| This research presents the proteomics analysis of the rectal cancer and adjacent tissues by using two-dimensional chromatography coupled with mass spectrometry, from which thirty-five differentially expressed proteins were detected. The eukaryotic Elongation Factor 2(e EF2) was selected as target protein for further invesigation through analyzing multiple parameters and reviewing a large number of documents.Three hundred amino acid sequence from the N terminal of e EF2 were selected as targetpolypeptides with the codon optimization for synthesis of the target gene fragment,which was then successfully cloned into the prokaryotic expression vector p ET30 a.The recombinant plasmid was transformed into competent cells to induce the expression of high purity-e EF2 fusion protein. The hybridoma was formed by the fusion of immunized mouse spleen cells and myeloma cells after the micewere immunizedby purified e EF2 fusion protein.And four strains of hybridoma cells that constantly secrete monoclonal antibody against human e EF2 werescreened by limited dilution method. The titer of monoclonal antibody with sub-type of Ig G was 1:240000 or above. The purity of the antibody reached more than 90%.Andthe most appropriate pair of antibody was selected for the double antibody sandwich ELISA according to the pairing screening fromthe four strains of e EF2 monoclonal antibody.Meanwhile, the double antibody sandwich ELISA method for detection ofe EF2 was successfully established. Finally, the researcher used the established method for detection of e EF2 expression in timorous patients’ serum.This study is divided into four parts as follows:Part 1: The research on differential proteomics between rectal cancer and paracancerous tissueMethods:1 、 Twenty samples of rectal cancer and paracancerous tissue ofadenocarcinoma on the Dukes B stage was selected and preparedforpeptide mixture. 2、The protein profile of cancer and paracancerous tissue was analyzed by the combination of two-dimensional chromatography and mass spectrometry. 3、The differentially expressed proteins in rectal cancer and paracancerous tissues were obtained by data analysis.Results: 1、For cancer samples: A total of 31618 peptide sequences were acquired and 813 proteins were identified. 2 、 For paracarcinoma samples: A total of23547 peptide sequenceswere obtained, and 537 proteins were identified. 3 、 Statistical analysis: Thirty-five proteins were detected as differentially expressed proteins in cancer tissues, in which eighteen proteins were up-regulated and seventeen proteins were down-regulated. 4、Theeukaryotic Elongation Factor 2(e EF2) was selected as target protein for further investigation through analyzing multiple parameters and reviewing a large number of documents.Part 2: Cloning and expression of e EF2 geneMethods: 1、The epitope of e EF2 protein amino acid sequence was analyzed, and 300 amino acids from the N terminal were determined as the target proteins with the codon optimization.Therefore, it is more suitable for prokaryotic expression. 2、The target gene was synthesised after the codon optimization.The Enzyme Nde I restricted digestion site was applied in the upstream and Xho I was applied in the downstream. 3 、 The target gene fragment of e EF2 was cloned into the prokaryotic expression vector p ET30 a and the prokaryotic expression plasmidp ET30a-e EF2 was constructed. 4、The recombinant expression plasmid was transformed into E.coli BL21 competent cells, and the fusion protein was induced and expressed.Results: 1 、 The prokaryotic expression plasmid p ET30a-e EF2 was successfully constructed. 2、The e EF2 fusion protein was successfully expressed and purified. Part 3:The preparation of monoclonal antibodyagainste EF2Methods: 1 、 The mice were immunized bythe purified e EF2 fusion protein with Freund’s adjuvant. 2、The hybridoma cells were prepared by the fusion of myeloma cells and spleen cells from the immunized mice. 3、The positive hybridoma cells were screened bythe determination of titer of ELISA method. 4、The positive hybridoma cell lines were subject to subtype identification, cell line establishment and cryopreservation by limited dilution method. 5、The ascites of mice were induced and prepared by inoculation of the positive hybridoma cells into mouse peritoneal ascites and the antibody titer was detected. 6、e EF2 monoclonal antibody was subject to purification, subtype analysis and titer detection. 7 、 The matched monoclonal antibody was screened by ELISA double antibody sandwich methodResults: 1 、 Four strains of hybridoma cells that constantly secrete monoclonal antibody against human e EF2 were screened by limited dilution method.2、The titer of monoclonal antibody was 1:240000 or above. The sub-type was Ig G, and the purity of the antibody reached more than 90%. 3、The most appropriate pair of antibody was selected for the double antibody sandwich ELISA according to the pairing screening fromthe four strains of e EF2 monoclonal antibody. And the double antibody sandwich ELISA for detection ofe EF2 was successfully established.Part 4:The expression of e EF2 in tumorous patients’ serumMethods: 1、The e EF2 concentration in the serum of various tumorpatients was detected by double antibody sandwich ELISA method with e EF2 monoclonal antibodywhich was developed in Part 3 in this study. 2、The p-e EF2 concentration in the serum of malignantpatients was detected by the commercial phosphorylated e EF2(p-e EF2) kit. 3、The concentrations of e EF2 and p-e EF2 in the serum of healthy people were detected as normal control simultaneously. 4、The tumor patients were divided into different groups, including the pretreatment group, and chemotherapy group, etc. The changes in both p-e EF2 and e EF2 among the varieties of tumors during the different stages were evaluated with the associated clinical data. 5、In order to evaluate the impact of storage timeon the clinical specimens,the specimens collected in 2014 and 2015 respectivelywere compared as separate groups.Results: 1 、 The concentration of e EF2 was significantly different among the pretreatment group, chemotherapy group for rectal cancer patients. Additionally, the concentration of e EF2 was significantly different among patients with different cancer types such as colon cancer, lung cancer, breast cancer, in comparison withthe healthy control group, respectively. 2、There was no significant difference of e EF2 concentration between the gastric cancer and the control group.And there was also no significant difference between the pretreatment and chemotherapyof rectal cancer group. 3、The concentration of p-e EF2 was significantly different in the pretreatment group、 chemotherapygroup of patients with rectal cancer, in comparison with the healthy control group respectively. 4、 The concentration of p-e EF2 was significantly different between the pretreatment group and chemotherapygroup of patients with rectal cancer. 5、The results indicated that there was no significant difference ofboth the p-e EF2 and e EF2 concentration between frozen specimens in 2014 and 2015 groups.Conclusions: 1 、 The differentially expressed protein profile of rectal cancer and paracancerous tissue was obtained by mass spectrometry-based proteomics analyses, from which eukaryotic Elongation Factor 2(e EF2) in rectal cancer was identified as up-regulated protein. 2、Anti-human e EF2 monoclonal antibody was successfully prepared with high titer and specificity by using the fragment of e EF2 fusion protein as immunogen at the N terminal 300 amino acid. The double antibody sandwich ELISA method for detection ofe EF2 was successfully established. It is concluded that the multiple antigen epitopes at N terminalcan be induced to generate different monoclonal antibodies for the preparation of e EF2 monoclonal antibody in the followingresearch. 3、According to the results from analysis of both e EF2 and p-e EF2 in a large number of serum samples, the author proposed that the e EF2 can be considered as a tumor marker for tumor examination screening. P-e EF2 can be applied assensitive indicator in monitoring tumor therapy. |
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