Tumorous specific antigen TSP70 and application thereof

A tumor antigen, tumor technology, applied in the direction of tumor-specific antigen, tumor rejection antigen precursor, anti-tumor drugs, etc., can solve the problem that the amino acid sequence of protein antigen cannot be directly inferred, and achieve high expression specificity and high expression positive rate. Effect

Inactive Publication Date: 2017-02-22
NANJING MARKERLINE BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the amino acid sequence of the protein antigen it targets cannot be directly deduced from known monoclonal antibodies

Method used

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  • Tumorous specific antigen TSP70 and application thereof
  • Tumorous specific antigen TSP70 and application thereof
  • Tumorous specific antigen TSP70 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Isolation and purification of TSP70 protein

[0030] 1. Preparation of human lung cancer cell line SPC-A1 cell lysate

[0031] 1) Cultivate SPC-A1 cells, discard the SPC-A1 cell culture medium, wash twice with Hanks solution, trypsinize for 2 minutes, add culture medium to resuspend the cells, and transfer the cell suspension to a 15mL (or 50mL) centrifuge tube , centrifuge at 1000rpm for 5 minutes, and discard the supernatant;

[0032] 2) Wash the cells once with pre-cooled PBS, centrifuge at 1000rpm for 5 minutes; transfer the cells to a 1.5mL EP tube, wash with PBS, and centrifuge at 12000pm for 5 minutes;

[0033] 3) Thaw the IP cell lysate on ice in advance. Take an appropriate amount of lysate (50uL / 10 6 cells), add 100mM protease inhibitors PMSF, PHI and PI in a ratio of 1:100;

[0034] 4) Add the lysate to the cell mass, and mix by pipetting;

[0035] 5) Incubate on ice for 30 minutes, vortex every 5 minutes;

[0036] 6) Centrifuge at 12000pm at ...

Embodiment 2

[0051] SDS-PAGE electrophoresis and Western blot identification of embodiment 2 TSP70 protein

[0052] 1 SDS-PAGE determination of protein molecular weight

[0053] The concentration of the separating gel is 12%, and the concentration of the stacking gel is 5%. After the sample is boiled for 3 minutes, the sample is added for electrophoresis, and the standard molecular weight is added as a control. The starting voltage is 80V, the sample is concentrated into a line and then increased to 120V after entering the separation gel, the total electrophoresis is about 2.5h, and Coomassie brilliant blue staining after stripping the gel. Electrophoresis results see figure 1 After electrophoresis, draw a molecular weight standard curve on semi-logarithmic coordinate paper according to the molecular weight of the standard protein and its relative mobility, and then find out that its molecular weight is 70Kda from the standard curve according to the relative mobility of the protein to be ...

Embodiment 3

[0056] Example 3 Determination of the amino acid sequence of the TSP70 protein

[0057] 1. Treat with 8mol / L urea to split the polypeptide chain.

[0058] 2. Determine the number of polypeptide chains by determining the relationship between the molar number of terminal amino acid residues and the molecular weight of the protein.

[0059] 3. In the presence of 8mol / L urea, treat with excess β-mercaptoethanol to break the disulfide bond and reduce it to sulfhydryl.

[0060] 4. Determine the amino acid composition of each polypeptide chain, and calculate the molecular ratio of the amino acid composition.

[0061] 5. Analysis of the N-terminal and C-terminal ends of the polypeptide chains.

[0062] 6. The polypeptide chain is broken into multiple peptides and separated.

[0063] 7. Determine the amino acid sequence of each peptide.

[0064] 8. Determine the order of the peptides in the polypeptide chain, and use the overlapping and overlapping of the amino acid sequences of tw...

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Abstract

The invention relates to a tumorous specific antigen TSP70 and application thereof. An amino acid sequence of the tumorous specific antigen TSP70 is shown as SEQ ID NO.1. The tumorous specific antigen TSP70 of a non-small cell lung cancer, a pancreatic cancer, an ovarian cancer, a breast cancer and the like are applied to preparation of tumor diagnosis reagents for the non-small cell lung cancer, the pancreatic cancer, the ovarian cancer, the breast cancer and the like. The novel tumorous specific antigen TSP70 has high positive expression rate and expression specificity in tumor cells of the non-small cell lung cancer, the pancreatic cancer, the ovarian cancer, the breast cancer and the like, an immuno-electron microscope proves that the tumorous specific antigen TSP70 is located in the cell membranes, cytoplasm and nuclei of cells, can serve as a standard product for preparing the tumor diagnosis reagents for the non-small cell lung cancer, the pancreatic cancer, the ovarian cancer, the breast cancer and the like, can be also used for preparing drugs for treating or preventing tumor diagnosis reagents for the non-small cell lung cancer, the pancreatic cancer, the ovarian cancer, the breast cancer and the like.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a tumor-specific antigen TSP70 and its application. Background technique [0002] Non-small cell lung cancer, pancreatic cancer, ovarian cancer and breast cancer are the most common malignant tumors with high malignant degree and high mortality in the world today. With the continuous growth of population and the aggravation of population aging, coupled with changes in lifestyle, society, economy, and environment, the incidence and mortality of malignant tumors, especially lung cancer, are on the rise. About 1.61 million lung cancer cases are diagnosed each year, accounting for 12.7% of malignant tumors, and about 1.38 million lung cancer-related deaths account for 18.2% of cancer-related deaths. Lung cancer is also one of the most common malignant tumors in my country, and its morbidity and mortality rank first in most areas of my country. Non-small cell lung cancer (NSCLC) accounts for...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K16/18A61K39/00A61P35/00G01N33/68G01N33/574
CPCC07K14/4748A61K39/00C07K16/18G01N33/57415G01N33/57423G01N33/57438G01N33/57449G01N33/57488G01N33/68
Inventor 潘世扬徐建
Owner NANJING MARKERLINE BIOMEDICAL TECH CO LTD
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