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Microtubule-associated protein and coding genes and application thereof

A protein and gene technology, applied in the field of microtubule-binding protein and its coding gene and application, can solve problems such as abnormal chromosome separation and microtubule depolymerization

Inactive Publication Date: 2010-08-25
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

MCAK exists in the form of dimers in the body. When MCAK is highly expressed, it will cause the depolymerization of microtubules and the abnormal separation of chromosomes.

Method used

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  • Microtubule-associated protein and coding genes and application thereof
  • Microtubule-associated protein and coding genes and application thereof
  • Microtubule-associated protein and coding genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. Acquisition of the gene encoding microtubule-binding protein TIP150

[0048] Using the cDNA of human cervical cancer HeLa cells (Stratagene, Cat.No.937248) as a template, PCR amplification was carried out, and the primers used for PCR amplification were as follows:

[0049] Upstream primer: 5'-gcgaattctatgagcgtcccagtgg-3' (sequence 3 of the sequence listing);

[0050] Downstream primer: 5'-gcgtcgactcatctgggtgttgtgc-3' (SEQ ID NO: 4 in the Sequence Listing).

[0051] Digest the PCR product with EcoRI and SalI, connect the digested PCR product to the vector pMD18-T (TaKaRa Company) after the same digestion, transform the ligated product into Escherichia coli (E.coli) DH5α competent cells, and screen positive Cloning and extracting the plasmid, the recombinant plasmid containing the target fragment was obtained, which was named pMD-TIP150.

[0052] Sequencing was performed on pMD-TIP150, and the sequencing results showed that the human TIP150 gene has the nucl...

Embodiment 2

[0054] The construction of the expression vector of embodiment 2, TIP150 and EB1 and deletion mutant thereof

[0055] 1. Construction of GFP-TIP150

[0056] pMD-TIP150 was digested with restriction endonucleases EcoRI and SalI, and the digested product was ligated with pEGFP-C1 (Clontech, Cat. No. 6084-1) digested in the same way to obtain GFP-TIP150.

[0057] 2. Construction of MBP-TIP150

[0058] pMD-TIP150 was digested with restriction endonucleases EcoRI and SalI, and the digested product was ligated with pMAL-c2x (New England Biolabs, Cat. No. N8076S) digested in the same way to obtain MBP-TIP150.

[0059] 3. Construction of flag-TIP150

[0060] pMD-TIP150 was digested with restriction endonucleases EcoRI and SalI, and the digested product was ligated with p3XFLAG-myc-CMV-24 (Sigma, Cat. No. E6151) digested in the same way to obtain flag-TIP150.

[0061] 4. Construction of GST-EB1

[0062] Using the cDNA of human cervical cancer HeLa cells as a template, carry out PCR...

Embodiment 3

[0111] Example 3, Identification of the interaction region between TIP150 and EB1

[0112] 1. In vitro pull-down detection of the interaction between TIP150 and EB1

[0113] MBP-TIP150 was expressed in Rossetta (DE) pLysS host bacteria at 30°C, and then purified with Amylose resin (New England Biolabs) according to standard procedures to obtain purified MBP-TIP150 fusion protein. GST-EB1 was expressed in Rossetta (DE) pLysS host bacteria at 30°C, and then purified with glutathione-Sepharose 4B (Amersham Biosciences) according to standard procedures to obtain purified GST-EB1 fusion protein. pMAL-c2x was expressed in Rossetta (DE) pLysS host bacteria at 30°C, and then purified with Amylose resin (New England Biolabs) according to standard procedures to obtain purified MBP protein. pGEX-6P-1 was expressed in Rossetta (DE) pLysS host bacteria at 30°C, and then purified with glutathione-Sepharose 4B (Amersham Biosciences) according to standard procedures to obtain purified GST pr...

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Abstract

The invention discloses a microtubule-associated protein and coding genes and application thereof. The microtubule-associated protein is protein of (a) or (b): (a) protein formed by an amino acid sequence as shown by a sequence one in a sequence table; and (b) protein derived by the sequence one, related to the inhibition of the microtubule de-polymerization activity of MCAK protein and formed ina way the amino acid sequence of the sequence one is substituted and / or deleted and / or added by one to ten amino acid residues. The protein of the invention participates in the regulation and the control of the forward end dynamics of microtubules through the mutual effect with EB1 and MCAK and stabilizes the microtubules through inhibiting the microtubule de-polymerization activity of the MCAK protein. After the coding genes of the low-interference RNA of microtubule-associated protein genes are introduced into a host, the microtubule positioning of the MCAK is seriously affected. The protein and the coding genes thereof play a great role in the medical field and the pharmaceutical field and thereby the application prospect is wide.

Description

technical field [0001] The invention relates to a microtubule binding protein and its coding gene and application. Background technique [0002] Microtubules are highly dynamic fibrous cytoskeleton structures that play key roles in cell mitosis, cell migration, and cell polarization. The growth and contraction of microtubules are regulated by a series of microtubule-binding proteins. Dysregulation of microtubule dynamics will cause cells to fail to perform mitosis accurately, endangering the survival and health of organisms. [0003] Among the microtubule-binding proteins, there is a class of proteins that specifically bind to the forward end of growing microtubules, such as EB1 / 2 / 3, CLIP-115 / 170, CLASP1 / 2, p150Glued, adenomatous polyposis coli (APC), MCAK, ACF7, LIS1, Melanophilin and STIM1. How these proteins organize and regulate microtubule dynamics is unknown. Most of these proteins directly interact with EB1. [0004] EB1, one of the three members of the EB family...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/63C12N5/10C12N1/00C12N15/11C12N15/09A61K38/17A61K48/00A61P35/00
Inventor 姚雪彪姜恺王建宇王志凯
Owner UNIV OF SCI & TECH OF CHINA
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