| Background and ObjectiveThe incidence and mortality of gastrointestinal cancers have increased significantly in past decades, and most of them have poor prognosis, such as gastric cancer, hepatocellular carcinoma (HCC) and pancreatic cancer, which have been severely threatening human life and health. At present, the serum biomarkers commonly used in clinical diagnosis of gastrointestinal cancers have lower specificity and sensitivity, which have limitations in improvement of early diagnosis of a certain cancer. Thus, it is very urgent to find specific and effective biomarkers for gastrointestinal cancers diagnosis. The recent research has shown that the body can produce autoantibodies to the tumor-associated antigens (TAAs) during the transition from chronic inflammatory diseases such as hepatitis, cirrhosis to malignant diseases, and some of the autoantibodies can be detected before the malignant transiton, then it provides a new idea for the early diagnosis of cancers. Ferritin light chain (FTL) is a subunit of ferritin, which is the major intracellular storage and transport of iron that is required for normal cell growth and proliferation. Previous studies have revealed that the FTL serum level of a variety of malignant tumours patients is significantly higher than normal individuals, which suggested that it may be a serum tumor biomarker for the diagnosis of gastrointestinal cancers. Our previous studies have successfully constructed the recombinant plasmid-pET30a-FTL with the correct target gene fragments by sequence analysis. In this study, the recombinant FTL plasmid DNA was transformed into E.coli BL21 firstly, and then FTL recombinant protein was expressed, which be subsequentlty used as an antigen to detect anti-FTL antibody in sera from patients with HCC, esophageal cancer, gastric cancer and patients with chronic liver diseases as well as sera from normal individuals, for further evaluating the diagnostic value of FTL as a biomarker in the detection of gastrointestinal cancers.Our preliminary results has provided the basis for subsequent studies on the development of a diagnostic method in the diagnosis of certain type of gastrointestinal cancers. Methods1. The recombinant expression plasmid with the FTL, gene fragments was transformed into E. coli BL21.2. The best IPTG concentration and induced time for the expression of FTL was determined.3. The induced product was treated by ultrasonic equipment and centrifuged, and then the quantity of target protein in supernatants and pellets were analyzed by SDS-PAGE.4. The purification of recombinant protein:The target protein with His tag was purified with Ni2+affinity chromatography. The concentration and the validity of the protein were detected by different methods.5. The serum level of anti-FTL antibody in different groups which included a variety of gastrointestinal cancers (primary hepatocelluar carcinoma patients, esophageal cancer patients, gastric cancer patients), chronic liver diseases patients and normal individuals was measured by enzyme linked immunosorbent assay (ELISA).6. The positive rates of anti-FTL antibody in sera from different groups which included various gastrointestinal cancer groups, chronic liver diseases and normal groups was calculated respectively on the basis of the pencentile P95 of normal group as a cutoff point, then the diagnostic value of FTL for gastronintestinal cancers was evaluated with the method of screening trial.ResultsThe recombinant expression vector was transformed successfully, and the most appropriate condition for expression of target protein is 0.05mmol/L IPTG for 5h. Purified recombinant protein was acquired by Ni2+affinity chromatography, and validated by Western blot.ELISA results showed that the anti-FTL antibody level in gastrointestinal cancer groups was higher than chronic liver diseases group and normal individuals. The sensitivity of anti-FTL antibody in the detection of gastrointestinal cancers(HCC, esophageal cancer, gastric cancer), normal individuals and chronic liver diseases patients was 17.6%,14.0%,12.0%,5.1%,9.8%, respectively, which indicated that there has diagnostic value definitely when used on its own, but it is low.ConclusionThe prokaryotic expression system of FTL and protein purification system were successfully constructed and the purified FTL recombinant protein was acquired. The concentration of anti-FTL antibody is improved in gastronintestinal cancer patients and it can be as a serum biomarker for gastronintestinal cancer, the diagnostic value is low when used on its own. This preliminary study also suggests that the mini-array of multilple TAAs including FTL can be used in future for detection of certain type of gastrointestinal cancers to increase the diagnostic sensitivity. |
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