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CMKLR1 antagonistic polypeptide and derivative and application thereof

A derivative and antagonistic technology, applied in the field of biotechnology and biomedicine

Active Publication Date: 2019-06-28
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As CMKLR1 is one of the members of GPCRs, there is still no drug that directly targets CMKLR1 to treat clinical cancer.

Method used

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  • CMKLR1 antagonistic polypeptide and derivative and application thereof
  • CMKLR1 antagonistic polypeptide and derivative and application thereof
  • CMKLR1 antagonistic polypeptide and derivative and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0172] Example 1: Panning, amplification, purification, sequencing and synthesis of CMKLR1 antagonistic polypeptide LRH12-C5.

[0173] This example is mainly for the purpose of screening positive phages that specifically bind to CMKLR1, and then by amplifying and purifying the positive phages, extracting phage single-stranded DNA (ssDNA) for sequencing, analyzing and comparing the obtained sequences, and finally synthesizing high-purity The antagonistic polypeptide LRH12-C5.

[0174] details as follows:

[0175]1. Establishment of 293T cell line with permanent high expression of CMKLR1: 293T-CMKLR1 + / + / LRH

[0176] ①Select vigorously growing luminescent human 293T cells, and the day before transfection, use 5×10 5 cells / well, inoculated in a 6-well plate, cultured until the second day, the cell fusion degree was 60%;

[0177] ② Transfect on the second day, take one culture well of a 6-well plate as a unit, dilute 3 μg of plasmid with 200 μL of opti-MEM medium, and dilute ...

Embodiment 2

[0194] Example 2 The CMKLR1 antagonistic polypeptide LRH12-C5 can effectively alleviate the inhibitory effect of chemerin on the cAMP signaling pathway.

[0195] (1) Cyclic adenosine monophosphate (cAMP) ELISA:

[0196] ① Cell plating: Wild-type 293T cells and 293T cells highly expressing CMKLR1 (293TCMKLR1 + / + ), with 5x10 5 Each well was inoculated in a 6-well cell culture plate, and the medium volume of each well was 1 mL. After being placed in an incubator for 24 hours, starved overnight, and LRH12-C5 polypeptides with different concentration gradients (3 μM, 0.3 μM, 0.03 μM) were added. , Fosklin (25μM) and chemerin (30nM) for 6h;

[0197] ②Sample preparation: Add 300 μL of cell lysate to each well, place at 4°C for 20 minutes, scrape and collect cells with a cell scraper, mix them upside down, centrifuge at 12,000 rpm for 10 minutes, and collect the supernatant;

[0198] ③ Determination of sample concentration: the sample concentration is determined by BCA method;

...

Embodiment 3

[0206] Example 3 CMKLR1 antagonistic polypeptide LRH12-C5 can effectively inhibit calcium (Ca 2+ ) inflow effect.

[0207] ① Cell plating: Wild-type 293T cells and 293T cells highly expressing CMKLR1 (293T CMKLR1 + / + ), with 5x10 3 Each cell / well was inoculated in a 96-well cell culture plate, the volume of medium in each well was 200 μL, placed in an incubator for 24 hours, and then starved overnight;

[0208] ②Reagent configuration: dissolve probenecid into 1mL buffer solution to prepare probenecid with a concentration of 250nM, shake well, add to fluorescent reagent for use;

[0209] ③Remove the cell culture medium, add LRH12-C5 polypeptide and chemerin (0.3nM) with different concentration gradients (30μM, 3μM, 0.3μM, 0.03μM, 0.003μM) for 30 minutes, and then add 100μL of the above fluorescent reagent to each well;

[0210] ④ Place at 37°C for 30 minutes, then at room temperature for 30 minutes;

[0211] ⑤Measure the fluorescence absorbance at excitation light 494nm and...

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Abstract

The invention discloses a CMKLR1 antagonistic polypeptide and a derivative and application thereof and particularly relates to a CMKLR1 antagonistic polypeptide shown in SEQ ID No.1-24 and a derivative thereof. The derivative of the binding peptide is a product obtained in the mode that conventional modification is conducted on a CMKLR1 binding peptide amino acid side chain group and an amino terminal or a carboxyl terminal of a CMKLR1 antagonistic polypeptide fragment or a product obtained in the mode that a label for detection or purification of polypeptide or protein is linked to the CMKLR1antagonistic polypeptide. The binding peptide and the derivative thereof can be combined with CMKLR1 in vitro, by stopping combination of chemerin / RvE1 and CMKLR1, increase of the cAMP concentrationis promoted, the calcium influx caused by chemerin and cell chemotaxis caused by chemerin are inhibited, proliferation of ovarian carcinoma cells can be inhibited, apoptosis of the ovarian carcinoma cells is promoted, and effective micromolecule treatment drugs are provided for female reproductive diseases such as ovarian cancer.

Description

technical field [0001] The present invention relates to the fields of biotechnology and biomedicine, specifically, the present invention is the female reproductive disease target CMKLR1 receptor antagonistic polypeptide LRH12-C5 and its derivatives and applications. Background technique [0002] Among female reproductive cancers, some are related to endocrine. For example, breast cancer (Breast Cancer), endometrial cancer (Endometrial Cancer) and ovarian cancer (Ovarian Cancer, OAC), etc. Among them, ovarian cancer is a malignant tumor that occurs in the ovary, 90% to 95% are primary ovarian cancers, and the other 5% to 10% are primary cancers in other parts that have metastasized to the ovary. Although the incidence of ovarian cancer in my country is not as high as that in Europe and the United States, radical surgery and cytotoxic chemotherapy are not effective in reducing the mortality of ovarian cancer. Due to lack of symptoms in the early stage of ovarian cancer, even...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/11A61K38/16A61P35/00A61P1/16A61P3/10A61P29/00
Inventor 张键代小勇陈杰黄晨肖天霞
Owner SHENZHEN INST OF ADVANCED TECH
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