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MSLN (Mesothelin) specific fluorescent probe and application thereof

A fluorescent probe and specific technology, applied in the field of MSLN-specific fluorescent probes, can solve the problems of limited application of fluorescent probes, and achieve the effects of no cytotoxicity, low immunogenicity and high sensitivity

Inactive Publication Date: 2019-04-26
TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems existing in the prior art, the present invention provides a MSLN-specific fluorescent probe and its application, which solves the problem of limited application of fluorescent probes in the diagnosis and treatment of ovarian cancer in the prior art

Method used

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  • MSLN (Mesothelin) specific fluorescent probe and application thereof
  • MSLN (Mesothelin) specific fluorescent probe and application thereof
  • MSLN (Mesothelin) specific fluorescent probe and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1 Expression of anti-MSLN single chain antibody, and expression condition screening

[0026] The sequence of the nucleic acid encoding the anti-MSLN single-chain antibody is shown in SEQ ID NO.1. The plasmid was transformed into Escherichia coli DH5α competent cells. After picking the monoclonal colony grown on the plate, extract the plasmid.

[0027] The above plasmids were transformed into Escherichia coli BL21D3, plated and cultured overnight. Pick a single colony, resuspend it with 5ml LB medium, and culture it overnight at 37°C. Transfer 1 ml of the above medium to a 100 ml LB medium flask at a ratio of 1:100, and culture it in a shaker at 37°C for 2 hours. Add IPTG induction at a ratio of 1:1000 (the calculated final concentration of IPTG is 0.5mM or 1mM) as the induction group, set up the control group without IPTG, and culture at 37°C or 25°C. Centrifuge at 9000 rpm for 5 min at 4°C to collect the cells. Wash once with 40ml of PBS buffer, and use...

Embodiment 2

[0028] Purification and renaturation of the expressed protein of embodiment 2

[0029] The bacteria were resuspended in urea-containing and urea-free buffers, and added to the Ni+ column at a ratio of 1:0.4 (the magnetic beads were washed with PBS buffer in advance), and combined by inversion at 4°C for 1 h. Centrifuge at 15,000 rpm for 3 minutes in a centrifuge, and take 20ul of the supernatant as Flow-through. Wash the magnetic beads twice with 3ml of washing solution containing or not containing urea. Wash 4 times with 2ml of 8M urea eluent containing or not containing urea to elute the target protein. For protein purification results, see figure 2 . The eluates from the 4 washes were combined. Under the condition of 4°C, the above eluate was placed in 5LPBS buffer and dialyzed for 48h. The inclusion body proteins refolded under different conditions were detected by SDS-PAGE and Western-blot of HIS-tag, the results are shown in image 3 , it can be seen that the puri...

Embodiment 3E

[0030]Embodiment 3ELISA detects the affinity of anti-MSLN single-chain antibody

[0031] Dilute the MSLN protein to 0.25 mg / μl, add 100 μl to each well, and incubate overnight in a refrigerator at 4°C. The next day, take out the 96-well plate, carefully suck off the liquid in the microplate with a sample gun, and then pat the microplate against the absorbent paper several times. No clearly visible liquid. Wash twice with 0.1% PBS, 10min each time, 350ul per well. Block with 1% BSA, 150ul per well, shake slowly at room temperature for 1h. Anti-MSLN single-chain antibody at appropriate concentration (0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM) was added to each well, and incubated at room temperature for 1 h. Wash twice again with 0.1% PBST, each 10min, 350ul per hole. Add anti-his-HRP and incubate at room temperature for 1h. Wash twice again with 0.1% PBST, 10min each time, 350ul per well. Add 100 μl TMB solution to each well, and incubate at 37°C for no more th...

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Abstract

The invention discloses an MSLN (Mesothelin) specific fluorescent probe and application thereof. The MSLN specific fluorescent probe comprises a single-chain antibody which can resist MSLN, wherein the single-chain antibody is an Anti-MSLN-ICG (IndoCyanine Green) antibody which is connected with fluorescent dye ICG-Sulfor-Osu; the fluorescent dye ICG is connected with an amino functional group-NH2on the single-chain antibody through a Sulfor group, and fluorescent labeling of the single-chain antibody is realized. The MSLN specific fluorescent probe provided by the invention is good in specificity and high in sensitivity and has no cytotoxicity, the single-chain antibody can be easily eliminated from serum as the molecular weight of the single-chain antibody is smaller, the immunogenicityis smaller, and specific reaction cannot be easily caused in a body; in addition, the MSLN specific fluorescent probe also has the advantages of small interference and strong penetrability and is suitable for clinical application.

Description

technical field [0001] The design of the invention relates to the field of optical molecular imaging, specifically to the field of fluorescent probes, in particular to a MSLN-specific fluorescent probe and its application. Background technique [0002] Ovarian cancer is a malignant tumor of the female reproductive system, and its mortality rate ranks first among female gynecological tumors. Due to its hidden location and lack of diagnostic methods, the 5-year survival rate of ovarian cancer patients is only 20%. Its early diagnosis is one of the important factors related to the patient's prognosis. [0003] Mesothelin (MSLN) is a tumor differentiation antigen expressed on the mesothelial cells of the pleura, peritoneum and pericardium. Ideal target. In about 70% of ovarian cancers, mesothelin is highly expressed. [0004] Optical imaging is widely used in the field of tumor research due to its advantages of non-invasiveness, real-time, and high resolution. It can perform...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30C09K11/06A61K49/00
CPCA61K49/0034A61K49/0058C07K16/30C09K11/06
Inventor 鲍会静霍茜瑜邵艳宏黄伦辉程优刘亚珊蔺天宇陈佳佳
Owner TIANJIN MEDICAL UNIV
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