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Wang H.,Beijing TB and Thoracic Tumor Research Institute | Lai B.,Beijing TB and Thoracic Tumor Research Institute | Li W.,Beijing TB and Thoracic Tumor Research Institute | Yang X.,Beijing TB and Thoracic Tumor Research Institute | And 3 more authors.
Chinese Journal of Lung Cancer | Year: 2010

Background and objective: The p53 as a transcription factor in cell stress was activated to regulate cell cycle and programmed cell death to inhibit tumor growth. Usually, p53 is kept in non-activated state through various mechanisms, including the action of p53 C-terminal negative regulatory sequences. The purpose of the study is to prepare the two types p53 recombinant adenoviruses that carry full-length p53 as well as deletion of negative regulatory sequences at p53 C-terminus and to detect exogenous GFP expression in human lung cancer cell infected-virus by FCM scatter plot. Methods: Using pAdEasy-Track vector system the p53 recombinant plasmids was constructed and the homologous recombinants in E.coli was produced. The three kinds of recombinant adenovirus in L293 cells was generated, sequencing proved. Exogenous GFP expression in human lung cancer 801D cells infected-virus was detected by FCM scatter plot. Results: p53 recombinant adenoviruses named Ad-p53(wtp), Ad-p53(del) and Ad-(empty carrier) were produced. Results of sequences indicate that the Ad-p53(del) was deletion of 111 bases before stop codon TGA and of 3 untranslated region at p53, the Ad-p53(wtp) no loss of any p53 base, the Ad-(empty carrier) no p53 sequence. FCM scatter plot indicate the percentage of 801D cells expressed GFP with three kinds of viral infection was almost same and was increased with the virus density. 801D contains ratio of cells with different fluorescence intensity. Conclusion: The preparation of recombinant adenovirus, Ad-p53(del), pA-p53(wtp) and Ad-(empty carrier). The cells expressed-GFP can be quantitatively detected by FCM scatter plot. It was provide that the reliability of the virus system and accurate method for selecting viruses density to infecting cells. Source


Li Y.,Beijing TB and Thoracic Tumor Research Institute | Yue W.,Beijing TB and Thoracic Tumor Research Institute | Wang Y.,Beijing TB and Thoracic Tumor Research Institute | Zhang L.,Beijing TB and Thoracic Tumor Research Institute | And 2 more authors.
Chinese Journal of Lung Cancer | Year: 2010

Background and objective: Autoantibodies as new tumor markers may play an important role in the early diagnosis and evaluating the prognosis of lung cancer. In this study, we detect epidermal growth factor receptor (EGFR) autoantibodies using peptide array and screen the epitopes which are recognized by EGFR autoantibodies. Methods: Peptide array covering the extracellular domain of EGFR protein was synthesized by a synthesizer (ASPSL) made by Intavis company. EGFR autoantibodies in the serums of non-small cell lung cancer patients was detected using peptide array. Results: Six of 20 patients were found to have EGFR autoantibodis. The positive rate is 30%. Nine high frequency spots were found in the 6 positive patients and 8 high frequency spots clustered in the III and IV domains. Conclusion: These findings will offer new clues for the futher studies of EGFR and EGFR autoantibodies. Source


Ma L.,Beijing TB and Thoracic Tumor Research Institute | Yue W.,Beijing TB and Thoracic Tumor Research Institute | Zhang L.,Beijing TB and Thoracic Tumor Research Institute | Wang Y.,Beijing TB and Thoracic Tumor Research Institute | And 2 more authors.
Chinese Journal of Lung Cancer | Year: 2010

Background and objective: Lung cancer is one of the most common cancers worldwide and causes more deaths per year than other cancers. Autoantibody was one hotspot in tumor marker research. But the clinical value of Survivin autoantibody is not clear in lung cancer patients at present. The aim of this study is to dertermine whether Survivin autoantibody could be used as a diagnostic factor of lung cancer and what the clinical value of Survivin autoantibody was in non-small cell lung cancer (NSCLC). Methods: Survivin cDNA sequence was obtained by RT-PCR and inserted into prokaryotic expression vector pET30a(+). Fusion protein of Survivin was expressed in E.coil BL21(DE3). SDS-PAGE and Western blot were used to confirm the fusion protein. The Survivin 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 patients and serums from 20 samples of nonmalignant lung disease patients as well as 89 samples of healthy persons were also detected as control. Results: The recombinant vector pET30a(+)/Survivin was contracted and the Survivin protein is successfully expressed in E.coil BL21(DE3) as inclusion body. 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 patients. The expression of Survivin autoantibody has correlation with the volume of rumor 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 of Survivin 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. Source

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