Study Of The Preparation And Application In Colorectal Cancer Of Monoclonal Antibodies Of Cytokeratin-20

Objective: To prepare the monoclonal antibody of cytokeratin-20 and explore the expression of cytokeratin-20 in clinical blood samples with colorectal cancer, providing objective reference.Methods: cDNA of cytokeratin-20 was amplified by RT-PCR using the total RNA from human epithelial cells as template and was cloned into the prokaryotic vector of pET28a. The recombinant plasmid was conformed by enzyme digestion and sequencing, and transformed into the bacteria BL21(DE3) competent cells. The cytokeratin-20 protein was purified through the Ni-NTA column after being induced under particular temperature and IPTG concentration. Balb/c mice were immunized by the purified cytokeratin-20 which was fully mixed with the Freund's adjuvant. After the booster immunization and detection of immune effect, the spleen cells and myeloma cells were fused and cell clones were screened by ELISA. The clones were identified by Western Blot assay. A simple enzyme-linked immunosorbent assay kit for cytokeratin-20 detection was prepared by using the specific monoclonal antibodies and clinical blood samples of normal people and patients with colorectal cancer.Results: The recombinant plasmid pET28a-CK20 was constructed and cytokeratin-20 was purified. 3 cell lines which secret anti-cytokeratin-20 monoclonal antibodies were successfully obtained. 10 normal serum samples and 40 serum samples from patients with colorectal cancer were detected with the ELISA kit preparing with the before-mentioned anti-cytokeratin-20 monoclonal antibody. Normal specimens were negative. 19 cases from patients with colorectal cancer specimens were positive with a detection rate of 47.5%, all belonging to the Dukes points class B, C, D period. The detection rates of the B, C, D periods respectively were 25%, 41.7%, 84.6% (3 / 12, 5 / 12, 11/13). It showed statistically significant difference between colorectal cancer group and normal control group (P﹤0.05). C and D phases of colorectal cancer also had significant difference comparing to the A and B phases (P﹤0.05).Conclusion: In this study, cytokeratin-20 was successfully expressed and purified in prokaryotic expression system. Three high titer monoclonal antibodies of anti-cytokeratin-20 monoclonal antibody were prepared. By using a self prepared enzyme-linked immunosorbent assay kit, 10 normal blood samples and 40 blood samples from patients with colorectal cancer clinical were determined. The results showed that normal specimens were negative. 19 cases of patients with colorectal cancer specimens were positive, belonging to the Dukes classification B, C, D period. The detection rate was 47.5%. Respectively, the detection rates in the three period were 25.0%, 41.7%, 84.6% (3/12, 5/12, 11/13). The above results suggested that cytokeratin-20 can also be chosen as a tumor marker, in addition to CEA. Through the detection of peripheral blood cells of patients with cytokeratin-20, high-risk patients can be quickly detected and identified, facilitating the monitoring of tumor development and prognosis.