mu.l DMSO (Sigma-Aldrich) was added to each well and after 20 minutes detection was performed at 570 nm. Three replicates were performed. The results were statistically analyzed using the software One-way ANOVA and Turkey test. The results are shown in the figure9, when the concentration of iron ions in NP-linker-78C is less than 2.4nM, no obvious reduction occurs in either MS1 cells or MS1-TEM1 cells, i.e., no obvious death tendency occurs in the cells; however, when the concentration of iron reached 2.4nM, the cell viability of both cells decreased to 50% of the original value, indicating that the magnetic nanoparticles had strong cytotoxicity at high concentration, so the concentration of iron ions in the TEM 1-specific nuclear magnetic probe of the present invention was 0.15-2.2nM, preferably 0.5-2nM, more preferably 1-1.5 nM.

Example 9 affinity assay of NP-linker-78C

And performing affinity detection by using an ELISA method. First, 96-well plates were plated with 50. mu.l each using 2% gelatin. Incubate at 37 ℃ for 30 to 60 minutes. MS1-TEM1 cells were added to 96-well plates at approximately 10 cells per well⁴-10⁵. Incubated at 37 ℃ overnight. Cells were washed 3 times with PBS. Add appropriate concentration (10) to each well^-2nM、10^-1nM, 1nM, 10nM, 100nM, 1mM) NP-linker-78C, incubated at 4 ℃ for 1 hour. The cells were washed again 3 times with PBS. anti-His-HRP secondary antibody was added and incubated at 4 ℃ for 1 hour. The cells were washed again 3 times with PBS. 100. mu.l of TMB was added to each well and the incubation time at 37 ℃ was not more than 30 minutes. Mu.l of stop solution was added to each well, and OD was measured using a bioteck colorimeter. It was confirmed by the above cell ELISA assay that the affinity of the final NP-linker-78C was 196.8nM when 200. mu.g of the single-chain antibody was contained in the reaction system (FIG. 10A), and that the affinity of NP-linker-78C was 56.72nM when 400. mu.g of the single-chain antibody was contained in the reaction system (FIG. 10B).

Example 10 specific detection of NP-linker-78C

75cm full of cells²6ml of Versene solution was added to the cell culture dish and digested at 37 ℃ for 10 minutes. 6ml of cell culture medium was added to resuspend the cells, and the cells were centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in 7ml of flow buffer (FACS buffer), gently pipetted well, and 1ml per tube into 7 flow tubes. After 2ml of buffer was added to each tube and mixed, the mixture was centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. Add 100. mu.l of 50mM antibody diluted in flow buffer. Binding was incubated at 4 ℃ for 1 hour (to avoid endocytosis of the antibody by the cells). 3ml of buffer solution was added and mixed, centrifuged at 2000 rpm for 5 minutes and the supernatant was discarded. Washed again 2 times with 3ml buffer. Mu.l of murine His antibody was added to each tube and incubated at 4 ℃ for 1 hour for binding. Wash 2 times with 3ml buffer. Mu.l of murine APC antibody was added to each tube and incubated at 4 ℃ for 1 hour for binding. Wash 2 times with 3ml buffer. Add 300. mu.l of the visual dead cell probe solution and mount it on the machine. As can be seen from FIG. 11, NP-linker-78C retains its ability to specifically bind to antigen, while the aminated SPIO does not bind to antigen, thus excluding the binding of antigen to NP-linker-78C due to the effects of nanoparticle adhesion, etc.

Example 11 detection of the endocytosis Capacity of NP-linker-78C

2 pieces of 24-well plates were prepared, and a cover slip was put into each well of the 24-well plates. 2X 10 addition per well⁴The individual cells were cultured overnight. The mouse His antibody and the mouse APC antibody which are combined in advance are added into each well, and 2 pieces of 24-well plates are respectively placed at 37 ℃ and 4 ℃ for incubation for 2 hours to combine the cells and the antibodies. Wash 3 times with PBS. Add 100. mu.l of 1% formaldehyde per well and incubate for 30 min at room temperature. Wash 3 times with PBS. Blocking was performed with 100. mu.l of 5% fetal Bovine Serum (BSA) at 37 ℃ for 1 hour. Mu.l of cell membrane green fluorescent probe (DIOc18(3)) was added to each well and incubated for 30 minutes at a final concentration of 10. mu.g/ml in DIOc18 (3). Wash 3 times with PBST for 10 min each. Mu.l of DAPI were added to each well and incubated at room temperature for 5 minutes, with a final concentration of 0.1. mu.g/ml of DAPI. Wash 3 times with PBST for 10 min each. The slides were mounted with 3. mu.l glycerol. As can be seen from FIG. 12, NP-linker-78C still retained endocytosis. FIG. 13 is a statistical analysis of three experiments demonstrating that NP-linker-78C has endocytosis.

In the experiment, a fluorescence confocal method is also applied to verify that the NP-linker-78C has an endocytosis effect. Firstly, the corresponding antibody is pre-reacted, then the binding experiment is carried out with the corresponding cell under the conditions of 4 ℃ and 37 ℃, and the results in FIG. 14 show that a plurality of fluorescent signals exist in the cell under the condition of 37 ℃, and no corresponding fluorescent signal exists in the cell under the condition of 4 ℃, so that the NP-linker-78C is proved to have the endocytosis effect again.

EXAMPLE 12 cellular assay for MRI

75cm full of cells (MS1, MS1-TEM1)²6ml of Versene solution was added to the cell culture dish and digested at 37 ℃ for 10 minutes. 6ml of cell culture medium was added to resuspend the cells, and the cells were centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in 7ml of flow buffer (FACS buffer), gently pipetted well, and 1ml per tube into 7 flow tubes. After adding 2ml of FACS buffer to each tube and mixing, the mixture was centrifuged at 2000 rpm for 5 minutes and the supernatant was discarded. Add 100. mu.l of aminated SPIO and NP-linker-78C diluted in FACS buffer at a concentration of 50 mM. Binding was incubated at 37 ℃ for 1 hour. 3ml of buffer solution was added and mixed, centrifuged at 2000 rpm for 5 minutes and the supernatant was discarded. Washed again 2 times with 3ml buffer. Mu.l of murine His antibody was added to each tube and incubated at 4 ℃ for 1 hour for binding. Wash 2 times with 3ml buffer. Mu.l of murine antibody APC was added to each tube and incubated at 4 ℃ for 1 hour for binding. Wash 2 times with 3ml buffer. Washed cells were resuspended in PBS and placed in a plate dedicated to cellular MRI.

Specificity of NP-linker-78C was examined on the MRI level using a cell-pellet assay. Respectively combining NP-linker-78C and anti-TEM 1 single-chain antibody which is not combined with the NP-linker with cells, reacting at 37 ℃, then removing the anti-TEM 1 single-chain antibody which is not combined with the cells by centrifuging and eluting the cells, then placing the rest cells on a corresponding cell plate, and then placing the cell plate in an MRI instrument for detection. In the experiment, the negative is bright color, the darker the color is, the more magnetic nanoparticles are bound to the cells are shown, fig. 15 is a corresponding MRI result graph, fig. 16 is a statistical analysis of the experiment result, and as can be seen from the result, NP-linker-78C has the property of specifically binding to the antigen, and the property is also detected on the MRI detection level.

The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its central concept. It should be noted that it would be apparent to those skilled in the art that various changes and modifications can be made in the invention without departing from the principles of the invention, and such changes and modifications are intended to be covered by the appended claims.

Sequence listing

<110> Tianjin medical university

<120> TEM1 specific nuclear magnetic probe and application thereof

<130>PP169733

<160>2

<170>PatentIn version 3.5

<210>1

<211>939

<212>DNA

<213> Artificial sequence

<220>

<223> coding sequence of anti-TEM 1 single-chain antibody

<400>1

gtgagcggat aacaattccc ctctagaaat aattttgttt aaactttaag aaggagatat 60

acatatgcat catcatcacc atcaccatgt ccagcctgtg ctgactcagc caccttccct 120

ctctgcatct cctggagcat cagccagtct cacctgcacc ttacgcagtg acatcaatgt 180

tggttcctac aggatatcct ggtaccagca gaagccaggg agtcctcccc agtatctcct 240

gagctacaaa tcagactcag ataagcagaa gggctctgga gtccccagcc gcttctctgg 300

atccaaagat gcttcggcca atgcagggat tttactcatc tctgggctcc agtctgagga 360

tgaggctgac tattattgta tgatttggca caacagcgct ggggtgttcg gcgggggcac 420

caagctgacc gtcctaggcg gtggttcctc tagatcttcc tcctctggtg gcggtggctc 480

gggcggtggt gggcaggtgc agctgcagga gtcgggggga accttggtac agcctggggg 540

gtccctgaga ctctcttgtg aagcctctgg attcaccttt agcaactatg ccatgggctg 600

ggtccgccag actccaggaa aggggctgga gtggctgtcg gctattcgta aaagtggcac 660

taccacatac tacgcggact ccgtgaaggg ccggttcatc atctccagag acaattccaa 720

gaacaccctg tatctgcaaa tgaataggct gagagtcggc gacacggcca cttattactg 780

tgcgactcac cccatcgcgg gctactgggg ccagggaacc ctggtcaccg tctcctccgg 840

aggttcttgc tagctcgaga tcgatgatat tcgagcctag gtataatcgg atccggctgc 900

taacaaagcc cgaaaggaag ctgagttggc tgctgccac 939

<210>2

<211>262

<212>PRT

<213> Artificial sequence

<400>2

Met His His His His His His His Val Gln Pro Val Leu Thr Gln Pro

1 5 10 15

Pro Ser Leu Ser Ala Ser Pro Gly Ala Ser Ala Ser Leu Thr Cys Thr

20 25 30

Leu Arg Ser Asp Ile Asn Val Gly Ser Tyr Arg Ile Ser Trp Tyr Gln

35 40 45

Gln Lys Pro Gly Ser Pro Pro Gln Tyr Leu Leu Ser Tyr Lys Ser Asp

50 55 60

Ser Asp Lys Gln Lys Gly Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

65 70 75 80

Lys Asp Ala Ser Ala Asn Ala Gly Ile Leu Leu Ile Ser Gly Leu Gln

85 90 95

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Met Ile Trp His Asn Ser Ala

100 105 110

Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly Ser

115 120 125

Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Gly Thr Leu Val Gln Pro Gly Gly Ser

145 150 155 160

Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Asn Tyr Ala

165 170 175

Met Gly Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Leu Ser

180 185 190

Ala Ile Arg Lys Ser Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val Lys

195 200 205

Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

210 215 220

Gln Met Asn Arg Leu Arg Val Gly Asp Thr Ala Thr TyrTyr Cys Ala

225 230 235 240

Thr His Pro Ile Ala Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

245 250 255

Ser Ser Gly Gly Ser Cys

260

Claims (12)

1. A TEM 1-specific nuclear magnetic probe comprising an anti-TEM 1 single-chain antibody and an aminated SPIO nanoparticle linked by a cross-linking agent, wherein the aminated SPIO nanoparticle is linked to the cross-linking agent by an amide bond, the C-terminus of the anti-TEM 1 single-chain antibody is cysteine and is linked to the cross-linking agent by a thioether bond;

the anti-TEM 1 single-chain antibody is encoded by a nucleic acid sequence shown in SEQ ID NO. 1.

2. The TEM 1-specific nuclear magnetic probe according to claim 1, wherein the amino acid sequence of the anti-TEM 1 single chain antibody is shown in SEQ ID No. 2.

3. A TEM 1-specific nuclear magnetic probe according to claim 1, wherein the aminated SPIO nanoparticles have a particle size of 60-120 nm.

4. The TEM 1-specific nuclear magnetic probe according to claim 3, wherein the aminated SPIO nanoparticles have a particle size of 70-90 nm.

5. A TEM1 specific nuclear magnetic probe according to claim 1, wherein the lateral relaxation rate of the aminated SPIO nanoparticles is 70-90mM^-1S^-1。

6. The TEM 1-specific nuclear magnetic probe according to any one of claims 1-5, wherein the cross-linking agent is SMCC or sulfo-SMCC.

7. A TEM 1-specific nuclear magnetic probe according to any one of claims 1-5, wherein the iron ions are contained at a concentration of 0.15-2.2 nM.

8. A TEM 1-specific nuclear magnetic probe according to claim 7, wherein the concentration of iron ions is 0.5-2 nM.

9. A TEM 1-specific nuclear magnetic probe according to claim 8, wherein the concentration of iron ions is 1-1.5 nM.

10. A method of making a TEM 1-specific nuclear magnetic probe of claim 1, comprising the steps of:

(1) synthesizing an anti-TEM 1 single-chain antibody with cysteine at the C terminal, and then purifying and renaturing;

(2) synthesizing aminated SPIO nanoparticles;

(3) the anti-TEM 1 single chain antibody and the aminated SPIO nanoparticles were linked by a cross-linker.

11. Use of a TEM 1-specific nuclear magnetic probe according to any one of claims 1-9 in the preparation of a reagent for diagnosing ovarian cancer.

12. A kit comprising a TEM 1-specific nuclear magnetic probe of any one of claims 1-9.

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