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Antibody type molecular chaperone capable of inhibiting Tau protein from being aggregated

A single-chain antibody, nucleic acid molecule technology, applied in the direction of antibodies, immunoglobulins, anti-animal/human immunoglobulins, etc., can solve the problems of the side effects of kinase inhibitors and the difficulties of kinase inhibitors.

Active Publication Date: 2017-05-31
BEIJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Drugs targeting Tau mainly include Tau hyperphosphorylation inhibitors and Tau protein aggregation inhibitors. The former is mainly an inhibitor of related protein kinases. However, it is very difficult to develop specific kinase inhibitors. Kinase inhibitors There will be various side effects, the main reason is that a kinase inhibitor can simultaneously inhibit the activity of multiple kinases in the organism

Method used

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  • Antibody type molecular chaperone capable of inhibiting Tau protein from being aggregated
  • Antibody type molecular chaperone capable of inhibiting Tau protein from being aggregated
  • Antibody type molecular chaperone capable of inhibiting Tau protein from being aggregated

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1. Prokaryotic expression, separation and purification of Tau protein

[0116] 1. Prokaryotic expression, separation and purification of Tau protein

[0117] 1. Construction of pET21a-Tau plasmid

[0118] Using pGEX-6p-1-Tau plasmid as a template for PCR amplification, the PCR product, which is the Tau gene fragment, was obtained; the PCR product and pET21a plasmid were double digested with restriction enzymes NdeⅠ and XhoⅠ, respectively After the Tau gene and pET21a plasmid backbone vector; ligate the digested Tau gene and pET21a plasmid backbone vector to obtain pET21a-Tau plasmid and verify it by sequencing.

[0119] Sequencing results show that the pET21a-Tau plasmid inserts the DNA fragment shown in sequence 1 into the pET21a plasmid (purchased from Merck Millipore, catalog number 69740-3CN) between the NdeI and XhoI restriction sites, and maintains other sequences of the pET21a plasmid The resulting plasmid is unchanged.

[0120] 2. Construction of recombinant bac...

Embodiment 2

[0149] Example 2. Preparation, separation and purification of single-chain antibody

[0150] 1. Screening and identification of phage antibodies

[0151] 1. Screening of phage antibodies

[0152] Using the phosphorylated Tau protein prepared in Example 1 as the antigen, a large-capacity human phage antibody library was used to screen and obtain scFv single-chain antibodies with chaperone function and inhibit the aggregation of phosphorylated Tau protein. Specific steps are as follows:

[0153] (1) Coating: Coat the immunotube with the phosphorylated Tau protein prepared in Example 1, at 4°C overnight;

[0154] (2) Sealing: Sealing with 30g / L skimmed milk powder for 1 hour;

[0155] (3) Incubation: Wash twice with phosphate buffer containing 0.05% Tween-20, add 1 mL of phage antibody library (entrusted to prepare by the Central Laboratory of Naval General Hospital, titer is 3×10 13 CFU / mL), incubate at 37°C for 2 hours;

[0156] (4) Washing: Repeated washing to remove unbound phage antibo...

Embodiment 3

[0217] Example 3. The effect of single-chain antibody scFv on the aggregation of phosphorylated Tau protein

[0218] 1. The effect of scFv on phosphorylated Tau protein aggregation in dilute solution

[0219] 1. The effect of different scFv on the aggregation of phosphorylated Tau protein

[0220] According to the difference of single chain antibodies, they are divided into the following groups to study the effect of single chain antibodies on phosphorylated Tau protein aggregation:

[0221] Group 1 (control): Phosphorylated Tau protein (prepared in Example 1, final concentration of 5μM), heparin (purchased from Sigma-Aldrich, catalog number H3149, final concentration of 1.25μM), DTT (purchased from Sigma -Aldrich, catalog number D0632, final concentration is 1mM) and Tris-HCl buffer (10mM, pH7.5), mix well, incubate at 37℃, and use ThT fluorescence method to detect the aggregation of phosphorylated Tau protein at different times;

[0222] Group 2 (scFv T1): mix scFv T1 (purified sampl...

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Abstract

The invention discloses an antibody type molecular chaperone capable of inhibiting Tau protein from being aggregated. The antibody type molecular chaperone scFv T1 can effectively inhibit Tau aggregation, is strong in specificity, high in efficiency and low in toxicity and easy to eliminate in the body, and has the advantages of being humanized, convenient for antibody screening, easy to prepare and the like compared with monoclonal or polyclonal antibody prepared by using the traditional method; compared with common immune globulin, the single-stranded antibody has smaller molecules, is easier to express in animal cells in-vitro or in-vivo cultured in model organism, can break through blood brain barrier, has low toxic and side effects, cannot cause immunoreactions easily, can facilitate the research on cell and animal levels and the clinical treatment and applications of Alzheimer disease, thus providing a new direction for treatment of Alzheimer disease, and having potential application values for treating AD, and good medical application prospects.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an antibody-type molecular chaperone that inhibits the aggregation of Tau protein. Background technique [0002] The study of protein folding is one of the current research frontiers in biology. The clarification of protein folding mechanism will reveal how one-dimensional information in life is transformed into three-dimensional information, and the interaction between protein and its ligand determines the mystery of protein function and life process , This is its theoretical significance. At the same time, the study of protein folding has important application value. For example, the abnormal spatial structure of proteins and the incorrect aggregation of proteins can cause diseases. These diseases are called "folding diseases", including mad cow disease, Creutzfeldt-Jakob disease, and Alzheimer's disease. And Parkinson's disease, etc., in-depth understanding of the relations...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13C12N5/10A61K39/395A61P25/28
CPCA61K2039/505C07K16/18C07K2317/56C07K2317/622C07K2317/76
Inventor 李森杨宇丰
Owner BEIJING NORMAL UNIVERSITY
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