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Application of 26S prolease regulatory subunit gene 1(non ATP enzyne)

A technique for regulating subunits and proteasomes, applied in the application field of protein molecular markers

Inactive Publication Date: 2012-01-04
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report on the association of 26S proteasome regulatory subunit 1 non-ATPase or 26S proteasome with hepatocellular carcinoma

Method used

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  • Application of 26S prolease regulatory subunit gene 1(non ATP enzyne)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 , Preparation of protein samples from hepatocellular carcinoma tissue and paracancerous tissue

[0023] Urea, 3-[(3-cholamidopropyl)-diethylammonium]-1-propanesulfonic acid (CHAPS), phenylmethylsulfonyl fluoride (PMSF), dithiothreitol used in this example (DTT) were purchased from Sigma Company.

[0024] In this example, protein samples of cancer tissue and paracancerous tissue of hepatocellular carcinoma were prepared by nonenzymatic sample preparation (NESP), as follows:

[0025] The surgically excised fresh tissue block is quickly placed on ice and quickly cut into several small pieces that are visible to the naked eye without areas of necrosis. Use pre-cooled glutamine-free RPMI1640 medium (5% fetal bovine serum, 0.2mM PMSF, 1mM EDTA, benzisoxazole penicillin 25mg / mL, gentamicin 50mg / mL, penicillin 100U / mL, Streptomycin 100mg / mL, Amphotericin B 0.25mg / mL, Nystatin 50U / mL) After washing the small tissue pieces several times, they were quickly ground int...

Embodiment 2

[0030] Example 2 , Screening of differentially expressed proteins

[0031] Urea, 3-[(3-cholamidopropyl)-diethylammonium]-1-propanesulfonic acid (CHAPS), sodium dodecylsulfonate (SDS), dithiothreitol used in this example (DTT) was purchased from Sigma; iodoacetamide (IAA), acrylamide, N, N-methylenebisacrylamide, etc. were purchased from Fluka; cleavable ICAT reagents were purchased from Applied Biosystems Framingham, MA.

[0032] Ammonium persulfate (AP), TEMED, Tri-n-butylphosphat (TBP), PDQuest software, etc. are Bio-Rad products.

[0033] Avidin affinity column was purchased from Applied Biosystems, Framingham, MA.

[0034] LCQ TM Deca XP system and ProteomeX TM Workstation was purchased from Thermo Finnigan Company.

[0035] The composition of the loading buffer used is: 0.6ml of 1mol / L Tris-HCl (pH6.8), 5ml of 50% glycerol, 2ml of 10% SDS, 0.5ml of mercaptoethanol, 1.9ml of distilled water, and a small amount of hexabromophenol blue.

[0036] First, the gel-enhan...

Embodiment 3

[0044] Example 3 , 26S Proteasome Regulatory Subunit 1 Non-ATPase Differential Expression Verification by Western Blot

[0045] In order to confirm the differential expression of 26S proteasome regulatory subunit 1 non-ATPase, the protein samples of cancer tissues and corresponding paracancerous tissues of 10 patients with hepatocellular carcinoma were collected (prepared by NESP method, 10 cases except 429 cases in Table 1) , using the purchased anti-26S proteasome regulatory subunit 1 non-ATPase antibody for western blot analysis, the specific process is briefly described as follows:

[0046] Take 20 μg protein samples from each sample and use 12% SDS-PAGE to separate, transfer to PVDF membrane (purchased from Amersham Biosciences), and use goat anti-human 26S proteasome regulatory subunit 1 non-ATPase monoclonal antibody (purchased from Abcam) as the primary antibody. Ltd, 1:250), incubate at room temperature for 2 hours, wash three times with TBST (each liter contains 2....

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Abstract

The invention selects the proteins with different expressions in cancer organization and cancer beside organism, to find one protein with high expression in cancer organization of liver cell cancer, wherein the immunity print test has proved that the 26S prolease adjustable subunit 1 (non ATP enzyme) has different expressions in cancer organism and cancer beside organism. Based on said relation, the protein can be used in protein molecule mark to detect liver cancer.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to the application of a 26S proteasome regulating subunit 1 non-ATPase as a protein molecular marker for detecting liver cancer. Background technique [0002] Liver cancer is a disease that seriously endangers human beings. The incidence of liver cancer in western developed countries is low, and the basic research on liver cancer in the world is still relatively weak. However, my country is a country with a high incidence of liver cancer, and the morbidity and mortality are on the rise, and the age of onset is younger. Medical expenses have increased greatly, and liver cancer has become the number one enemy that seriously endangers the safety of people's life and property in our country, and is an important factor affecting social and economic development. It is of strategic significance to intensify the basic research of liver cancer in my country, and to separate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/574G01N33/532G01N33/68
Inventor 曾嵘袁新雨李辰周晓
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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