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Functional research and development of BACE1 subtype for inhibiting accumulation of Abeta in brain and application of BACE1 subtype

A technology of BACE1 and inhibition, which is applied in the functional development and application of BACE1 subtypes that inhibit the accumulation of Aβ in the brain, and can solve the problems of not paying attention to the molecular cutting function of APP and low expression levels

Active Publication Date: 2017-12-15
NORTHEAST NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Functional research and development of BACE1 subtype for inhibiting accumulation of Abeta in brain and application of BACE1 subtype
  • Functional research and development of BACE1 subtype for inhibiting accumulation of Abeta in brain and application of BACE1 subtype
  • Functional research and development of BACE1 subtype for inhibiting accumulation of Abeta in brain and application of BACE1 subtype

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction of 47KD subtype BACE1 eukaryotic expression plasmid and its expression detection

[0036] 1. Construction of 47KD isoform BACE1 eukaryotic expression vector

[0037] The full length of the BACE1 gene was obtained from Genebank, and the gene sequence number is NM 001207048. D-type I-432 (47KD) BACE1 compared with A-type I-501 (55KD) BACE1 deletion 146-214aa【 figure 2 ], using pcDNA3.1-BACE1 (55KD) as the template, the upstream primer is 5'-ACCTGCTTTGTGGTGCTGGCTTCCCCCTCA-3', and the downstream primer is 5'-CAAAGCAGGTCGGTGCCCAGCTCCCCTTCC-3' to carry out PCR. The PCR product was transformed with DH5α competence, and the transformed colonies were cultured in a small amount, and then the bacterial liquid of the small amount of culture was extracted by alkaline lysis method. Finally, the extracted plasmids were sequenced and compared. , the mutation is successful [ image 3 ].

[0038] 2. Expression detection of 47KD isoform BACE1 eukaryotic expres...

Embodiment 2

[0040] Example 2: Functional assay of 47KD isoform BACE1

[0041] 1. ELISA to detect the effect of 47KD isoform BACE1 on Aβ in AD cell model 1-40 and Aβ 1-42 production inhibition of

[0042] The cell supernatant of the AD cell model SHSY5Y was used as the sample, and the mutant plasmid of BACE1 and the APP wild-type plasmid or APP were co-transfectedSWE After 48 hours of mutant plasmid, the cell culture medium was centrifuged, and 1 / 100 of it was taken as a sample to detect the cleavage effect of 47KD subtype BACE1 on APP molecules, and the sample concentration was calculated according to the standard concentration and absorbance curve. Compared with the blank control group, whether overexpressing APP wild type or overexpressing APP SWE Mutant, only 47KD of BACE1 can make Aβ 1-40 The concentration of the Figure 7 ]; for Aβ 1-42 The same result was obtained in the generation detection experiment of [ Figure 8 ].

[0043] Therefore, it can be determined that D-type BAC...

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Abstract

The invention belongs to the technical field of DNA recombination in bioengineering, relates to the technical field of bioengineering and particularly relates to a 47KD subtype BACE1 which is recombined from a gene based on a BACE1 alternative splicing mechanism and is capable of antagonizing APP pathological incision and inhibiting Abeta. The promotion effects of the 47KD subtype BACE1 to Abeta1-40 and Abeta1-42 are detected by virtue of an ELISA experiment, the correction effect of the 47KD subtype BACE1 on the memory impairment of an AD mouse is verified by virtue of a behavioral experiment, and finally, the correction effect of the 47KD subtype BACE1 on the formation of Abeta in a brain is verified by virtue of a pathological experiment. According to the 47KD subtype BACE1, the basis and the clues are provided for the deep research on AD molecular targets and the research and development of AD drugs.

Description

technical field [0001] The invention belongs to the technical field of DNA recombination in bioengineering, in particular to gene recombination based on the BACE1 alternative splicing mechanism to recombine a BACE1 subtype that has an antagonistic effect on APP pathological cleavage and an inhibitory effect on Aβ formation, and verifies that it has an effect on memory damage in AD mice And the corrective effect of Aβ formation in the brain, and it provides basis and clues for the in-depth study of AD molecular targets and AD 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 that threaten 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 APP (amyloid precursor) molecular pathological cleavage...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/47
CPCC07K14/4703
Inventor 李晓萌王娅李佳乐汪小莞刘孜育姜波李江
Owner NORTHEAST NORMAL UNIVERSITY
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