Screening and authentication of polypeptide binding specificly to tumour tranferring cell

A tumor metastasis and specific technology, applied in peptides, material inspection products, biological tests, etc., can solve the problems of complex, unknown and non-specific components on the cell surface

Inactive Publication Date: 2006-04-26
NANKAI UNIV
View PDF0 Cites 21 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Our solution overcomes some problems in previous cell screening, mainly that the cell surface components are complex and unknown, the non-specificity of cell screening is large, the background value is high, and the cell surface difference between metastatic and non-metastatic tumor cells is small

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Screening and authentication of polypeptide binding specificly to tumour tranferring cell
  • Screening and authentication of polypeptide binding specificly to tumour tranferring cell
  • Screening and authentication of polypeptide binding specificly to tumour tranferring cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Obtaining recombinant phage capable of selectively binding to the surface of SW620 cells after four rounds of screening

[0029] The first round: Dissolve 10.4g of RPMI1640 medium powder (Gibico company) and 2.0g of sodium bicarbonate in 800ml of double distilled water, add 10ml of 1mol / L HEPES solution, and then distill the total volume to 1000ml with double distilled water, 0.22 Store at 4°C after sterilizing with a μm filter membrane; add fetal bovine serum (FBS, purchased from Tianjin Institute of Blood Diseases) and penicillin-streptomycin double antibody to the above medium to make the content reach 10% and 1% respectively , stored at 4°C; 2ml of the above-mentioned medium was added to each well of a 6-well plate, and SW620 cells (ATCC deposit number: CCL-228) were inserted to make the cell density 10 6 cells / well, cultivated in a carbon dioxide cell incubator for about 48 hours; discard the medium, and wash the cells with 1×PBS (10×PBS: sodium chloride...

Embodiment 2

[0037] Example 2: Determination of phage binding ability to SW480 and SW620

[0038] SW480 and SW620 respectively with 10 6 Spread the cells / well into a 6-well plate, culture in a carbon dioxide cell incubator at 37°C for about 48 hours (the medium uses RPMI1640, 10% FBS, 1% penicillin-streptomycin double antibody), discard the medium, and wash with PBS , add blocking solution (PBS containing 10mg / ml BSA) and incubate at room temperature for 1 hour; at the same time block the phage, discard the blocking solution, and add 2ml of phage to each of the SW480 and SW620 wells (the titer is about 1×10 11 ), incubated at room temperature for 2 hours; discarded the phage, washed 10 times with PBST (PBS containing 0.1% Tween), washed twice with PBS, eluted, and measured their respective titers: the original library phage was used as a control, and the above-mentioned Test; the phage mixed library obtained in the fourth round has a binding ability to SW620 that is 100 times higher than ...

Embodiment 3

[0043] Example 3: Isolation and preparation of monoclonal phage

[0044] Randomly pick 105 monoclonal phages after 4 rounds of screening, add them to Escherichia coli ER2738 in mid-logarithmic phase, and culture them on a shaker (250 rpm) at 37°C for 4.5 to 5 hours; centrifuge the amplified product at 10,000 rpm For 10 minutes, take the supernatant and add 1 / 6 volume of PEG / NaCl solution, precipitate overnight at 4°C; centrifuge at 10,000 rpm for 15 minutes, discard the supernatant: resuspend the pellet in 200 μl of PBS, and centrifuge at 10,000 rpm for 5 minutes to remove the remaining Bacterial precipitation; measure the phage titer in the supernatant by agar stacking method, add an equal volume of glycerol, and store at -20°C.

[0045] Complete cell SW480 or SW620 was detected by ELISA with 105 monoclonal phages. Into a 96-well plate, add 10 5 For SW480 or SW620 cells / well, add 200 μl of blocking solution [5% (w / v) BSA in PBS] to each well after two days of culture, and b...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The present invention relates to four dodecapeptide sequences capable of binding specifically with surface antigen of tumor metastasis cell, and three dodecapeptides capable of binding specifically with tumor metastasis cell have the following conservative: Pro-Trp-X1-Glu-Pro-X2-Tyr, where X1 and X2 represses any one amino acid each. Owing to the characteristic of specific binding with tumor metastasis cell, these dodecapeptides and their derivatives may be used as the marker for tumor metastasis diagnosis and as the precursor for developing tumor metastasis inhibiting medicines.

Description

technical field [0001] The present invention relates to a method for using phage display library technology to screen polypeptides that can specifically bind to surface antigens of tumor metastasis cells. Specifically, the differential screening of random 12-peptide phage display libraries using in situ tumors and tumor metastasis cell lines is used to obtain peptides that can be used Specific 12-peptide sequence as tumor metastasis marker. Background technique [0002] Tumor cells invade lymphatic vessels, blood vessels or body cavities from the original site, migrate to other places and continue to grow, forming tumors of the same type as the primary tumor, which is called metastasis [Yang Guanghua et al. "Pathology" (fifth edition), People's Health Press, 2001, P87). [0003] Finding the abnormal expression of various molecules on the surface of tumor cells from the molecular level is of great significance for the identification of tumor metastasis and development. VEGFR...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08G01N33/68
Inventor 曹又佳李鑫张宏恺张翠竹
Owner NANKAI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap