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Anti-human amyloid beta-peptide nano antibody and application thereof

A nanobody and amyloid technology, applied in the fields of biochemistry and molecular biology, can solve the problems of increasing the cost of antibody preparation

Inactive Publication Date: 2016-05-04
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Either strategy further increases the cost of antibody preparation

Method used

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  • Anti-human amyloid beta-peptide nano antibody and application thereof
  • Anti-human amyloid beta-peptide nano antibody and application thereof
  • Anti-human amyloid beta-peptide nano antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Establishment of Nanobody Library

[0022] The phage display library used in the present invention is a non-immune library with T7 phage as the carrier, and the establishment steps are as follows:

[0023] (1) Total RNA was extracted from peripheral blood lymphocytes of 2-year-old male alpaca ( PuerLinkTMRNAMiniKit, LifeTechnologies: 12183018A); With UPprimer1 as a primer, the extracted total RNA was reverse-transcribed into cDNA, and two rounds of nested PCR were used to amplify the VHH gene; wherein the first round of PCR was based on the transcribed cDNA as a template, with UPprimer1 and DOWNprimer1 is the primer. After PCR amplification, a band with a size of 650-750bp is recovered, and then used as a template for the second round of PCR amplification. The upstream and downstream primers are UPprimer2 and DOWNprimer2 respectively, and a PCR product of 450-500bp is recovered. ;

[0024] UPprimer1:5'-GGTACGTGCTGTTGAACTGTTCC-3'

[0025] DOWNprimer1:5'-CTT...

Embodiment 2T7

[0030] The liquid amplification of embodiment 2T7 bacteriophage

[0031] (1) Activation of host bacteria: Inoculate Escherichia coli BLT5403 in 50ml of LB medium (containing 0.1mg / mL carbenicillin, referred to as Carb-LB), and culture overnight at 37°C and 170r / min to obtain the first-generation host bacteria;

[0032] (2) Inoculate 1% of the first-generation host bacteria in Carb-LB liquid medium, culture on a shaker, and reach OD 600 0.5-1.0, get the second generation host bacteria;

[0033] (3) Infect the second-generation host bacteria with T7 phage, culture at 200 r / min at 37°C for 2-3 hours, observe the bacteriolysis situation, and stop the cultivation immediately if the bacteriolysis is complete. The lysate was centrifuged at 8000 g for 10 min, and the supernatant was recovered, which was the amplified phage. Determine the titer.

Embodiment 3T7

[0034] The mensuration of embodiment 3T7 phage titer

[0035] (1) Pour the Carb-LB bottom medium into the culture dish, cool and solidify;

[0036] (2) Dissolve the top culture medium, cool it down, add Carb and keep it warm at 45°C;

[0037] (3) The amplified phages are diluted in gradient with sterile LB liquid medium;

[0038] (4) Mix 300 μL of the second-generation host bacteria with 50 μL of phage diluted in different gradients, add 4 mL of preheated Carb-top medium, quickly pour it into a plate with the bottom medium, and shake the plate to make it evenly distributed;

[0039] (5) Incubate upside down at 37°C for 3-4 hours until moderately sized plaques are seen;

[0040] (6) Select a plate with 50-500 plaques to count the plaques and calculate the phage titer.

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Abstract

The invention discloses an anti-human amyloid beta-peptide nano antibody (Abeta) and application thereof. A bacteriophage display technique is utilized to carry out multi-screening on an alpaca bacteriophage antibody library to enrich the Abeta-specific bacteriophage; the bacteriophage is cultured to prepare the antibody; identification is carried out to obtain a positive clone; sequencing is carried out to obtain a corresponding coding sequence; and expression is carried out in Escherichia coli to obtain a soluble antibody segment. The antibody disclosed by the invention has the amino acid sequence disclosed as SEQ ID NO.8. The antibody disclosed by the invention has the specific combining capacity with Abeta, and can be used for preparing Abeta-targeted drugs for reagents for disease therapy and diagnosis.

Description

technical field [0001] The invention belongs to the field of biochemistry and molecular biology, and relates to a nanobody against human amyloid β peptide (amyloid β, Aβ) and application thereof. Background technique [0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system characterized by progressive cognitive dysfunction and behavioral impairment. In September 2015, the Alzheimer'sdisease international, (ADI) report data showed that: at present, about one person suffers from Alzheimer's disease every 3.2 seconds, and 60-70% of the patients with Alzheimer's disease are Alzheimer's disease dementia. With the aging population As the base and proportion continue to increase, AD will bring more and more serious personal pain to patients and social and family pressure. [0003] Amyloid β peptide (amyloidβ, Aβ) is a natural product present in the human brain, which is involved in the development of nerve cells (HeoC, et al. J Neurochem. 2007; 102...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13A61K39/395A61P25/28G01N33/68
CPCC07K16/18C07K2317/22C07K2317/565C07K2317/567C07K2317/74
Inventor 徐丽刘红静贾凌云王从刚任军
Owner DALIAN UNIV OF TECH
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