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Colon cancer specific antigenic peptide and colon cancer detection kit

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, failure of early diagnosis of colorectal cancer, difficult acceptance by patients, etc., and achieve high sensitivity and high specificity

Active Publication Date: 2011-01-19
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these examinations all have deficiencies, such as the limited range of digital rectal examination, poor sensitivity and specificity of fecal occult blood test, colonoscopy is painful and difficult for patients to accept, and imaging examination is not sensitive to early tumors
Currently commonly used tumor markers such as carcinoembryonic antigen (CEA), CA19-9 and other indicators lack sensitivity and specificity, and cannot be used for early diagnosis of colorectal cancer

Method used

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  • Colon cancer specific antigenic peptide and colon cancer detection kit
  • Colon cancer specific antigenic peptide and colon cancer detection kit
  • Colon cancer specific antigenic peptide and colon cancer detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of colorectal cancer phage display peptide library

[0044] Extract total RNA from 30 fresh colorectal cancer tissues, mix the same amount into 1 mg, and extract mRNA. Use oligodT primers to reverse transcribed into cDNA. The ends of cDNA are flushed, and EcolⅠ and Hind Ⅲ linkers are added. The fragments are then connected to the arms of T7 phage (NOVAGEN) and packaged in vitro to construct a colorectal cancer phage display peptide library. The optical picture is as follows figure 1 Shown. Determine the capacity of the constructed phage display peptide library to be 3.0×10 6 pfu, the recombination rate is 60%, which meets the requirements of the late screening test.

Embodiment 2

[0045] Example 2 Affinity screening of colorectal cancer specific antigen peptides

[0046] Take 10μl of A / G agarose beads and place them in a 1.5ml centrifuge tube, wash twice with 500μl PBS (pH7.4), and block with 1% BSA at 4°C for 1h. The agarose beads were incubated with 15μl of colorectal cancer patients and control patients' serum at 4°C overnight. The control patients were defined to exclude any malignant tumors and precancerous lesions, as well as patients with adenomatous polyposis and hereditary non-polyposis colorectal cancer. After washing with 500μl of PBS for 3 times, the enriched antibody was dissolved with 10μl of PBS. 10 tubes of colorectal cancer antibodies and 10 tubes of non-colorectal cancer antibodies are each combined into one tube. Take 20μl of the phage library amplification solution and incubate it with 20μl of non-colorectal cancer antibody for 1h. The unbound supernatant is then combined with 20μl of colorectal cancer antibody. The phage bound to the ...

Embodiment 3

[0047] Example 3 Serological screening of molecular markers for early detection of colorectal cancer

[0048] 3.1 Preliminary screening of mixed blood samples

[0049] The selected peptide library uses LB liquid medium 1:10 8 After dilution, take 100μl of phage solution, 250μl of freshly shaken BLT5403 bacteria, 3ml of top agar insulated at 55°C, and immediately pour it into a plate covered with LB after mixing. After cooling, incubate in a 37°C incubator for 4 hours. A single plaque is formed. Add 1ml of freshly shaken bacteria BLT5403 to each 1.5ml centrifuge tube. Use a sterile toothpick to randomly pick a single, clear-bounded phage into a 1.5ml centrifuge tube. A total of 2000 phage clones are randomly selected. Number in order. Place the selected phage in a constant temperature shaker at 37°C and shake for more than 4 hours until the liquid in each tube becomes clear. Store the selected phage at 4°C. Use T7 primers (synthesized by Shanghai Shenggong Biological Co., Ltd.) ...

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Abstract

The invention discloses a colon cancer specific antigenic peptide, an expression vector and a preparation method thereof, a colon cancer specific autoantibody enzyme-linked immune detection kit comprising the expression vector and an operation method thereof. The preparation method comprises the following steps of: showing a colon cancer cDNA library onto the surface of a phage by utilizing the phage; screening an expression vector of the colon cancer specific antigenic peptide from the colon cancer cDNA library through affinity screening and serology analysis as a main component; and formingthe detection kit by the main component with a negative control, an elisa plate, a color developing agent, a stop solution, and the like. The expression vector of the colon cancer specific antigenic peptide effectively expresses the colon cancer specific antigenic peptide, is coated on the elisa plate and has the capacity of detecting autoantibodies generated by tumor simulation in the blood serum of a colon cancer patient so as to achieve the purposes of early detecting and screening colon cancer patients.

Description

Technical field [0001] The invention relates to the technical field of medical detection reagents, and relates to a colorectal cancer-specific antigen peptide expression vector and a construction method, a colorectal cancer-specific autoantibody enzyme-linked immunoassay detection kit and an operation method. Background technique [0002] Colorectal cancer is one of the most common malignant tumors, and its global incidence rate ranks third. In my country, it has a rising trend year by year, and it ranks second in Shanghai. According to statistics from the Ministry of Health of China in 2007, the incidence rate is the 3rd among all malignant tumors, and the mortality rate from colorectal cancer is 10.25 / 100,000, which is the 5th among cancer deaths. Colorectal cancer can be divided into stages Ⅰ, Ⅱ, Ⅲ, and Ⅳ according to the different TNM stages. The prognosis of each stage of colorectal cancer is very different. The recurrence and metastasis rate of early colorectal cancer is lo...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/63C40B50/06G01N33/574
Inventor 曹广文傅传刚常文军吴玲玲曹付傲刘岩高显华李小攀
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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