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Novel polypeptide targeting multiple tumor cells and application of novel polypeptide

A tumor cell, targeting technology, applied in the field of biomedicine, can solve the problems of strong toxic and side effects, large trauma, lack of efficient treatment methods, etc., and achieve the effects of weak immunogenicity, high efficiency, and simple synthesis and purification.

Active Publication Date: 2020-06-09
中国医科大学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The treatment of malignant tumors is still a difficult problem worldwide. The three traditional treatment methods of surgery, chemotherapy and radiotherapy have serious defects such as large trauma and strong side effects. At present, there is a lack of efficient and low-toxic treatment methods

Method used

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  • Novel polypeptide targeting multiple tumor cells and application of novel polypeptide
  • Novel polypeptide targeting multiple tumor cells and application of novel polypeptide
  • Novel polypeptide targeting multiple tumor cells and application of novel polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Three rounds of subtractive screening of positive polypeptides specifically binding to liver cancer cells using phage display technology.

[0039] 1.1 Recovery and cultivation of host bacteria E.coli ER2738.

[0040]To prepare an E. coli plate, take the LB-TET culture plate and preheat it in a 37°C incubator for 1 hour. After the E.coli ER2738 bacterial liquid melts, use an inoculation loop to dip a small amount of bacterial liquid evenly on the culture plate, and then place it upside down at 37°C. overnight in a constant temperature incubator. Prepare the host bacterial solution, pick a single colony from a well-grown culture plate, place it in LB bacterial culture solution containing tetracycline, and cultivate overnight at 37°C with shaking at 180rpm, so that the bacteria are in the logarithmic growth phase. The prepared LB-Tet plate containing Escherichia coli was stored in a 4°C refrigerator for later use, and the host bacterial solution was stored in a ...

Embodiment 2

[0062] Example 2 Enzyme-linked immunosorbent assay detection of target affinity between positive phage clones and liver cancer cells

[0063] 2.1 Purification of positive phage clones.

[0064] 2.1.1 Amplification of positive phages: Add 20ml LB / Tet liquid medium to the Erlenmeyer flask, then add Escherichia coli liquid and phage to be amplified at a ratio of 1:100, place at 37ºC, shake vigorously in a constant temperature shaker for 4.5 h, the amplification solution of phage was obtained.

[0065] 2.1.2 Purification of positive phage: centrifuge the phage amplification solution obtained through the above steps at 4ºC and 12000r / min for 10min, take the supernatant, add 1 / 6 volume of PEG-NaCl to precipitate overnight, centrifuge at 12000r / min for 15min, discard Remove the supernatant, dissolve the precipitate with TBS buffer, give 1 / 6 volume PEG-NaCl again, and incubate on ice for 1 h. 4ºC, 14000r / min, centrifuge for 15min, discard the supernatant, dissolve the obtained preci...

Embodiment 3

[0078] Example 3 Determination and analysis of DNA sequences of positive phage clones.

[0079] 3.1. Selection of positive monoclonal phages.

[0080] The phage liquid obtained after the third round of screening was titrated and spread on LB plates. On the plates with less than 100 growing spots, 20 well-growing blue spots at an interval of 5 mm were randomly selected. Add 20 randomly picked blue spots to 1ml pre-logarithmic host bacterial solution (same as phage amplification), and amplify at 37°C and 200rpm with rapid shaking for 4.5 hours.

[0081] 3.2 Positive monoclonal phage single-stranded DNA extraction.

[0082] Centrifuge the amplified monoclonal phage liquid at 4°C and 14,000 rpm for 30 seconds, transfer the supernatant to a new tube, centrifuge at 4°C and 1,000 rpm for 30 seconds, take 80% of the supernatant and transfer to a new nuclease-free centrifuge In the tube, take 300ul of bacterial liquid and add 300ul of glycerol at a ratio of 1:1, and store it in a -20 d...

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Abstract

The invention belongs to the field of biomedicine, relates to a polypeptide in targeted binding with tumor cells and particularly relates to the polypeptide in specific binding with multiple tumor cells as well as application of the polypeptide in tumor prevention, treatment and diagnosis. An amino acid sequence of the polypeptide is selected from an amino acid sequence shown in one of SEQ ID NO.1-SEQ ID NO.5. The polypeptide and bioactive fragments and derivatives thereof are applied in diagnosis and treatment of tumors as molecular image probes and medicine target heads. The polypeptide hasa function of specifically targeting the multiple tumor cells and is high in specificity and weak in side effects, so that a series of earlier-stage diagnosis reagents and targeted therapy medicines for tumors are developed, and a new direction is opened for design, research and development of novel tumor targeted medicines.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a polypeptide that binds to tumor cells in a targeted manner, in particular to a polypeptide that specifically binds to various tumor cells and its use for tumor prevention, treatment and diagnosis. Background technique [0002] Tumor is a major disease that seriously threatens human life and social development. According to WHO statistics, more than 8 million people die of tumors worldwide every year. The number of cancer patients in my country ranks first in the world, and the morbidity and mortality continue to rise. With the transformation of disease patterns and the trend of population aging, the burden of cancer diagnosis and treatment in my country is increasing. The treatment of malignant tumors is still a difficult problem worldwide. The three traditional treatment methods of surgery, chemotherapy and radiotherapy have serious defects such as large trauma and strong side effects...

Claims

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Application Information

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IPC IPC(8): C07K7/08C12N15/11A61K47/64A61K51/08A61K49/00A61P35/00
CPCC07K7/08A61K47/64A61K51/08A61K49/0056A61P35/00
Inventor 魏敏杰于丽凤余涧坤赵琳
Owner 中国医科大学
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