Four Kinds Of Tumor Markers In Vitro Expression And The Clinical Significance Of Survivin Antibodies In NSCLC Patients

Background:Lung cancer is the most common cancer worldwide and causes more deaths per year than any other cancer. Early diagnosis of lung cancer has became the serious challenge to the scientific community. Currently, there have many clinical method to detect lung cancer. Tumor markers attracted large scholars interest because its have many advantages to lung cancer early diagnosis and possess potential applications for risk population screening.With the progress of the molecular biology and the human genome project, more and more specific tumor markers were found.Autoantibody was one hotspot as tumor markers in continuous in-depth study, Ideal tumor marker has not been found, The single tumor marker for cancer detection sensitivity and specificity is not high , Combined detection of tumor markers become a trend. Objective: Fusion protein of Survivin, EGFR, CyclinB1 and CCNY was successfully expressed in E.coil BL21(DE3). The research purpose is to discuss the clinical significance and value of Survivin autoantibodies in lung cancer. Methods: cDNA sequence was obtained by RT-PCR and inserted into prokaryotic expression vector pET30a ( + ). Fusion protein was expressed in E.coil BL21(DE3). SDS-PAGE and Western blotting technique was used to confirm the fusion protein. The fusion protein was purified by Ni-NTA agarose affinity chromatography. At last, indirect ELISA was used to detect Survivin autoantibody level in the serums of 215 samples from NSCLC patient and serums from 20 samples of nonmalignant lung disease patient as well as 89 samples of healthy persons were also detected as control. Results: The recombinant vector pET30a ( + )/survivin,pET30a( +)/egfr,pET30a(+)/cyclinb1 and pET30a( +)/ccny were contructed and the fusion protein is successfully expressed in E.coil BL21(DE3) as inclusion body,especially Survivin protein accounted for 50% of the total bacterial protein. Using nickel ion affinity chromatography to purify four kinds of fusion protein, the purity of fusion protein is above 80%, in particular the purity of Survivin protein is over 90%.Indirect EILSA was done to detect Survivin autoantibody in these serums using purified Survivin protein. It was shown that the positive rate of Survivin autoantibody was 19.5 % with a specificity of 88.9 % in NSCLC patient.The expression of Survivin autoantibody have correlation with the volume of tumor and the metastasis of tumor in NSCLC patients, P < 0.05. In our research, positive detection rate of NSCLC was much improved by detecting Survivin autoantibody combined with CEA compared to other tumor markers combined with CEA. Conclusion: In this study, purified fusion protein was successfully obtained. Indirect ELISA for detecting Survivin autoantibody in serum of NSCLC patients using purified Survivin protein was established. Our results indicated that Survivin autoantibody as a latent value of tumor marker could be used for clinical diagnosis in NSCLC patients..