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Fusion polypeptide and application of fusion polypeptide in preparing anti-tumor medicines

A technology for fusing polypeptides and drugs, applied in the field of biomedicine, can solve problems such as affecting activity, and achieve the effect of inhibiting migration and promoting growth

Active Publication Date: 2014-12-10
SHANDONG UNIV QILU HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no literature reporting whether DYRK1A phosphorylates NFAT2 or affects the latter's activity

Method used

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  • Fusion polypeptide and application of fusion polypeptide in preparing anti-tumor medicines
  • Fusion polypeptide and application of fusion polypeptide in preparing anti-tumor medicines
  • Fusion polypeptide and application of fusion polypeptide in preparing anti-tumor medicines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Preparation and purification of fusion polypeptide TAT-NFAT2

[0050] (1) Determining the DYRK1A action site on NFAT2

[0051] 1. The substrate of DYRK1A (the substrate refers to the protein that DYRK1A can phosphorylate, that is, the target protein) contains a conserved RPX(S / T)P domain (ie arginine-proline-any amino acid-serine or amino acid sequence of threonine-proline). Such as figure 1 As shown in A, the sequence analysis of NFAT2 protein by ClustalW2 software shows that it has three similar RPX(S / T)P domains. In order to determine the action site or target of DYRK1A on NFAT2, construct NFAT2 vector (sequence shown in SEQ ID NO.27) and NFAT2 deletion vector 1-272aa (sequence shown in SEQ ID NO.28), 1-308aa (sequence shown in SEQ ID NO.29), 1-433aa (sequence shown in SEQ ID NO.30), 307-716aa (sequence shown in SEQ ID NO.31) and 424-716aa (sequence shown in SEQ ID NO.31) and 424-716aa (sequence shown in SEQ ID NO.31) shown in NO.32).

[0052] The NF...

Embodiment 2

[0079] Example 2: The fusion polypeptide TAT-NFAT2 inhibits the effect of DYRK1A on NFAT2 in tumor cells

[0080] (1) DYRK1A increases the expression of NFAT2 protein in tumor cell HEK293

[0081] 1. pGFP-V-RS-shDYRK1A (it is the knockout vector of DYRK1A, which can express a 20-length nucleotide sequence, which can degrade the mRNA of DYRK1A and inhibit the protein expression of DYRK1A) was constructed by our laboratory (EST negative feedback loop regulates DYRK1A. Journal of Biochemistry (2011) 286, 10755-10763 (see literature Lu, M., Zheng, L., Han, B., Wang, L., Wang, P., Liu, H., and Sun, X. (2011) The Journal of biological chemistry 286, 10755-10763).

[0082] Using Liposome 2000 (11668-027; Invitrogen), p-NFAT2mycflag was co-transfected with pCMV-DYRK1A and pGFP-V-RS-shDYRK1A into HEK293 cells, respectively, at 37°C, 5% CO 2 Cells were harvested after 48 hours of culture in the environment, and the cells were lysed with RIPA lysate containing 0.1% SDS to obtain the...

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PUM

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Abstract

The invention discloses a fusion polypeptide. The fusion polypeptide is prepared from polypeptide I and polypeptide II, wherein the polypeptide I is a specific amino acid sequence on NFAT2, and is selected from one or combination of sequences as shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4; the polypeptide II is an HIV-TAT protein transduction domain, and the sequence of the polypeptide II is as shown in the SEQ ID NO.1. The fusion polypeptide is a competitive inhibitor against DYRK1A phosphorylated NFAT2; in-vitro tests indicate that the fusion polypeptide is capable of lowering the protein level of NFAT2 in tumor cells, so that metastasis of the tumor cells can be inhibited. According to the fusion polypeptide, a new target is provided for design of novel anti-tumor medicines, and a new thought is provided for reverse of multidrug resistance of tumors.

Description

technical field [0001] The invention relates to a fusion polypeptide capable of inhibiting the effect of DYRK1A on the expression and transcription activity of T cell nuclear factor (nuclear factor of activated T cell, NAFT) protein, and its application, belonging to the technical field of biomedicine. Background technique [0002] Tumor is the number one killer of human health. The rapid growth, invasion and distant metastasis of malignant tumor cells to surrounding tissues and organs are the main reasons why tumors lead to the death of patients. The protein encoded by the tumor suppressor gene has the functions of inhibiting tumor growth and tumor invasion and metastasis, and has become a candidate drug for antitumor drugs. Peptide drugs have attracted widespread attention due to their advantages such as small molecules, easy synthesis, easy structure-activity relationship research, easy modification, strong pertinence, and obvious curative effects. In recent years, wit...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K38/10A61K47/48A61P35/00
Inventor 刘恒孙秀莲
Owner SHANDONG UNIV QILU HOSPITAL
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