Difunctional tumor diagnosis reagent and method thereof
A tumor diagnosis, dual-function technology, applied in the intersection of immunology and biomedicine, nanotechnology, and biomimetic fields, it can solve the problems of complex steps and long operation time, and achieve the effect of simple operation.
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Embodiment 1
[0029] The preparation of embodiment 1 bionic ferritin
[0030] By genetically recombining the full-heavy chain human ferritin shell, using the principle of human biomineralization, iron ions are transferred into the protein shell, and then oxidized into iron oxide nanoparticles. Ferritin, named biomimetic ferritin, for tumor detection. The specific operation is as follows: First, construct the recombinant plasmid pET12b-HFn (Santambrogio P. et al., (2000), Protein Expr. Purif., 19: 212-218) containing the gene sequence of human full heavy chain subunit ferritin. The human skeletal muscle total cDNA library was purchased from Invitrogen (D8090-01, Carlsbad, CA, USA). The full heavy chain human ferritin gene was isolated and amplified from the cDNA using PCR technology. Two primers were designed as follows: forward PCR primer: 5'-A GTC GCC CAT ATG ACG ACCGCG TCC-3', the restriction site is NdeI (underlined), with 7 protection bases; reverse PCR primer: 5'-GCC GGA TCC TTA...
Embodiment 2
[0036] The peroxidase activity of embodiment 2 bionic ferritin
[0037] Based on the report of our research group, magnetic iron oxide nanoparticles have catalytic activity similar to that of peroxidase (Gao L, et al. (2007) Nature Nanotech., 2: 577-583.), because biomimetic ferritin is wrapped with The inner core of iron oxide should therefore have oxidase-like activity. We use the substrate of horseradish peroxidase to detect the enzymatic activity of biomimetic ferritin, the specific method is as follows: add 30% H to biomimetic ferritin 2 o 2 And TMB or DAB, observe the color change. Such as figure 1 As shown in B, when biomimetic ferritin and H 2 o 2 After that, it turns dark blue, and DAB is added with biomimetic ferritin and H 2o 2 After that, it turns dark brown, and the same color reaction as horseradish peroxidase appears, indicating that biomimetic ferritin has peroxidase-like activity.
Embodiment 3
[0038] Example 3 Biomimetic Ferritin Binding to Tumor Cells
[0039] In order to study the binding of biomimetic ferritin to human tumor cells, common human tumor cells were selected to incubate with fluorescent molecularly labeled biomimetic ferritin, and cell flow cytometry was used to detect the binding of biomimetic ferritin to each tumor cell.
[0040] The experimental method is as follows: according to the labeling method provided in the manual, NHS-activated Cy5.5 (Cy5.5-NHS, purchased from GE Healthcare) was labeled on biomimetic ferritin. Culture each cell line to 1×10 5 Left and right, trypsinize, wash the cells three times with 0.3% BSA / PBS, add 50 μg / ml Cy5.5-labeled biomimetic ferritin, and incubate at 4°C for 45 minutes. Then the cells were washed three times with 0.3% BSA / PBS, and finally resuspended in PBS, and the fluorescence of the samples was detected by flow cytometry.
[0041] The results are shown in Table 1. Among the 12 tumor cell lines tested, biomi...
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