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Rectal tumor marker and application thereof and kit

A rectal tumor and kit technology, which is applied in the field of biomedicine, can solve the problems of limited rectal tumor-specific biomarkers, difficult prevention and treatment of rectal tumors, etc.

Inactive Publication Date: 2017-07-14
深圳人仁生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art, and provide a rectal tumor marker and its application and kit, aiming at solving the technical problems of limited specific biomarkers for rectal tumors and difficulties in the prevention and treatment of rectal tumors

Method used

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  • Rectal tumor marker and application thereof and kit
  • Rectal tumor marker and application thereof and kit
  • Rectal tumor marker and application thereof and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Simulation of Beclin1 Phosphorylation

[0030] The interaction between Beclin1_WT, Beclin1_229E233E and autophagy regulator Atg14L was determined by constant temperature calorimetric titration, and the binding force between Beclin1 phosphorylation and autophagy activation factor Atg14L was simulated by Beclin1 mutant protein. The specific process is as follows:

[0031] First realize the soluble expression of human Beclin1_WT, Beclin1_229E233E, Atg14L proteins in Escherichia coli, and separate and purify enough target proteins; Beclin1_WT, Beclin1_229E233E and Atg14L were all dialyzed into buffer (containing 50mM Tris, 150mM NaCl; pH 8.0 ); In the titration syringe of the isothermal calorimetric titrator, load the Beclin1_WT (concentration 12mg / ml) of sample 40μl and the Beclin1_229E233E (concentration 12mg / ml) of 40 μ l respectively, and the Atg14L of sample 220 μ l ( Concentration 500μg / ml), use the isothermal calorimeter iTC200 (MicroCal) to measure the th...

Embodiment 2

[0033] Example 2 Conditional test of Beclin1 phosphorylation

[0034] The Beclin1 wild-type (Beclin1_WT) and mutant (mutated for 229, 233 tyrosine sites) were heterologously expressed in Escherichia coli, isolated and purified, and the phosphorylation conditions were tested. Under the action of EGF and EGFR, Beclin1_WT and three mutations (Beclin1mutant A: Beclin1_229E, only mutate the 229 tyrosine site; Beclin1mutantB: Beclin1_233E, only mutate the 233 tyrosine site; Beclin1mutant C: Beclin1_229E223E, For 229 and 233 two tyrosine sites were mutated), phosphorylation tests were carried out at 4°C, 16°C, and 30°C, respectively, and Western Blot was used for characterization, wherein the primary antibody was anti-phosphorylation Tyrosine antibody.

[0035] Experimental results such as figure 2 Shown: the tyrosine of Beclin1_WT can be phosphorylated by EGFR, and it is less affected by temperature; while the signal of Beclin1_229E and Beclin1_233E is obviously weakened, which i...

Embodiment 3

[0036] Example 3 Analysis of the total level and phosphorylation level of LC3-I / II and Beclin1 in clinical rectal tumors

[0037] Obtain five clinical samples of rectal tumors (Phase III, samples obtained from surgery, codes A, B, C, D, E), and use Western Blot to detect the total levels of LC3-I / II and Beclin1 in the five samples. Characterization: first extract the corresponding target protein from paraffin tissue, and directly detect it with the corresponding antibody; at the same time, also characterize the phosphorylation level of Beclin1 in the five samples: first use anti-Beclin1 antibody for co-immunoprecipitation, Then detected with anti-phosphotyrosine antibody.

[0038] Test results such as image 3 Shown: The results showed no significant association between late autophagy levels and total Beclin1 levels in rectal tumors. Combined with the clinical chemotherapy facts of the five samples, the chemotherapy drugs affected the phosphorylation regulation pathway of Be...

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Abstract

The invention belongs to the technical field of biological medicine and specifically relates to a rectal tumor marker which is Beclin1 protein, and application of a reagent for detecting expression level and / or phosphorylation level of the Beclin1 protein in the preparation of a kit used for detection and / or diagnosis of rectal tumor or used for treatment and / or prognostic evaluation of rectal tumor patients. The kit for detection and / or diagnosis of rectal tumor or treatment and / or prognostic evaluation of rectal tumor patients comprises the reagent for detecting expression level and / or phosphorylation level of the Beclin1 protein. Through joint evaluation of total level and phosphorylation level of the marker, the marker can be used as the reference for early detection and / or diagnosis of rectal tumor as well as the reference for medication monitoring and drug resistance prediction for rectal tumor patients.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a rectal tumor marker and its application and kit. Background technique [0002] Autophagy is a "self-eating" phenomenon of multicellular organisms. Cells wrap the cytoplasmic content and transport it to the lysozyme system for degradation to recycle the degradation products for normal activities. This phenomenon has evolutionary conservation sex. Autophagy is an important defense and protection mechanism of the body, and plays complex and pleiotropic roles in many physiological and pathological processes. When cell survival is threatened, such as hypoxia or nutrient deficiency, autophagy can degrade survival non-emergency proteins and organelles through lysosomes, and recycle metabolic nutrients to promote cell survival; autophagy can also degrade and eliminate toxic substances. Old proteins and protein aggregates tend to form a protective mechanism against neu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/574
CPCG01N33/68G01N33/574G01N33/57446G01N2800/065G01N2800/52
Inventor 李小花张佳霄周志林李芸芳
Owner 深圳人仁生物医药科技有限公司
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