Screening Of Molecular Markers For The Early Diagnosis Of Colorectal Cancer By Bacteriphage Display

[Objective]Establish a colorectal cancer phage-peptide library and select biomarkers for early detection of colorectal cancer.[Method](1)Cinstruction of phage display library derived from colorectal cancer tissue. Collected 30 cases of colorectal cancer tissue samples, which was frozen immediately after surgery by 1st affiliated Hospital of Second Military Medical University. Total RNA was extracted by using Trizol method. Equal amounts of total RNA from 30 tissues were pooled together and poly (A) RNA was purified from the total RNA pool using the mRNA extracting protocol. OrientExpression cDNA Synthesis and Cloning System were used for the construction of the T7 phage colorectal cancer cDNA libraries including reverse transcription, end Modification, Ligation to Directional EcoRⅠ/HindⅢLinkers, EcoRⅠ/HindⅢdigesting, inserting into T7Selectl-1 EcoRⅠ/HindⅢVector Arms and in vitro packaging. We successfully constructed a colorectal cancer phage-peptide library.(2) Enrichment for colorectal cancer specific phage-peptide clones. To enrich for phage clones that bind to IgGs specifically associated with colorectal cancer, we performed 5 round of positive and negative selection.(3)Marker primary screening by ELISA. Randomly selected 2000 phage clones after 5 round of positive and negative selection. To find the clear colorectal cancer specific markers further experiment of ELISA was explored. The 1:10000 diluted T7 tailed antibody was coated by 1×PBS in 96 well plates overnight at 4℃. After washing, closed, washing, add phage solution, add serum, washing, add HRP coupled goat anti-human IgG, washing, color, termination and other steps, and read at 450nm using an microplate reader. The phages with the ration of OD>2.0(colorectal cancer plasma pool v.s. control plasma pool)were selected as the potential makers for colorectal cancer diagnosis.(4)DNA sequencing and protein function prediction. The insert of positive phages selected by ELISA were sequenced. The gene alignment was blasted at NCBI to find the exact or most similar knowen gene. Then the relation between these gene and cancer was predicted. (5) Ssecondary screening by ELISA. After sequencing and compared with the latter, a total of 20 clones with 30 cases of colorectal cancer patients and 30 controls were patients with serum ELISA screening. Screening by the ELISA method.(6) Verification. After re-screening ELISA, the selected representative of the five clones, with 60 cases of colorectal cancer patients and 60 control patients with serum ELISA validation. Screening by the ELISA method.(7) Immunohistochemistry. COX6B1 for immunohistochemistry, using EnVision two-step method.[Result](1) The quality and diversity of the phage library was tested by PCR amplification and get electrophoresis of 20 randomly selected phage colonies and the recombination rate was 60% and its average titre is 3.0×106pfu. (2)22 phage colonies were selected totally by ELISA and were sequenced. Amplification of phage 871 was failed. There are two gene sequencing failed.5 of the rest of them are novel gene. Of the gen rescarched before 11 have been reported have relation with cancer. Phage 73 and phage 1048 is the same gene. (3) After re-screening ELISA selected clones representative of five, there are NO 95,149, 174,396 and 1009 phages. (4)Though ELISA validation, we can see No 95,149,174,396, 1009 of phage can be used to diagnose colorectal cancer. The area under the ROC curve is 0.763,0.669,0.790,0.742,0.796, respectively (all P<0.05). Their joint detection of colorectal cancer had a sensitivity of 95%(P＜0.001), specificity of 69%(P<0.001), the ROC area under the curve is 0.886,95% confidence interval is (0.827,0.945). (5) Immunohistochemistry showed COX6B1 expression in colorectal cancer was significantly higher than the normal mucosa. The statistics between the two significant differences (P <0.001).[Conclusion](1)The cDNA library constructed by T7 phage could well display little abudance peptide. (2)The phages which displayed colorectal cancer special peptide were enriched after five cycles of affinity selection. (3)These gene selected by ELISA can be biomakers for detection of colorectal cancer.