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A kind of polypeptide with antitumor activity and its application

A technology of anti-tumor activity and anti-tumor drugs, applied in the field of polypeptides with anti-tumor activity, to achieve the effect of short polypeptide sequence, broad clinical application value and prospects, and significant anti-tumor activity

Active Publication Date: 2022-03-11
江苏莱森生物科技研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of reports on LHRHR targeted drug research by phage display technology in the prior art

Method used

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  • A kind of polypeptide with antitumor activity and its application
  • A kind of polypeptide with antitumor activity and its application
  • A kind of polypeptide with antitumor activity and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: pET-30a / (His) 6 - Construction of LHRHR protein expression vector

[0020] Such as figure 1 Construct pET-30a / (His) as indicated 6 -LHRHR, search the gene sequence of LHRHR (1-125AA, NM_000233.4) from the Genbank database, design PCR primers, the upstream primer sequence is: 5'-CG GGATCC ATGAAGCAGCGGTTCTCGGC-3' (BamHI), the downstream primer sequence is: 5'-CC AAGCTT GCTCCGGGCTCAATGTATCT-3' (HindⅢ), the underlined part is the restriction site sequence. The PCR product and the vector pET-30a were digested with BamHI and Hind III at 37°C for 3 h, then ligated with T4 DNA ligase at 16°C for 12 h. Transform the ligation product into DH5α competent cells, then spread the transformation product on a kanamycin-resistant (50 μg / ml) LB plate and culture until a single colony grows, pick a single colony, and extract the plasmid for enzyme digestion verification , the recombinant plasmid was sequenced to obtain the recombinant plasmid pET-30a / (His) 6 - LHRHR.

Embodiment 2

[0021] Example 2: (His) 6 - Expression, purification and validation of LHRHR

[0022] The recombinant plasmid pET-30a / (His) prepared in Example 1 6 -LHRHR was transformed into BL21 (DE3) strain, and the recombinant plasmid was screened on a kanamycin-resistant LB plate, and cultured to OD in 10 ml LB liquid medium (containing 50 μM kanamycin) 600 After about 0.5, the culture was inoculated in multiple bottles of LB liquid medium at a volume ratio of 1: 10, and cultured to OD at 37°C with vigorous shaking 600 About 0.5, add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 1 mM and induce at 37 °C for 10 h to obtain a bacterial solution.

[0023] Centrifuge the bacterial liquid, remove the supernatant, and resuspend the bacterial pellet in the lysate (50 mM Tris–HCl, 20 mM imidazole, 100 mM NaCl, 10% glycerol , 1% Triton, 1 mM protease inhibitor PMSF, 1 mg / ml lysozyme, pH 8.0), placed on ice for 30 min, sonicated, and centrifuged at 12000 g for 30 min to...

Embodiment 3

[0025] Example 3: Phage display panning for bioactive peptides specifically binding to LHRHR

[0026] (1) Immobilize the target protein: mix 600 μl of the target protein solution with a concentration of 17 μg / ml (0.1 M NaHCO 3 pH8.6) was added to a six-well plate, placed on a shaker with slight shaking, and incubated overnight at 4°C. After washing 6 times with TBST (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% [v / v] Tween-20 ), blocked with blocking solution (0.1 M NaHCO 3 pH 8.6, 5 mg / ml BSA, 0.02% NaN 3 ) closed for 1 h.

[0027] (2) Screening for specifically binding phages: Wash the six-well plate 10 times with TBST. The amplified phage was diluted with TBST to a copy number of 10 9 ~10 11 In between, the diluted phage was added to the six-well plate to allow it to bind to the target protein, and incubated at room temperature for about 60 min. Wash 10 times with TBST and pat dry after each wash. Add eluent to collect phages that specifically bind to the target protei...

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Abstract

The invention belongs to the field of biotechnology, and specifically relates to a polypeptide with antitumor activity and its application. In the present invention, through the process of constructing protein expression vector, expressing and purifying target protein, verifying target protein, phage display and panning biologically active peptides specifically binding to target protein, in vitro detection experiment of polypeptide anti-tumor effect, etc., the finally screened products with Peptides with antitumor activity. The polypeptide provided by the invention takes the luteinizing hormone-releasing hormone receptor (LHRHR) as the target molecule, and is obtained through three rounds of panning by phage display technology. The provided polypeptide sequence is relatively short, easy to synthesize and realize large-scale production, and has important application value in the research and development of antitumor drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a polypeptide with antitumor activity and its application. Background technique [0002] Luteinizing hormone-releasing hormone (LHRH), also known as gonadotropin-releasing hormone (GnRH), is a nomethyl peptide secreted by the pituitary gland and plays an important role in the reproduction of mammals. Luteinizing hormone-releasing hormone receptor (LHRHR) is a seven transmembrane G protein-coupled protein that is activated by GnRH binding and releases luteinizing hormone (LH) from the pituitary gland through stimulation of tyrosine phosphatase. Recent studies have shown that ovarian cancer is an LHRH-dependent tumor. The expression of LHRHR in ovarian cancer cells is higher than that in normal tissues, and LHRHR becomes a promising therapeutic target in ovarian cancer. [0003] Phage display technology is to insert the DNA sequence of foreign protein or polypeptide into t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/23C07K1/22C12N15/70A61K38/09A61P35/00
CPCC07K7/23C12N15/70A61P35/00A61K38/00
Inventor 刘晗青屠志刚
Owner 江苏莱森生物科技研究院有限公司
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