A kind of erk1/2 protein inhibitory polypeptide, its preparation method and application

A protein inhibition, recombinant plasmid technology, applied in the field of bioengineering, can solve the problems of restricting ERK1/2 protein research, influence, and no specific ERK1/2 inhibitor, etc., to achieve in vivo research, broad application prospects, cell less toxic effect

Inactive Publication Date: 2019-02-26
金鑫
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no specific inhibitor against ERK1 / 2 at present, so the purpose of inhibiting ERK1 / 2 activity can only be achieved indirectly by inhibiting its upstream proteins, such as MEK protein, etc.
Since one of the most important characteristics of cell signal transduction is to form a complex signal network system with highly nonlinear characteristics, there is a phenomenon of cross-talking between different signal pathways in cells, and the Ras signal pathway As one of the most important signaling pathways inside the cell, the upstream protein of ERK1 / 2 is usually the conduction node of other signaling pathways, so the inhibition of its upstream protein will have a wide impact on the activities inside the cell, and thus cannot specifically target ERK1 / 2 protein research, which greatly limits the research on ERK1 / 2 protein

Method used

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  • A kind of erk1/2 protein inhibitory polypeptide, its preparation method and application
  • A kind of erk1/2 protein inhibitory polypeptide, its preparation method and application
  • A kind of erk1/2 protein inhibitory polypeptide, its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 contains the interaction verification and preliminary functional verification of FBPe4 and IQGAP1

[0042] S11. Main experimental materials:

[0043] 1), cells

[0044] In this experiment, the pancreatic cancer cell line PANC-1 was selected. The pancreatic cancer cell line is a K-ras mutant cell line, and pERK1 / 2 can be detected under normal conditions, so the changes of pERK1 / 2 can be observed after FBPe4 treatment, which is convenient for observing the experimental effect.

[0045] PANC-1 was purchased from ATCC (American Type Culture Collection, Manassay, VA, USA). PANC-1 cells used DMEM medium (CORNING, Manassa, VA, USA) containing 10% fetal bovine serum (Life Technologies), 100 units / ml penicillin and 100 μg / ml streptomycin (Life Technologies). Cultured in a cell incubator with a culture condition of 5% CO 2 , 37°C, 95% humidity.

[0046] 2), plasmid

[0047] Flag-FBP1, FBPe4 and FBPΔE4 were loaded into pcDNA3.1 / V5-His-TOPO (Invitrogen, 460083) vect...

Embodiment 2

[0070] Embodiment 2 Containing the construction of the recombinant plasmid of FBPe4 and the expression of FBPe4

[0071] According to the base sequence shown in SEQ ID No: 2, this sequence was cloned into the expression vector pcDNA3.1 / V5-His-TOPO (containing Fag tag, which can be detected by Flag antibody).

[0072] S21. Amplify the target DNA by PCR, and recover the target DNA by electrophoresis

[0073] Utilize the FBP1 plasmid (sequence shown in SEQ ID No:3) as template amplification to obtain the gene sequence of FBPe4 (i.e. SEQ ID No:2), the PCR amplification reaction system is:

[0074]

[0075] Among them, the FBPe4 upstream primer is:

[0076] 5-CGGATATCAAGGATGCTCTGCAACCAGGCCGG-3';

[0077] FBPe4 downstream primers:

[0078] 5'-CGCTCGAGGGCAAGGACCAGCATGGTGGCACT-3'.

[0079] The PCR reaction procedure is:

[0080]

[0081] After the completion, the amplified product was subjected to agarose electrophoresis, and the target band of about 100 bp was cut out, and t...

Embodiment 3

[0101] Example 3 FBPe4 inhibits the phosphorylation of ERK1 / 2

[0102] S31. Using the pcDNA3.1 / V5-His-TOPO-FBPe4 recombinant plasmid to construct the FBP1G260R ​​plasmid.

[0103] Primers constructed by FBP1G260R ​​(glycine at position 260 replaced with arginine):

[0104] Upstream primers:

[0105] 5'-CATCGCACTCTGGTCTACAGAGGGATATTTCTGTAC-3'

[0106] Downstream primers:

[0107] 5'-AACATCAGCCACCATGGAGCCCACATACCGGGCCC-3'

[0108] FBP1G260R ​​lost the enzymatic activity of synthesizing glycogen.

[0109] The structures of FBP1G260R ​​and FBP1ΔFBPe4 mutant proteins are as follows: image 3 As shown in A.

[0110] S32. The FBP1G260R ​​and FBP1ΔFBPe4 plasmids were transformed into PANC-1 cells and expressed, and the cell lysate was collected and used for co-immunoprecipitation experiments with Flag antibody (the recombinant proteins expressed by FBP1G260R ​​and FBP1ΔFBPe4 plasmids all had Flag tags).

[0111] The experimental results show that FBP1, FBP1G260R, and FBPe4 can ...

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Abstract

The invention relates to the field of bioengineering, in particular to an ERK1 / 2 protein inhibiting polypeptide which can competitively inhibit combination of ERK1 / 2 and IQGAP1 to inhibit phosphorylation of ERK1 / 2.The ERK1 / 2 protein inhibiting polypeptide only has 21 amino acids, both artificial synthesis of the polypeptide and preparation of the polypeptide through host cell expression are very convenient and easy to operate, and therefore large-scale utilization and popularization are facilitated; in addition, the polypeptide is derived from inherent protein in an organism, immune elimination cannot be caused when the polypeptide is used for an animal experiment, most of inhibitors are generally artificially synthesized compounds, and by contrast, the protein polypeptide has less toxicity to cells and is more special.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to an ERK1 / 2 protein inhibitory polypeptide, its preparation method and application. Background technique [0002] Ras protein is a small G protein, and the RAS signaling pathway is a very common and important cell molecular signal transduction pathway. The signaling pathway is: Ras→Raf(MAPKKK)→MAPKK(MEK)→MAPK(ERK1 / 2)→entering the nucleus→transcription factor→gene expression. The RAS pathway is closely related to inflammation, tumor and so on. Therefore, it has become a target for the treatment of inflammation and tumors. [0003] At present, a variety of inhibitors have been developed for multiple links of the Ras pathway. However, there is no specific inhibitor against ERK1 / 2 at present, so the purpose of inhibiting ERK1 / 2 activity can only be achieved indirectly by inhibiting its upstream proteins, such as MEK protein. Since one of the most important characteristics of cell sign...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/70C12N1/21C12R1/19
CPCC12N9/16
Inventor 金鑫黄浩杰
Owner 金鑫
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