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Preparation method and application of BACE1 shearing high-titer antibodies

A cutting type and antibody technology, applied in the field of DNA recombination, can solve problems such as research difficulties

Inactive Publication Date: 2016-11-09
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there have been no more reports and studies on the functions of these splicing types, the mechanism of action with Aβ, and the relationship with AD. Difficult, so there is an urgent need for technical means that can distinguish these types of shearing

Method used

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  • Preparation method and application of BACE1 shearing high-titer antibodies
  • Preparation method and application of BACE1 shearing high-titer antibodies
  • Preparation method and application of BACE1 shearing high-titer antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Anti-GST-BACE1 190-214aa and GST-BACE1 146-189aa In preparation of spliced ​​sera

[0043] 1. PCR-PMD18-T / BACE1 190-214aa and BACE1 146-189a Construction of recombinant plasmids

[0044] The full length of the BACE1 gene was obtained from Genebank, and the sequence number of the gene is NM 001207048. Using pCDNA3.1-Flag-BACE1 as template, 190-214aa upstream primer is GGA TCC ATG CCT GAC GAC TCC CTG GAG CCT TTCTTT GAC TCT CTG GTAAAG (including Bamh1 restriction site); downstream primer is 5'-CTC GAG CTA CTG CAG GGAGGA GAG GTT GGGAAC GTG GGT CTG CTT TAC CAG (with XhoI restriction site). 146-189aa The upstream primer is 5'-GGATCC ATG GTA AGC ATC CCC CAT GGC (containing the Bamh1 restriction site); the downstream primer is 5'-CTC GAGCTA CCT GGC AAT CTC AGC ATA (containing the XhoI restriction site). The DNA sequences corresponding to 75bp and 132bp of 25 and 44 amino acids on the BACE1 gene were successfully amplified by PCR.【 figure 2 】. The amplified f...

Embodiment 2

[0053] Example 2: GST-BACE1 190-214aa and GST-BACE1 146-189a Antibody analysis and application

[0054] 1. GST-BACE1 190-214aa and GST-BACE1 146-189a Determination of antibody titer

[0055] with GST-BACE1 190-214aa and GST-BACE1 146-189a New Zealand white rabbit serum before fusion protein immunization was used as a control, and the purified GST-BACE1 190-214aa and GST-BACE1 146-189a The antibody was first diluted 10 times and then doubled, and then the titer of the antibody was determined by indirect ELISA. The results showed that the anti-fusion protein GST-BACE1 was not detected in the rabbit serum before immunization 190-214aa and GST-BACE1 146-189a Antibody to GST-BACE1 190-214aa and GST-BACE1 146-189a The titer of the antibody is as high as 1:100000 or more【 Figure 6 】.

[0056] 2. GST-BACE1 190-214aa and GST-BACE1 146-189a Application of antibody to detection of BACE1 spliced ​​protein in cells

[0057] Two high titer sera of rabbit anti-mouse GST-BACE...

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Abstract

The invention belongs to the technical field of bioengineering, and relates to BACE1 shearing high-tilter antibodies prepared by using a GST expression system, and an application of the antibodies. BACE1 shearing gene is cloned into a prokaryotic expression vector by using a gene engineering technology, enzymatic detection identification is carried out, Escherichia coli is converted by using recombinant plasmids, IPTG induction is carried out to generate GST-BACE1146-189aa and GST-BACE1190-2144aa fusion proteins, purified fusion proteins are used to immunize New Zealand rabbits in order to prepare antiserum, and the antiserum is purified by using ProteinA / G. The two anti-BACE1 shearing polyclonal antibodies have good titer and specificity, can be applied to the detection of different BACE1 shearing types 52 KD and 49 KD of in cells and the distinguishing of four shearing types, can effectively detect the differential expression of the BACE1 shearing types in different cells, and is of great significance to systemically research the functions of the BACE1 and the action mechanism of the BACE1 in AD.

Description

technical field [0001] The invention belongs to the technical field of DNA recombination in bioengineering, and in particular relates to the preparation of a polyclonal antibody of GST-BACE1 fusion protein by using a GST prokaryotic expression system and its application. Background technique [0002] Alzheimer's disease (AD), commonly known as Alzheimer's disease, is a fatal central nervous degenerative disease mainly characterized by progressive cognitive impairment and memory impairment. One of the main pathological features of AD is the Amyloid plaques formed by β-amyloid protein (Aβ), Aβ is produced by APP (Amyloid Precursor Protein) under the sequential action of β-cleaving enzyme (BACE1) and γ-cleaving enzyme complex. [0003] BACE1 protein, also known as β-site APP lyase 1, is a membrane-bound aspartic acid protease and is the key enzyme for cleaving APP to produce Aβ. It is expressed in various tissues, with the highest expression in the brain and pancreas . The ac...

Claims

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Application Information

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IPC IPC(8): C07K16/40C12N15/13C07K16/06G01N33/573
CPCC07K16/40G01N33/573G01N2333/96425
Inventor 李晓萌王娅沈晓延汪小莞李晓春李江
Owner NORTHEAST NORMAL UNIVERSITY
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