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CAMSAP1 protein and application of gene thereof as epileptic drug target

A protein and target technology, used in drug combinations, compound screening, peptide/protein components, etc., can solve problems such as weak depolymerization ability

Inactive Publication Date: 2020-03-06
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the function of CAMSAP2 and CAMSAP3 to stabilize the negative end of non-centrosomal microtubules, although CAMSAP1 can be located at the negative end of non-centrosomal microtubules, its ability to resist the depolymerization of microtubules by MCAK is weak, and CAMSAP1 can not be very A well-stabilized microtubule negative end inhibits the polymerization and depolymerization of the negative end, and CAMSAP1 is functionally different from CAMSAP2 and CAMSAP3

Method used

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  • CAMSAP1 protein and application of gene thereof as epileptic drug target
  • CAMSAP1 protein and application of gene thereof as epileptic drug target
  • CAMSAP1 protein and application of gene thereof as epileptic drug target

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0205] Embodiment 1, the impact of CAMSAP1 protein on mouse body weight and survival time

[0206] 1. Construction of CAMSAP1 knockout mice

[0207] To construct CAMSAP1 knockout mice, the 13th exon of CAMSAP1 was selected as the target site and knocked out by homologous recombination ( figure 1 Middle A).

[0208] The vector used for homologous recombination (targeting vector) is: DT-A-pA / loxP / PGK-Neo-pA / loxP, such as figure 1 As shown in A, the vector contains the homologous sequences (5'arm and 3'arm) on both sides of the Camsap1 gene mutation site, and the inserted loxP-Neo-loxP sequence. The expression of CAMSAP1 was terminated prematurely due to the insertion of Neo.

[0209] 2. Detection of CAMSAP1 in CAMSAP1 knockout mice

[0210] 1. The CAMSAP1 gene was detected in wild-type mice, CAMSAP1 knockout mice and heterozygous mice by Southern hybridization. The probe sequences for Southern hybridization are shown in SEQ ID NO: 3 (5'probe) and SEQ ID NO: 4 (3'probe), res...

Embodiment 2

[0232] Embodiment 2, the distribution of CAMSAP family protein in cerebral cortex and neuron

[0233] 1. Expression of CAMSAP family proteins detected by Western blot

[0234] Previous studies have shown that CAMSAP family proteins are expressed in the rat brain, but CAMSAP2 is mainly expressed, CAMSAP1 and CAMSAP3 are less expressed, especially the expression of CAMSAP1 is extremely weak, and the expression of CAMSAP1 is not detected in the hippocampus. Therefore, firstly, whether CAMSAP1 protein is expressed in the mouse central nervous system was detected.

[0235] The total protein of the cerebral cortex, hippocampus of different periods and hippocampal neuron cells cultured for different periods were extracted respectively, and detected by Western blot using CAMSAP1 antibody, CAMSAP2 antibody and CAMSAP3 antibody respectively.

[0236] Figure 4 The upper panel in middle A shows the results of immunoblotting of the cerebral cortex, and the lower panel shows the percenta...

Embodiment 3

[0266] Example 3, CAMSAP1 participates in regulating the polarity establishment of neurons in vitro

[0267] 1. CAMSAP1 is involved in regulating the polarity establishment of hippocampal neurons in vitro

[0268] 1. The inventors of the present invention found through the co-staining of the neuron axon marker protein Tau-1 antibody and the dendrite marker protein Map2 antibody that compared with the cultured wild-type hippocampal neurons, the neurons after knocking out CAMSAP1 grew out The proportion of neurons with single axons (only one protrusion is Tau-1 positive) decreases and the proportion of neurons with multiple axons (multiple protrusions are Tau-1 positive) increases ( Figure 9 Middle A), the ratio of the first and second longest neurites, the longest neurite length, the total length of all neurites and the total number of neuron neurites all changed ( Figure 9 Middle C, D, E and F). This suggests that knocking out CAMSAP1 does alter neuronal polarity.

[0269...

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Abstract

The invention discloses a CAMSAP1 protein and the application of a gene thereof as an epileptic drug target. The 13th exon of CAMSAP1 is selected as a target site, and a wild mouse is knocked out through a homologous recombination method to acquire a knocked-out mouse. Experiments prove that compared with wild type mice, the knocked-out mouse is small in size and light in weight, and the death rate is increased. The CAMSAP1 protein is knocked out to change the polarity of neurons. The knocked-down CAMSAP1 protein can influence the transformation of neurons from multipolar cells to bipolar cells, resulting in abnormal migration of neurons along radial glial cells (the migration of neurons is influenced), and abnormal layered arrangement of neurons in cerebral cortex. The knocked-out CAMSAP1protein can cause epilepsy. Therefore, the CAMSAP1 protein can maintain normal growth of neurons and prevent and / or treat epilepsy. The CAMSAP1 protein and the application, which are provided by theinvention, have an important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the use of CAMSAP1 protein and its gene as epilepsy drug targets. Background technique [0002] As a cytoskeleton, microtubules participate in many life activities such as cell division, establishment of cell polarity, cell migration, and intracellular transport. Structurally, microtubules are assembled longitudinally into linear protofibrils by heterodimers formed by negatively charged α-tubulin and β-tubulin end-to-end, and then 10-15 protofibrils pass through the side The walls are aligned to form a hollow tubular structure with a diameter of 24nm. The microtubule is a highly dynamic structure, and its two ends are always in the dynamic process of polymerization and depolymerization. Among them, the end with β-tubulin and faster assembly speed is called the positive end, while the end with α-tubulin The end of the protein with a relatively slow assembly speed is calle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/561A61K49/00A61K38/17A61K45/00A61P25/08
CPCA61K38/17A61K45/00A61K49/0004A61K49/0021A61K49/006A61P25/08G01N33/561G01N33/6893G01N2500/04G01N2800/2857
Inventor 孟文翔周峥嵘徐鸿林陈荣清
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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