Polypeptide of specific efficient affinity membrane type I substrate metal protease (MT1-MMP), protein and use
A specific and affinity membrane technology, which is applied in the direction of medical preparations containing active ingredients, peptide/protein ingredients, non-active ingredients of polymer compounds, etc., can solve the problem of restricting the wide application of antibody reagents, prone to immunogenicity, Problems such as short half-life
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Embodiment 1
[0012] Example 1. Biotin-His-Trp-Lys-His-Leu-His-Asn-Thr-Lys-Thr-Phe-Leu has specific selection and high affinity for membrane type I matrix metalloproteinases.
[0013] Experimental steps:
[0014] Use 100-200 μl 100 μg / ml target molecules MT1-MMP, MMP-1, MMP-7, MMP-13, MMP-16, MMP-26 and MMP-28 (dissolved in 0.1M pH8.6 NaHCO 3 Middle) Coat each well of the ELISA plate, one row of coated wells for each clone to be identified. Incubate overnight at 4°C in a sealed wet box (such as a sealed plastic box lined with wet paper towels).
[0015] Shake off excess target molecule solution, invert the plate and pat on paper towels to remove residual liquid. Fill each well with blocking solution. In addition, a row of uncoated wells of each clone to be identified was also added with blocking solution to detect the binding force of the selected sequence to the BSA-coated plastic plate. Before adding the peptide to the target molecule-coated plate, another microtiter plate is prepared...
Embodiment 2
[0028] Example 2.Biotin-His-Trp-Lys-His-Leu-His-Asn-Thr-Lys-Thr-Phe-Leu has no inhibition on membrane type I matrix metalloproteinase (MT1-MMP) on enzymatic and cellular levels effect.
[0029] Experimental steps:
[0030] Enzymatic detection of the inhibition of MT1-MMP by Biotin-His-Trp-Lys-His-Leu-His-Asn-Thr-Lys-Thr-Phe-Leu:
[0031] The substrates we used were DQ-Gelatin and P126; measured at room temperature, the total reaction volume was 100ul. When the inhibitor is added, the enzyme and the inhibitor should be incubated in the buffer system for 30 minutes at 37°C and then added to the substrate assay. All detection instruments are FLX800 fluorescence microplate reader (Bio-Tek). For DQ-Gelatin, the excitation wavelength is 495nm, the emission wavelength is 515nm, and for P126, the excitation wavelength is 328nm, and the emission wavelength is 393nm. The measured reaction time is 8 minutes, and the slope of the line formed by the fluorescence value is taken as the ve...
Embodiment 3
[0036] Example 3. On the cellular level, Biotin-His-Trp-Lys-His-Leu-His-Asn-Thr-Lys-Thr-Phe-Leu marks several cells with different expression levels of MT1-MMP:
[0037] Experimental steps:
[0038] Take out the cells that have been cultured to about 80%, digest them with trypsin, add 5ml of medium, repeatedly blow the cell solution, blow the cells into single cells, take 200ul of the cell suspension and drop it on the sterilized coverslip, and store the cells at 37°C Incubate overnight in the incubator.
[0039] After washing the coverslip three times with PBS, add DMEM / FBS and incubate for 30mins.
[0040] Add Biotin-His-Trp-Lys-His-Leu-His-Asn-Thr-Lys-Thr-Phe-Leu at room temperature and incubate overnight at 4°C.
[0041] Wash with PBS three times, add FITC-Avidin, and incubate for 30min in the dark
[0042] Wash three times with PBS, fix with 95% ethanol, and cover with glycerol.
[0043] Observation under fluorescence microscope
[0044] attached Figure 4 It is the...
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