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The application of the complex of cysteine ​​protease inhibitor sn and cathepsin L1 as a diagnostic marker for gastric cancer

A cysteine ​​protease and inhibitor technology, applied in the field of medical diagnostics, to achieve the effect of improving the detection rate and tissue specificity

Active Publication Date: 2021-01-22
SHANGHAI LIANGRUN BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although CST1 has a certain degree of discrimination in gastric cancer, due to its high sequence homology with the same family of cystatins S (CST4), CST1 also has certain limitations as a gastric cancer target. sex

Method used

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  • The application of the complex of cysteine ​​protease inhibitor sn and cathepsin L1 as a diagnostic marker for gastric cancer
  • The application of the complex of cysteine ​​protease inhibitor sn and cathepsin L1 as a diagnostic marker for gastric cancer
  • The application of the complex of cysteine ​​protease inhibitor sn and cathepsin L1 as a diagnostic marker for gastric cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Expression and purification of embodiment 1CST1-CTSL recombinant protein

[0056] Protein expression: Synthesize the gene according to the amino acid sequence of SEQ ID NO: 1 in the sequence table and optimize the codons for mammalian expression. The gene was inserted into the pcDNA3.1 vector containing 6×His tag to obtain pcDNA3.1-CST1-CTSL. Then pcDNA3.1-CST1-CTSL was transformed into DH5α, and after positive clones were picked and cultured in large quantities, the recombinant plasmid pcDNA3.1-CST1-CTSL was extracted with a high-purity plasmid extraction kit. The recombinant plasmid was transferred into 293t cells, and the pcDNA3.1 empty vector was transfected at the same time as a negative control. 2 After culturing for 72 hours under the same conditions, the supernatant was collected and filtered with a 0.22 µm filter membrane.

[0057] SEQ ID NO:1MNPTLILAAFCLGIASATLTFDHSLEAQWTKWKAMHNRLYGMNEEGWRRAVWEKNMKMIELHNQEYREGKHSFTMAMNAFGDMTSEEFRQVMNGFQNRKPRKGKVFQEPLFYEAPRSV...

Embodiment 2

[0059] Example 2 Activity Verification and Antibody Pairing of CST1-CTSL Recombinant Protein

[0060] Activity analysis: Coat the chemiluminescence plate with 1ug / ml recombinant CST1-CTSL protein carbonate buffer (pH9.5) in a volume of 100ul at 4°C overnight, and serially dilute the capture antibody and enzyme-labeled antibody (concentration: 0~1ug / ml), add goat anti-mouse IgG-HRP (100ng / ml). The test found that the luminescence value of the capture antibody and the detection antibody at 100ng / ml was not less than 200,000, and the reaction curve between the protein and the antibody was R 2 >0.99, the reactivity of the protein meets the requirements.

[0061] Antibody pairing: Coat the chemiluminescence plate with 1ug / ml capture antibody, add 100ul of CST1-CTSL calibrator with different concentrations (5~1000pg / mL), incubate at 37°C for 60min, add 100uL of horseradish with a concentration of 100ng / ml after washing Peroxidase-labeled detection antibody, and incubated at 37°C ...

Embodiment 3

[0062] Embodiment 3 CST1-CTSL calibration curve drawing

[0063] Calibration curve drawing: First, coat the capture antibody on the chemiluminescence plate overnight at 4°C with a concentration of 1 μg / mL, and dilute the recombinant human CST1-CTSL calibrator protein with protein stabilizer to 0pg / mL, 10pg / mL, 50pg / mL , 100pg / mL, 200pg / mL, 500pg / mL, 1500pg / mL, 100μL per well, then add horseradish peroxidase-labeled detection antibody at a concentration of 5ng / ml, 100μL per well, and incubate at 37°C for 1 hour. Wash 3 times with PBST, add chemiluminescence substrate and measure the luminescence intensity of each well. The CST1-CTSL content of the tested samples was calculated from the calibration curve. The linear range of the calibration curve is 10~1500pg / mL, attached figure 2 It is the calibration curve of the CST1-CTSL detection kit, where the Y-axis represents the logarithmic value of the luminescence value, and the X-axis represents the logarithmic value of the concen...

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Abstract

The invention relates to the field of medical diagnostics, in particular to the application of a cysteine ​​protease inhibitor SN and Cathepsin L1 complex as a gastric cancer diagnostic marker. The detection of the CST1-CTSL complex can effectively improve the tissue specificity of the marker, and can also effectively improve the detection rate of early gastric cancer.

Description

technical field [0001] The invention relates to the field of medical diagnostics, in particular to the application of a cysteine ​​protease inhibitor SN and Cathepsin L1 complex as a gastric cancer diagnostic marker. Background technique [0002] Gastric cancer refers to malignant tumors of epithelial origin that originate in the stomach. Studies have found that in the process of tumor invasion and metastasis, tumor cells must pass through the surrounding matrix, and the degradation of the matrix is ​​mainly related to various cathepsin hydrolytic enzymes. Studies have shown that the ability of tumor cell invasion and metastasis is closely related to the ability of cathepsins to degrade the extracellular matrix . [0003] Cathepsin L1 (Cathepsin L1, CTSL) is a lysosomal cysteine ​​protease widely present in human tissue cells. The enzyme is an endopeptidase, but it also has exonuclease activity. Enzymes are usually synthesized as inactive precursors whose endopeptidase act...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/574G01N33/573G01N33/577
CPCG01N33/68G01N33/57446G01N33/57488G01N33/573G01N33/577G01N2333/8139G01N2333/96466
Inventor 王力军孙玉龙杨亚云王弢
Owner SHANGHAI LIANGRUN BIOMEDICINE TECH CO LTD
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