tem1 specific fluorescent probe and its application
A fluorescent probe and specific technology, applied in the field of TEM1-specific fluorescent probes, can solve the problems of clinical symptoms, atypical signs, lack of early diagnosis methods, hidden onset, etc., achieving no cytotoxicity and not easy to induce specific reactions , less immunogenic effect
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Embodiment 1
[0027] Example 1 Vector construction and expression, purification and renaturation of anti-TEM1 single-chain antibody
[0028] The sequence of the nucleic acid encoding anti-TEM1 single-chain antibody is shown in SEQ ID NO.1, and the 3' end is a codon encoding a cysteine (Cys), which was cloned into the restriction endonuclease Nsi I / Xho I. On the PET302 vector, a complete plasmid was constructed. The plasmids were transformed into E. coli competent cells, plated and grown overnight. After the colony has grown on the plate, extract the plasmid, and perform PCR with one primer on the foreign gene and another primer on the carrier. The results of cloning verification are as follows: figure 1 As shown, except for clone 2, which has no specific band, all other clones have specific bands. Sequencing results also proved that the vector was constructed successfully.
[0029] The above plasmids were transformed into E. coli BL21D3, plated and grown overnight. A single colony was...
Embodiment 2
[0031] Example 2 ELISA detects the affinity of anti-TEM1 single-chain antibody
[0032] Plate 96-well plates with 2% gelatin, 50 μl per well. Incubate at 37°C for 30 to 60 minutes. MS1-TEM1 cells (mouse pancreatic islet endothelial cells transfected with TEM1 plasmid to express TEM1 protein) were added to a 96-well plate, and the number of cells in each well was about 10 4 -10 5 . Incubate overnight at 37°C. Cells were washed 3 times with PBS. Appropriate concentrations (0.0001nM, 0.001nM, 0.01nM, 0.1nM, 1nM, 10nM, 100nM, 1mM, 10mM) of anti-TEM1 single chain antibody were added to each well and incubated at 4°C for 1 hour. Cells were washed 3 times with PBS again. Anti-His-HRP secondary antibody was added and incubated at 4°C for 1 hour. Cells were washed 3 times with PBS again. Add 100 μl of TMB solution to each well, and incubate at 37°C for no more than 30 minutes. 100 μl of stop solution was added to each well, and the OD value was detected by a bioteck colorimete...
Embodiment 3
[0033] Example 3 Binding of fluorescent dye ICG to anti-TEM1 single-chain antibody (78C-PEG-ICG)
[0034] It is reported in the literature that the quenching phenomenon occurs when ICG binds to the antibody. The present invention innovatively introduces a linker so that there is a certain distance between the dye and the antibody, thereby avoiding the quenching of the fluorescent dye. Linker is a maleimide group (MAL) and an amino group (NH) at both ends. 2 ) of polyethylene glycol (PEG), abbreviated as MAL-PEG-NH 2 . The cysteine (cys) located in the tail of the anti-TEM1 single-chain antibody will specifically bind to the MAL group on the linker, and the amino group on the linker will specifically bind to the NHS group on the fluorescent dye, thereby binding the anti-TEM1 single chain antibody. The chain antibody (78C for short) is combined with the fluorescent dye to form an ICG fluorescent system specific for TEM1. Figure 5 It is a schematic diagram of the above reac...
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