Cathepsin D antigen polypeptide and application thereof as well as detecting kit containing polypeptide

An antigen polypeptide and kit technology, applied in the field of bioengineering, can solve the problems of low five-year survival rate of lung cancer, difficult early diagnosis, difficult treatment, etc., and achieve good application prospects and market prospects, high sensitivity, high specificity Effect

Inactive Publication Date: 2010-06-16
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the diagnosis and treatment of lung cancer have made great progress, but the five-year survival rate of lung cancer is still less than 15%.
At present, the diagnosis of human tumors mainly relies on imaging and pathological techniques. Most patients are diagnosed after they have obvious space-occupying lesions and clinical symptoms. Most patients have reached the middle and late stages, and it is difficult to receive effective treatment.
Moreover, there is currently a lack of effective monitoring indicators for the development of tumors clinically, which leads to difficulties in early diagnosis

Method used

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  • Cathepsin D antigen polypeptide and application thereof as well as detecting kit containing polypeptide
  • Cathepsin D antigen polypeptide and application thereof as well as detecting kit containing polypeptide
  • Cathepsin D antigen polypeptide and application thereof as well as detecting kit containing polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The preparation of embodiment 1 Cathepsin D protein

[0034] 1. Serum-free culture of M-BE cells

[0035] Three passages (P40, P140 and P200) of M-BE cells were cultured in MCDB 151 medium (serum-free) supplemented with growth factors and pituitary extract, at 37°C, 4% CO 2 Incubator cultivation. When the cell density reaches 80%, after washing the cells three times with PBS, replace the MCDB 151-conditioned medium without growth factors and pituitary extract, and continue at 37 °C, 4% CO 2 Incubator cultivation. After 48 hours, the conditioned medium was collected and centrifuged at 1000g at 4°C for 10 minutes to remove cell debris; the cells were washed three times with PBS before protein extraction.

[0036] 2. Protein Extraction from Serum-Free Medium

[0037]The collected conditioned medium was dialyzed in a NaCl concentration gradient solution using a 3500Da dialysis membrane. The dialysate concentration and time were: 100mM 2h; 50mM 4h; 25mM 8h; 10mM 10h; 0mM...

Embodiment 2

[0053] Embodiment 2 Cathepsin D antigen polypeptide polyclonal antibody preparation

[0054] The full-length Cathepsin D antigen purified by a nickel column was fully dialyzed with PBS, and then concentrated to a concentration of 1.0 mg / ml. Mix 1.0 mg antigen concentrate with an equal volume of complete Freund's adjuvant, fully emulsify, and then inject it subcutaneously at multiple points on the back of New Zealand white rabbits. Before the initial immunization, the blood was collected from the leg vein of the rabbit to separate the serum, which was used as the pre-immune serum control; two weeks later, the first booster immunization was carried out, and 500 μg of antigen concentrate and an equal volume of incomplete Freund’s adjuvant were mixed evenly, fully emulsified, and placed in New Zealand Multiple subcutaneous injections on the back of white rabbits. Afterwards, a booster immunization was performed every 2 weeks, and blood was collected from the rabbit's leg vein 7 d...

Embodiment 3

[0057] Example 3 Cathepsin D-specific antibody preparation kit components

[0058] The basic steps of antibody kit preparation are as follows:

[0059] (1) Select a suitable polystyrene ELISA plate to ensure that the difference between the absorbance value of a single well and the average value of the absorbance value of 96 wells is within 10%;

[0060] (2) Coating the anti-Cathepsin D antibody (100ng / ml) of horseradish peroxidase in each hole with a micro-spotter; Antibody (100ng / ml);

[0061] (3) blocking with 0.5% bovine serum albumin;

[0062] (4) Carry out drying treatment and vacuum packing to plate and coated antibody.

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Abstract

The invention provides a tumor marker Cathepsin D antigen polypeptide which has an amino acid sequence shown as SEQ IDNo.1 in a sequence table. The invention also discloses a method for cloning and expressing the protein by a gene, preparing a specific antibody of the protein and judging gastric cancer biological behavior. The Cathepsin D antigen polypeptide and the antibody thereof prepared by the method can be used for preparing the tumor detecting kit and the research and development of a drug target.

Description

technical field [0001] The invention relates to a Cathepsin D antigen polypeptide, more specifically a tumor marker Cathepsin D antigen polypeptide, the application of an autoantibody in preparing a tumor detection kit and a detection kit containing the polypeptide, belonging to the field of bioengineering. Background technique [0002] In my country, lung cancer is the most common cancer, ranking first in both morbidity and mortality. In recent years, the diagnosis and treatment techniques of lung cancer have made great progress, but the five-year survival rate of lung cancer is still lower than 15%. At present, the diagnosis of human tumors mainly relies on imaging and pathological techniques. Most patients are diagnosed after they have obvious space-occupying lesions and clinical symptoms. Most patients have reached the middle and late stages, and it is difficult to receive effective treatment. Moreover, there is currently a lack of effective monitoring indicators for th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C07K16/18G01N33/574A61K39/395A61P35/00
Inventor 娄晓敏张军张聚吕有勇徐宁志刘斯奇
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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