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Variable region sequence for novel anti-beta amyloid polypeptide antibody and coding sequence thereof

A kind of variable region, antibody technology

Inactive Publication Date: 2009-09-23
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, full antibodies will increase the difficulty of crossing the blood-brain barrier, but studies have shown that some antibodies can indeed cross the blood-brain barrier, and even antibodies that remain in the blood circulation can bind to and clear Aβ in the blood. After the Aβ in the blood is reduced, the Aβ monomers in the brain will continue to penetrate into the blood vessels, thereby reducing the Aβ substances in the brain and also reducing the Aβ lesion substances in the brain [DeMattos RB, Bales KR, Cummins DJ, et al.Peripheral anti-Aβantibody alters CNS and plasma Aβclearance and decreases brain Aβburden in a mouse model of Alzheimer's disease.Proc Natl Acad Sci USA, 2001, 98(15):8850-8855]

Method used

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  • Variable region sequence for novel anti-beta amyloid polypeptide antibody and coding sequence thereof
  • Variable region sequence for novel anti-beta amyloid polypeptide antibody and coding sequence thereof
  • Variable region sequence for novel anti-beta amyloid polypeptide antibody and coding sequence thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Screening of monoclonal phage antibodies:

[0025] Multiple rounds of enrichment and screening of human phage single-chain antibody libraries were performed using phage display technology, and four rounds of "adsorption-elution-amplification" enrichment screening were performed using purified Alzheimer's disease Aβ as the antigen. The recovery rate of rounds of screening showed an increasing trend, indicating that after screening, the phages with Aβ specificity were highly enriched (Table 1). Methods as below:

[0026] (1) Amplification of the phage antibody library: Take 100 μl of the original library bacteria solution, inoculate it into 100ml 2×YT-AG (2×YT containing 100 μg / ml ampicillin, 1% glucose) medium, and culture it with shaking at 37°C until Logarithmic period. plus 10 12 The pfu (plaque forming unit) helper phage M13KO7 was placed in the culture medium, bathed in water at 37°C for 30min, centrifuged at 4°C and discarded the supernatant. Resuspe...

Embodiment 2

[0032] Example 2: Detection of monoclonal phage antibodies:

[0033] 80 single bacterial clones were randomly picked from the TYE-AG plate cultured overnight after the fourth round of screening, and cultured to prepare phage antibodies. ELISA method for detection: Antigen Aβ was coated on an enzyme plate (0.4 μg / well), and after blocking, 100 μl of monoclonal phage antibody supernatant was added to each well, and incubated at 37° C. for 2 hours. After washing, 1:5000 HRP-labeled mouse anti-M13 antibody was added and incubated at 37°C for 1h. The positive control was the primary antibody (mouse anti-human Aβ), the secondary antibody (goat anti-mouse IgG); the negative control only added 2% MPBS. With OPD as substrate, 2M sulfuric acid was used to terminate the reaction, and detected by OD490nm of microplate reader. When the OD value was greater than 2 times of the negative control, it was judged as positive. Detection results of monoclonal phage antibody binding to Aβ antige...

Embodiment 3

[0035] Example 3: Expression and identification of soluble anti-Aβ scFv fragments:

[0036] (1) The expression of positive phagemids was induced in Escherichia coli HB2151, and the phages displaying antibodies specifically binding to Aβ were infected with logarithmic phase Escherichia coli HB2151, and left in a water bath at 37°C for 30 minutes. Inoculate on SOBAG-N plates and culture at 30°C until a single colony grows. Pick one clone from each plate with different clone sources, inoculate them in 5ml 2×YT-AG medium respectively, and shake overnight at 30°C. Add 5ml of overnight bacteria to 50ml of fresh 2×YT-AG medium, shake at 30°C for 1h. Centrifuge, discard the supernatant, resuspend the pellet in 50ml of fresh 2×YT-AI (I:IPTG, 1mM) medium, induce expression at 30°C for 20h. After centrifugation, the supernatant was sterilized by filtration and stored at -20°C. Resuspend one tube of pellet with 0.5ml ice-cold 1×TES, then add 0.75ml ice-cold 1 / 5×TES, vortex, and ice-bat...

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Abstract

The invention relates to a variable region sequence for a novel anti-beta amyloid polypeptide antibody and a coding sequence thereof. The invention utilizes phage display technology to perform multiple screening and enrichment on a human-originated phage single-chain antibody library to obtain phages with Abeta specificity, prepares a phage antibody by culturing the phages, and identifies the acquisition of positive clone. Then an anti-Abeta scFv segment which obtains the solubility is expressed in Escherichia coli, and a corresponding coding sequence of the anti-Abeta scFv segment is obtained through the sequence measurement. Therefore, the invention provides the novel anti-Abeta scFv segment and the coding sequence thereof.

Description

technical field [0001] The present invention relates to an anti-beta-amyloid polypeptide antibody, and further relates to a new anti-beta-amyloid polypeptide specific scFv antibody, and the amino acid sequence and DNA coding sequence of its variable region. Background technique [0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system characterized by progressive memory impairment and mental decline in the elderly. Senile plaques, neurofibrillary tangles, and neuronal loss are the three main pathological features of AD. The main component of senile plaques is the fibers formed by the aggregation of β-amyloid peptide (Aβ), and Aβ is considered to be the main pathogenic substance that causes neuronal damage and cognitive function decline [Frost D. Co-incorporation of A beta 40 and A beta42 to formmixed pre-fibrillar aggregates. Eur J Biochem, 2003, 270(4): 654-663]. Aβ mainly includes two molecules, Aβ40 and Aβ42, which are composed of 39-43 a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/63C12N1/21C12R1/19
Inventor 梁平
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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