Colorectal cancer specific antigenic peptide and test kit for detecting colorectal cancer
A colorectal cancer, specific technology, applied in the field of colorectal cancer-specific autoantibody enzyme-linked immunosorbent assay kits, can solve the problems of lack of sensitivity and specificity, inability to diagnose colorectal cancer early, and unacceptable to patients, etc. and high specificity
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Embodiment 1
[0044] Example 1 Construction of colon cancer phage display peptide library
[0045] Extract total RNA from 30 cases of fresh colorectal cancer tissues, mix equal amounts to 1 mg, extract mRNA, use oligo dT primers to reverse transcribe into cDNA, blunt the ends of cDNA, add EcolⅠ and Hind III adapters, and remove after double enzyme digestion The small fragments were then inserted into the arms of T7 phage (NOVAGEN), packaged in vitro, and constructed into a colon cancer phage display peptide library. The optical picture is as follows: figure 1 shown. The capacity of the constructed phage display peptide library was determined to be 3.0×10 6 pfu, the recombination rate was 60%, meeting the requirements of the screening test.
[0046] Example 2 Affinity Screening of Colorectal Cancer Specific Antigen Peptides
[0047] Take 10 μl of A / G agarose beads and place them in a 1.5ml centrifuge tube, wash twice with 500 μl of PBS (pH7.4), and block with 1% BSA at 4°C for 1 hour. Th...
Embodiment 3
[0048] Example 3 Serological screening of molecular markers for early detection of colorectal cancer
[0049] 3.1 Preliminary screening of mixed blood samples
[0050] LB liquid medium for the screened peptide library 1:10 8 After dilution, take 100 μl of phage liquid, 250 μl of freshly shaken BLT5403 bacteria, and 3ml of top layer agar kept at 55°C, mix well and immediately pour it into a plate covered with LB. After cooling, incubate for 4 hours in a 37°C incubator. Individual plaques form. Add 1ml of freshly shaken bacteria BLT5403 to each 1.5ml centrifuge tube, use a sterilized toothpick to randomly pick a single, well-defined phage in a 1.5ml centrifuge tube, randomly select 2000 phage clones, and Numbered sequentially. The selected phages were shaken in a constant temperature shaker at 37°C for more than 4 hours until the liquid in each tube became clear. The selected phages were stored at 4°C. Use T7 primers (synthesized by Shanghai Sangon Biotechnology Co., Ltd.) ...
Embodiment 4
[0057] Example 4 Application of training group serum to establish detection model
[0058] 4.1 Establish a joint detection mathematical model
[0059] In order to improve the sensitivity of the detection kit, a joint detection method of different phage clones was adopted. Summarize the response data of 16 different phage clones and 30 pairs of training groups (30 cancer patients and 30 controls), and use Binary Regression Models Version 2.0 software in the Matlab 7.0 environment to establish a Bayesian bivariate for screening colorectal cancer Mathematical model (Bayesian binary regression) to detect the probability of the tester suffering from colorectal cancer. 30 cases of cancer patients belong to category 1, 30 cases of controls belong to category 0, the cut-off value is 0.5, greater than or equal to 0.5 is positive, and less than 0.5 is negative, such as image 3 As shown, this model can be successfully applied to distinguish 30 pairs of cancer patients and healthy cont...
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