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Novel polypeptide specifically bound with multiple tumor cells and application thereof

A tumor cell-specific technology, applied in the field of biomedicine, can solve the problems of easy uptake by the phagocytic system, poor cell membrane penetration, and application limitations, and achieve small relative molecular weight, simple synthesis and purification steps, and immunogenicity weak effect

Active Publication Date: 2020-07-10
中国医科大学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibodies or their fragments are commonly used molecular targeting drugs so far, but their application is greatly limited due to their large molecular weight, poor cell membrane penetration ability and easy uptake by the phagocytic system

Method used

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  • Novel polypeptide specifically bound with multiple tumor cells and application thereof
  • Novel polypeptide specifically bound with multiple tumor cells and application thereof
  • Novel polypeptide specifically bound with multiple tumor cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Three rounds of subtractive screening of intestinal cancer cell-specific binding-positive polypeptides using phage display technology.

[0038] 1.1 Recovery and cultivation of host bacteria E.coli ER2738.

[0039] To prepare an E. coli plate, take the LB-TET culture plate and preheat it in a 37-degree 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 liquid, put a single colony picked from a well-grown culture plate into the LB bacterial culture medium containing tetracycline, and cultivate overnight at 37°C and 180rpm shaking, and stop the shaking when the bacteria are in the logarithmic growth phase. The LB-Tet plate containing E. coli was prepared and stored in a 4°C refrigerator for later use, and the host bacterial solution was sto...

Embodiment 2

[0061] Example 2 Enzyme-linked immunosorbent assay was used to detect the affinity of positive phage clones to intestinal cancer cells.

[0062] 2.1 Purification of positive phage clones.

[0063] 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.

[0064] 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 pr...

Embodiment 3

[0077] Example 3 Determination and analysis of DNA sequences of positive phage clones.

[0078] 3.1. Selection of positive monoclonal phages.

[0079] The phage liquid obtained after the third round of screening was titered and spread on LB plates. On the plate with less than 50 plaques, 20 blue spots with good growth at intervals 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.

[0080] 3.2 Positive monoclonal phage single-stranded DNA extraction.

[0081]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, and transfer 80% of the supernatant 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 degree refri...

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Abstract

The invention belongs to the field of biomedicine, and particularly relates to a novel polypeptide specifically bound with multiple tumor cells and application thereof. The amino acid sequence of thepolypeptide is selected from an amino acid sequence shown as one of SEQ ID NO. 1 to SEQ ID NO. 5. The polypeptide and bioactive fragments and derivatives thereof are applied to tumor diagnosis and treatment as molecular imaging probes and drug target heads. The polypeptide disclosed by the invention has the effects of specifically targeting various cancer cells, and is high in specificity and small in side effect, so that a series of tumor early diagnosis reagents and targeted treatment drugs are developed, and a new direction is opened up for designing and researching novel tumor targeted drugs.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a novel polypeptide specifically combined with various tumor cells and its application. Background technique [0002] The number of cancer patients in China ranks first in the world. With the transformation of disease patterns and the trend of population aging, its morbidity and mortality continue to rise, and 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. At present, there is a lack of efficient and low-toxic treatment methods. In view of the refractory nature of tumor diseases and the urgency of clinical needs, anti-tumor drugs are the hottest and most active field of drug development and marketing in the world. Therefore, it ...

Claims

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Application Information

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IPC IPC(8): C07K7/08C12N15/11G01N33/68G01N33/574A61K51/08A61K47/64A61K47/42
CPCC07K7/08G01N33/68G01N33/57484G01N33/57438G01N33/57423G01N33/57415G01N33/57449G01N33/57446A61K51/08A61K47/64A61K47/42G01N2410/00
Inventor 魏敏杰于丽凤余涧坤赵琳王欣瑀王佳琪牛延新李梓楠
Owner 中国医科大学
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