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Lung carcinoma cell specificity conjugation oligopeptide and application thereof

A lung cancer cell and specific technology, applied in the field of protein peptides, can solve the problem of few reports related to screening lung cancer targeting ligands, and achieve the effect of small molecular weight, high specificity and good tissue penetration

Inactive Publication Date: 2014-05-14
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, this technology has successfully screened targeting peptides targeting liver cancer, prostate cancer, breast cancer and other human tumors, but there are few reports on screening targeting ligands for lung cancer

Method used

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  • Lung carcinoma cell specificity conjugation oligopeptide and application thereof
  • Lung carcinoma cell specificity conjugation oligopeptide and application thereof
  • Lung carcinoma cell specificity conjugation oligopeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Screening of Lung Cancer Specific Binding Polypeptides

[0023] In this example, a phage display random 12-peptide library is used for subtractive screening of polypeptides that specifically bind to lung cancer cells, and the specific steps are as follows:

[0024] After digesting human lung cancer cells (A549) and human lung normal cells (MRC-5) with trypsin, adjust the cell density, inoculate in a culture dish pre-coated with poly-lysine, and wait until the cells grow to 85%-90% Screening experiments were performed for fusion.

[0025] Take the above-mentioned A549 cells, culture them with serum-free DMEM, add bovine serum albumin BSA to block, then add 10 μl phage peptide library, after incubation for 1.5 h, pour to remove unbound phage, and shake upside down to remove the residual solution. Rinse 3 times with washing solution 0.1% (v / v) TBST buffer, add non-specific buffer 0.2M glycine-hydrochloric acid (pH2.1) 1ml, suck out the eluate, add 150μl 1M Tris-...

Embodiment 2

[0029] Example 2 The amount of phage after screening and amplification

[0030] In Example 1, the phage obtained in each round of screening was diluted 100 times with LB medium, and 10 μl of the diluted phage was mixed with 200 μl of E. After the LB top layer agar, quickly pour it on the LB solid plate containing IPTG / Xgal, overnight, count the number of spots on the plate where the phage can grow, and then multiply this number by the dilution factor to get the plaque forming unit (pfu) drops per 10 μl phage Spend.

[0031] Enrichment of phage polypeptides specifically bound to lung cancer cells: As shown in Table 1, the recovery rate of phage clones positive for A549 was increased by 100 times after three rounds of selection.

[0032] Table 1 The recovery rate of positive phage after three rounds of screening

[0033]

Embodiment 3

[0034] Embodiment 3ELISA identification phage polypeptide

[0035] Identification of positive clones of phage polypeptides specifically binding to lung cancer: In Example 1, after three consecutive rounds of subtractive screening of the phage peptide library, 12 phage clones were randomly selected, and the phage clones were preliminarily identified for A549, A549, Affinity for H1299.

[0036] Press A549, H1299 by 1×10 5 The density per well was seeded in a 96-well plate and placed in CO 2 After culturing in the incubator for 24 hours, the cells were treated with serum-free for 1 hour, washed, fixed with paraformaldehyde, washed with PBS, treated with Triton X-100, blocked with PBS-BSA, added phage monoclonal, incubated for 2 hours; added HRP- Anti M13 antibody, incubate at 37°C for 1 h; use TMB for color development (50 μl TMB per well, color development for 10-15 minutes), add an equal volume of 1N HCl or 2N H 2 SO 4To terminate the reaction, the reaction solution in the ...

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Abstract

The invention provides an oligopeptide capable of performing specificity conjugation with lung carcinoma cells. The oligopeptide provided by the invention is high in conjugation specificity with the lung carcinoma cells, and can be used for early-phase clinical diagnosis and evaluation and prognostic judgment of a treatment effect.

Description

technical field [0001] The invention belongs to the technical field of protein polypeptides, and in particular relates to a polypeptide capable of specifically adhering to lung cancer cells and its preparation method and application. Background technique [0002] Lung cancer is one of the malignant tumors with the fastest increasing morbidity and mortality and the greatest threat to the health and life of the population. In the past 50 years, many countries have reported that the morbidity and mortality of lung cancer have increased significantly. The incidence and mortality of lung cancer in men rank first among all malignant tumors, and the incidence and mortality of lung cancer occupy the second place in women. The cause of lung cancer is still not fully understood. Therefore, the early diagnosis of lung cancer is of great significance for adopting reasonable treatment methods, prolonging the survival time of patients and reducing mortality. [0003] At present, there a...

Claims

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Application Information

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IPC IPC(8): C07K7/08G01N33/68G01N33/574
Inventor 孙绪德高昌俊张莲花任鹏程吕苗苗张玉明孙美艳杨璐
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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