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Specific combined polypeptide for lung cancer, preparation and uses thereof

A technology that combines peptides and specificity, applied in the field of protein peptides, can solve the problems of limited value and non-specificity of lung cancer, and achieve good tumor targeting, high specificity, and sensitive tumor diagnosis

Inactive Publication Date: 2009-07-29
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The occurrence and development of lung cancer have a very complex mechanism. Under the action of carcinogenic factors, a variety of adhesion molecules and their receptors or ligands can be expressed in the body of patients, and the levels of markers released in different stages of tumor development are also different. These The vast majority of indicators are non-specific. There is no marker with high sensitivity and specificity for each tissue type, but only reflects changes in certain aspects of the disease. The value of a single marker detection for lung cancer is limited

Method used

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  • Specific combined polypeptide for lung cancer, preparation and uses thereof
  • Specific combined polypeptide for lung cancer, preparation and uses thereof
  • Specific combined polypeptide for lung cancer, preparation and uses thereof

Examples

Experimental program
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Embodiment 1

[0045] Example 1 Screening of polypeptides specifically binding to lung cancer cells

[0046] 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:

[0047] After human lung cancer cells (NCI-H1299) and human embryonic lung fibroblasts (MRC-5) were digested with trypsin, the cell density was adjusted, and seeded in 60×15mm cells pre-coated with poly-lysine 2 In the culture dish, when the cells grow to 80%-90% confluence, they are used for screening.

[0048] Take the above NCI-H1299 cells, culture them with serum-free DMEM, add bovine serum albumin BSA to block, and then add 10 μl phage peptide library (Ph.D.-7 of NEW ENGLAND Biolabs TM Phage Display Peptide Library Kit phage original library), after incubation for 1 h, remove the supernatant, wash 3 times with washing solution TBST (Tween-20 concentration is 0.1 volume %), add glycine elu...

Embodiment 2

[0053] The mensuration of embodiment 2 phage titers

[0054] 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. LB top layer agar (top layer agar: each liter contains 10g Bacto-Tryptone, 5g yeast extract, 5g NaCl, 1g MgCl2.6H20 and 7g agar powder, after autoclaving, it will be divided into 3ml / tube, and it will be heated and melted when used) and then quickly poured into LB solid plates containing IPTG / Xgal, overnight, count blue plaques.

[0055] Calculation formula: phage titer (pfu / 10 μL) = number of plaques × dilution factor, that is, phage forming units per 10 μL.

[0056] Enrichment of phage polypeptides specifically bound to lung cancer cells: As shown in Table 1, there were about 10 positive phage clones binding to NCI-H1299 after three rounds of screening. 2 double enrichment phenomenon.

[0057] Table 1 Enrichment effect of subtractive screening on positive ph...

Embodiment 3

[0059] The ELISA identification of embodiment 3 phage polypeptides

[0060] Identification of positive clones of phage polypeptides specifically binding to lung cancer cells: In Example 1, after three consecutive rounds of subtractive screening of the phage peptide library, 9 phage clones were randomly selected, and the conventional method in the field-ELISA method was used to preliminarily identify the phage clones against NCI. - Affinity for H1299, A549.

[0061] NCI-H1299, human lung adenocarcinoma cells (A549) were divided into 1×10 4 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 the phage monoclonal of Example 1, and incubated for 2 hours ; Add HRP-anti M13 antibody, incubate at 37°C for 1 h; develop color with TMB (50 μl TMB per well, 8-15 mi...

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Abstract

The invention discloses a polypeptide that can be specifically combined with lung cancer cells and a preparation method and application thereof. The polypeptide specifically combined with lung cancer cells shows peptide library reducing screening by a bacterial virus so as to obtain bacterial virus clone specifically with lung cancer; the bacterial virus clone having stronger affinity with lung cancer is identified by Elisa and an immunohistochemical method; and sequencing is sent so as to obtain a 12 polypeptide, and the amino acid sequence is shown as SEQ ID NO: 1. The 12 polypeptide screened by the invention not only has the advantages of traditional antibody molecule but also has small molecular weight, strong penetrating power and easy arriving to tumor tissue; the 12 polypeptide has good tumor targeting effect and more sensibility of tumor diagnosis, and can be used for preparing a kit for tumor diagnosis and drug for curing tumors.

Description

technical field [0001] The invention belongs to the technical field of protein polypeptides, and in particular relates to a polypeptide capable of specifically binding to lung cancer cells and its preparation method and application. Background technique [0002] Lung cancer is one of the most common and deadly malignant tumors in the world, and its incidence rate is also the highest among all malignant tumors. Due to the insidious onset of lung cancer, there is still a lack of effective screening and early diagnosis methods. Most patients are late when symptoms appear, and the prognosis is poor. The overall five-year survival rate does not exceed 15%, and symptoms are ≤ 10%. At present, the 5-year survival rate (clinical cure rate) of cancer patients in my country is about 20%-30%, of which lung cancer is only 10%, and liver cancer is about 3%. For a long time, the treatment of malignant tumors has been a worldwide problem. [0003] However, in patients with early-diagnosed...

Claims

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Application Information

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IPC IPC(8): C07K16/18G01N33/53A61K39/395A61P35/00
Inventor 臧林泉石磊郭姣潘雪刁潘琴巫玮
Owner GUANGDONG PHARMA UNIV
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