Application of cst1-ctsh complex as a diagnostic marker for esophageal cancer
A technology of CST1-CTSH and complex is applied in the application field of CST1-CTSH complex as a diagnostic marker of esophageal cancer, and achieves the effect of improving tissue specificity and improving detection rate
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Embodiment 1
[0054] Example 1 Expression and purification of CST1-CTSH recombinant protein
[0055] Protein expression: The gene was synthesized according to the amino acid sequence of SEQ ID NO: 1 in the sequence listing and optimized for mammalian expression codons. The gene was inserted into pcDNA3.1 vector containing 6×His tag to obtain pcDNA3.1-CST1-CTSH. Then pcDNA3.1-CST1-CTSH was transformed into DH5α, positive clones were picked and cultured in large quantities, and the recombinant plasmid pcDNA3.1-CST1-CTSH 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 72h under conditions, the supernatant was collected, and the supernatant was filtered through a 0.22 μm filter.
[0056] SEQ ID NO: 1
[0057] MWATLPLLCAGAWLLGVPVCGAAELCVNSLEKFHFKSWMSKHRKTYSTEEYHHRLQTFASNWRKINAHNNGNHTFKMALNQFSDMSFAEIKHKYLWSEPQNCSATKSNYLRGT...
Embodiment 2
[0059] Example 2 Activity verification and antibody pairing of CST1-CTSH recombinant protein
[0060] Activity analysis: Coat the chemiluminescence plate with 1ug / ml of recombinant CST1-CTSH protein carbonate buffer (pH9.5) in 100ul volume at 4°C overnight, and dilute the capture antibody and enzyme-labeled antibody (concentration: 0~1ug) / ml), add goat anti-mouse IgG-HRP (100ng / ml). The detection found that the luminescence value of the capture antibody and the detection antibody at 100ng / ml were not less than 200,000, and the reaction curve of 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 CST1-CTSH calibrator of different concentrations (5-1000pg / mL), incubate at 37°C for 60min, and add 100uL horseradish with a concentration of 100ng / ml after washing The detection antibody labeled with peroxidase was incubated at 37° C. for...
Embodiment 3
[0062] Example 3 CST1-CTSH calibration curve drawing
[0063] Calibration curve drawing: First coat the capture antibody on a chemiluminescent plate at 4°C overnight at a concentration of 1 μg / mL, and dilute the recombinant human CST1-CTSH calibrator protein with protein stabilizer to 0pg / mL, 10pg / mL, 50pg / mL , 100pg / mL, 200pg / mL, 500pg / mL, 1500pg / mL, 100 μL per well, then add horseradish peroxidase-labeled detection antibody at a concentration of 5ng / ml, 100 μL per well, and incubate at 37°C for 1 hour. After washing 3 times with PBST, chemiluminescent substrate was added and the luminescence intensity of each well was measured. The CST1-CTSH content of the tested samples was calculated from the calibration curve. The linear range of the calibration curve is 10~1500pg / mL, with attached figure 2 is the calibration curve of the CST1-CTSH detection kit, wherein the Y-axis represents the logarithmic value of the luminescence value, and the X-axis represents the concentration l...
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