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Application of small molecule peptide nfib

A small molecule polypeptide and DNA sequence technology, which is applied to the small molecule polypeptide NFIBΔ and its carrier and in the application field of anti-lung cancer proliferation, can solve problems such as poor prognosis and achieve the effect of inhibiting cell proliferation

Active Publication Date: 2019-08-30
太仓泽鑫生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Despite great advances in lung cancer diagnosis and multimodal treatment including chemotherapy in recent years, the prognosis remains poor

Method used

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  • Application of small molecule peptide nfib
  • Application of small molecule peptide nfib
  • Application of small molecule peptide nfib

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Co-immunoprecipitation of NFIB full-length protein and SOX2 in lung cancer tissues and cells

[0020] Materials: Protein A / G beads, cell lysate, NFIB antibody, SOX2 antibody, IgG.

[0021] Method: Homogenize small pieces of fresh tissue; wash cells twice with pre-cooled 0.01M PBS; add 1mL PBS, scrape cells on ice, transfer to 1.5mL centrifuge tube, collect A549 cells by centrifugation at 1000g×5min Add 500 μL of the prepared cell lysate and place on a rotator at 4°C for 30 minutes to completely lyse the cells, centrifuge at 12000g at 4°C for 10 minutes, and transfer the supernatant to a new EP tube. Add 20 μL of mixed Protein A / G beads for pre-clarification, rotate on a DNA mixer for 1 hour, centrifuge at 900g for 5 minutes (4°C), transfer the supernatant to a new centrifuge tube, and discard the beads. Take 40-60 μL supernatant as Input, add an equal amount of 2×SDS loading buffer, cook in boiling water for 5-10 minutes, centrifuge and freeze. The remainin...

Embodiment 2

[0023] Embodiment 2: Small molecule polypeptide NFIB Δ Cloning vector construction and co-immunoprecipitation identification with SOX2

[0024] The invention provides a small molecular polypeptide capable of competing for binding to SOX2 and inhibiting the proliferation of tumor cells. The small molecular polypeptide is coded by its coding gene to design corresponding primers, synthesized by polymerase chain reaction (PCR) and then inserted into an expression plasmid. Then the expression vector was transformed into Escherichia coli BL21, cultured in LB medium, and induced to express at 37°C. Take the supernatant after breaking the bacteria, and use the DNA extraction kit from OMEGA to obtain the purified target DNA, which is named NFIB Δ ; Its nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence of the corresponding encoded protein is shown in SEQ ID NO.1. Afterwards, lung cancer A549 cells were transfected, and NFIB was detected by co-immunoprecipitation...

Embodiment 3

[0033] Example 3: Small molecule polypeptide NFIB Δ Detection of anti-lung cancer function

[0034] 1) PI staining flow cytometry to demonstrate NFIB Δ Inhibition of lung cancer cell cycle

[0035] Materials: A549 cells, serum-free medium, PBS, trypsin, low-speed shaker, propidium iodide, pipette tips.

[0036] Method: Cells were pressed 4 x 10 per well 5 The density of each was inoculated into a 60mm cell culture dish, and after overnight culture, different expression plasmids (3XFLAG-CMV-13 plasmid expressing the wild full-length form of NFIB; empty plasmid control; expressing 231 amino acid small molecule polypeptide NFIB Δ Type p3XFLAG-CMV-13 plasmid) was transfected into A549 cells, and the cell cycle was detected by PI staining flow cytometry. Cells were collected by trypsinization, and the remaining cells were washed once with 1 mL of PBS buffer, all added to a 15 mL tube. Centrifuge at 800 rpm for 5 minutes, remove the supernatant, add 5 mL of PBS buffer to resusp...

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Abstract

The invention discloses an application of a micro-molecular polypeptide NFIB<delta>. With adoption of a bioengineering technology, a segment of NFIB truncated gene is subjected to genetic recombination into a polypeptide with the length of 231 amino acids, and the polypeptide can be specifically bound with SOX2 and block interaction between endogenous full-length NFIB and SOX2; biological experiments prove that the micro-molecular polypeptide NFIB<delta> has an effect of inhibiting lung cancer cell proliferation. New experimental basis is provided for targeting therapy of lung tumor, and the polypeptide can have important clinical application value in research and development of drugs for targeting therapy of the lung tumor on the basis of the characteristics of high efficiency and low toxicity.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to small molecule polypeptide NFIB Δ Its carrier and its application in anti-proliferation of lung cancer. Background technique [0002] Lung cancer is one of the most common malignant tumors in the world, and it has ranked first among the main causes of cancer-related death in many countries. Although the diagnosis and multimodal treatment of lung cancer, including chemotherapy, have made great progress in recent years, the prognosis is still not optimistic. The occurrence of lung cancer is the accumulation of gene damage. With the continuous discovery and in-depth research of new lung cancer oncogenes, the treatment of targeted drugs targeting the corresponding genes has gradually entered the clinic. [0003] With the rapid development of biotechnology, peptide drugs continue to emerge. It has attracted attention for its simple and easy-to-obtain raw materials, high drug...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/85A61K38/17A61P35/00
Inventor 季俐俐沈爱国刘琨王铄
Owner 太仓泽鑫生物科技有限公司
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