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Function development and application of subtype BACE1 (beta-secretase 1) capable of promoting A-beta accumulation in brain

A BACE1, promoting effect technology, applied in the functional development and application field of BACE1 subtype that promotes the accumulation of Aβ in the brain, can solve the problems of not paying attention to the molecular cutting function of APP and low expression level

Inactive Publication Date: 2017-12-29
NORTHEAST NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most widely expressed in the brain is A / type I-501, while the other three forms have very low expression in the brain and are mainly expressed in tissues such as the pancreas, so the researchers did not pay attention to the effects of the other three forms on Cleavage function of APP molecule

Method used

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  • Function development and application of subtype BACE1 (beta-secretase 1) capable of promoting A-beta accumulation in brain
  • Function development and application of subtype BACE1 (beta-secretase 1) capable of promoting A-beta accumulation in brain
  • Function development and application of subtype BACE1 (beta-secretase 1) capable of promoting A-beta accumulation in brain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Construction of 52KD subtype BACE1 eukaryotic expression plasmid and its expression detection

[0035] 1. Construction of 52KD subtype BACE1 eukaryotic expression vector

[0036] The full length of the BACE1 gene was obtained from Genebank, and the sequence number of the gene is NM 001207048. Type B I-476 (52KD) BACE1 is missing 146-190aa compared with Type A I-501 (55KD) BACE1【 figure 2 ], using pcDNA3.1-BACE1 (55KD) as a template, the upstream primer is 5'-CCAGGCTTTGTGGTGCTGGCTTCCCCCCTCA-3', and the downstream primer is 5'-CAAAGCCTGGCAAATCTCAGCATAGGCCAGC-3' for PCR. The PCR product was transformed with DH5α competent, and the transformed colony was cultured in a small amount, and then a small amount of the cultured bacterial solution was extracted by alkaline lysis method, and finally the extracted plasmid was sequenced and compared , the mutation succeeds【 image 3 】.

[0037] 2. Expression detection of 52KD subtype BACE1 eukaryotic expression plasmid...

Embodiment 2

[0039] Example 2: Functional detection of 52KD subtype BACE1

[0040] 1. ELISA detection of 52KD subtype BACE1 on Aβ in AD cell model 1-40 and Aβ 1-42 production inhibition

[0041]The AD cell model SHSY5Y cell supernatant was used as a sample, and the BACE1 mutant plasmid and APP wild-type plasmid or APP were co-transfected SWE After 48 hours of the mutant plasmid, the cell culture medium was centrifuged, and 1 / 100 was taken as a sample to detect the cleavage effect of the 52KD subtype BACE1 on the APP molecule, and the sample concentration was calculated according to the concentration and absorbance curve of the standard. Compared with the blank control group, whether overexpression of APP wild type or overexpression of APP SWE Mutant type, 52KD BACE1 can significantly increase the concentration of Aβ1-40【 Figure 7 ]; for Aβ 1-42 The same result was also obtained in the generation detection experiment [ Figure 8 】.

[0042] Therefore, it can be determined that B-typ...

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Abstract

The invention belongs to the technical field of DNA (deoxyribonucleic acid) recombination of bioengineering and relates to the technical field of bioengineering, particularly to a 52KD subtype BACE1 for APP (amyloid protein precursor) pathological cutting and A-beta formation recombined on the basis of BACE1 alternatively slicing mechanism. ELISA (enzyme linked immunosorbent assay) detects that the 52KD subtype BACE1 achieves promoting effects on generation of A-beta1-40 and A-beta1-42, and behavioral experiments verifies the damage effects of the 52KD subtype BACE1 on memory of AD (Alzheimer's disease) rats, and pathological experiments proves the promoting effects of the 52KD subtype BACE1 on formation of A-beta in brain. The 52KD subtype BACE1 provides basis and clues for diagnosis of AD, deep research of molecular targets and development of drugs.

Description

technical field [0001] The invention belongs to the technical field of DNA recombination in bioengineering, specifically, based on the gene recombination of the BACE1 alternative splicing mechanism, a BACE1 subtype that can promote the pathological cutting of APP and the formation of Aβ is produced, and its effect on memory damage and Aβ formation in AD mice is verified. The exacerbation of Aβ formation in the brain, and its ability to provide evidence and clues for the diagnosis of AD, in-depth research on molecular targets, and drug development. Background technique [0002] Alzheimer's disease (AD) is an important disease in an aging society and has become one of the four major killers threatening the health of the elderly. Amyloid Aβ is continuously generated and accumulated in the brain, causing significant amyloid plaque formation and secondary nerve damage, which is an important pathogenesis of AD. Aβ is formed by pathological cleavage of APP (amyloid precursor) mole...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N9/64G01N33/573
CPCC12N9/6478C12Y304/23046G01N33/573
Inventor 李晓萌王娅李佳乐汪小莞刘孜育王译唱李江
Owner NORTHEAST NORMAL UNIVERSITY
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