Application of drebrin (DBN1) to preparing neurodegenerative disease diagnosis medicines
A neurodegenerative disease and protein technology, applied in the field of medicine and biology, can solve problems such as difficulties in clinical application, patient burden, and assess the degree of nerve damage, and achieve important clinical significance, social and economic benefits, and convenient detection.
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Embodiment 1
[0040] Embodiment 1 discloses the purification of antigen and the preparation process of antibody;
[0041] 1. Expression, purification and identification of DBN1 protein:
[0042] The DBN1 protein is derived from the genetically engineered Saccharomyces cerevisiae in the previous study, induced overexpression by galactose, separated and purified by agarose affinity medium (glutathione), and identified by Western-Blotting.
[0043] 2. Preparation of serum samples:
[0044] Centrifuge the whole blood sample at 1000g for about 20 minutes after standing at room temperature for 2 hours or overnight at 4°C, and take the supernatant for immediate detection; or aliquot and store the sample at -20°C or -80°C, but avoid Freeze and thaw repeatedly. Thawed samples should be centrifuged again prior to testing. The tested samples cannot contain NaN3, because NaN3 inhibits the activity of horseradish peroxidase (HRP).
[0045] 3. Preparation methods of various buffers and reagents for E...
Embodiment 2
[0070] The preparation of embodiment 2 experimental model and the verification of result
[0071] Rat optic nerve injury animal model: cut the bulbar conjunctiva on the temporal side of the eyeball of the rat, bluntly dissect the periocular tissue, and clamp the optic nerve with No. 5 surgical forceps for 5 seconds at the 2 mm directly behind the eyeball to make an animal model of optic nerve injury.
[0072] Retinal tissues were taken after optic nerve clipping in rats, and immunoblotting and immunofluorescence staining were performed to detect the expression of retinal DBN1 protein, which was quantified by Image J software.
[0073] Neuro-2A cells were treated with 100uM NMDA for 24 hours, and the level of Drebrin was detected by immunofluorescence assay.
[0074] Most of the DBN1 protein is mainly distributed in the retinal ganglion cell layer and the inner plexiform layer. The results of Western Blot experiments on the optic nerve clipping 3d, 7d, 14d and the retina of the...
Embodiment 3
[0076] Example 3: Exemplary diagnostic applications of the diagnostic kit according to the present invention
[0077] From October 2016 to December 2017, we collected 232 glaucoma patients in the Eye Hospital of Wenzhou Medical University, including 164 primary angle-closure glaucoma (closed blue, PACG), 46 primary open-angle glaucoma Angular glaucoma (Kaiqing, POAG) and 22 patients with glaucomatous cyclocytitis syndrome (blue eyelashes, PS), and 50 controls.
[0078] By the diagnostic kit provided by the present invention, the enzyme-linked immunosorbent assay (ELISA) kit is used to detect the plasma Drebrin expression level, and the clinical examination results such as the patient's intraocular pressure, OCT and visual field are collected and analyzed, and the results are as shown in Table 1:
[0079]
[0080]
[0081] The visual field index in Table 1 is close to 100 as a normal value, and the smaller the value is, the visual field damage is serious; the average vari...
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Abstract
Description
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Application Information
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