Cloning And Expression Of Ornithine Decarboxylase Gene & Prepration Of Monoclonal Antibodies Against Human ODC Protein

Ornithine decarboxylase(ODC, 4.1.1.17) is the first rate-limiting enzyme in polyamine biosynthesis. The polyamines are naturally occurring aliphatic polycations found in almost all living cells. It contains putrescine, spermidine, and spermine, which play an important role in cell proliferation, differentiation, and transformation. They are positively charged at neutral pH and the charge is distributed along the length of the molecule. This facilitates their interaction with anionic molecules such as DNA and RNA. Polyamines can interact with membranes, which generally leads to an decrease in the fluidity of the membrane. This has important consequences for the movement of proteins and lipids within the membrane. In the early 1970s, the late Diane Russell first observed that patients with malignant disease excreted higher amounts of polyamines in their urine than normal individuals. As with other tumours, polyamine content of colorectal cancers is increased when compared to the equivalent normal tissue.In addition to changes in polyamine content, Over-expression of ODC activity is a well-recognized feature of many cancers. As the first and rate-limiting enzyme, ODC is the most extensively studied enzyme of polyamine metabolism. The ODC protein is 50kDa as an monomer and about lOOkDa when forming the active dimmer. ODC synthesis is dramatically induced by different growth stimuli, such as hormones, growth factors,carcinogens, viruses and oncogens. The regulation can occur at the levels of transcription, translation and protein degradation. The alteration in enzyme can occur very quickly and change polyamine level in the end. ODC gene is considered as an immediate early gene, and contains response elements for several trans-acting factors, including a CAMP response element, a possible insulin response element and several Sp1 binding sites. In addition, ODC gene expression is tightly linked to transformation by activated ras, v-src and myc. So, recently, ODC gene is postulated as an oncogene that is essential for cell transformation. As to colorectal cancer ODC is found in very limited amounts in quiescent cell and its activity is found to increase significantly in colon adenocarcinoma and prostate tissue compares to normal tissue from the same patients. Colorectal carcinoma is a major health problem in the west and become more and more popular in the east. Colorectal adenoma increases sharply in the six decade of life and occur in more than half the population by the ninth decade of life, but colorectal cancer develops in only small part of the population. With the improvement of surgical techniques, more effective adjuvant therapy and earlier diagnosis, mortality has been decreasing over the last decade. However, older individuals still need to improve the survival rate by molecular approaches.The same as other cancers, colorectal cancer is a mutifactorial disease with about 75%of cases being sporadic and only 25% being linked to other conditions such as chronic inflammatory disease, and family history of colonrectal neoplasia. Many risk factors have been identified. In addition to advancing age and conditions mentioned above, the diet such as rich in fat, low fiber and calcium, tobacco use and sedentary lifestyle have play an important role in the disease initiation and progression. Laboratory testing and screening has the potential favorable effect in patients outcome and survival rate. Studies have been directed onidentifying markers for later development of colorectal cancer. Polyamine content as well as ODC activity has been found to be increased significantly in adenocarcinoma tissue compared to paired nomal tissue. Colorectal cancer tissue had approximately 4 times the polyamine content of the colonic mucosa from patients with non-malignant disease and Serum polyamine levels were also increased in patients with colorectal carcinoma. It was shown that wild-type APC decreased the levels of ODC RNA and the ODC promoter activity. These research results suggested that ODC might be used as an identifying marker for later development of additional primary tumors.The present study examined the ODC gene expression in human colorectal carcinoma and normal colon mucous. ODC mRNA was extracted from human colon cancer tissues and ODC mRNA were detected by RT-PCR. The ODC cDNA was inserted into expression vector and the ODC gene expression vector pQE-ODC has been established. The vector has a 6-His tag which made the expression product ODC protein of about 95% pure was used as a good immunogenic agent to immunize the BALB/c mouse. Monoclonal anti-ODC antibody is produced by cell hybrids between hypoxanithine phosphoriboxyltransferase deficient myeloma cells and spleen cells of immunized mouse. The antibody is a IgG2a type.ELISA and western blotting showed that the mAb could combine the ODC protein. Staining the colorectal tissues with ODC mAb showed significant difference between normal and tumor mucous. The deep brown color staining could be found mostly in gland cells of the tumor while there are no stains or weak stains in stoma cells. The results suggested that there was high concentration of ODC in tumor cell cytoplasm. Moderately differentiated carcinoma cells demonstrate most cells and deepest staining than others. More samples should be tested. RT-PCR showed that the ODC mRNA was much higher in colon tumor tissues than in normal tissues. These changes didnot correlate with gender, histological grade or Duck' s stage, which confirmed that ODC was both early and late events in the colon carcinoma sequence in all patients. The continuously change in ODC content play an important role in the diagnosis of human colorectal carcinoma. Part One: Cloning, Expression and Purify of Human ODC Protein METHODS:1. According to the sequence of the human ornithine decarboxylase gene, a pair of primers specific for amplifying the complete ODC cDNA were designed and synthesized.2. Total RNA were extracted from normal and cancer tissues, respectively. The method of RNA extraction was similar to the Trizol RNA extraction protocol. ODC cDNA was synthesized by RT-PCR3. The product of PCR was inserted into a T-A clone vector pMD18-T.4. The recombinant was transformed into E. coli. DH5 a , Then the plasmid was extracted, purified and digested with BamH I and Sal I . The insert fragment was collected.5. The expression vector pQE30 were digested by BamH I and Sal I . The large fragment was linked with the target DNA fragment. The recombinant plasmid was identified arid sequenced.6. The positive recombinant was transformed into an expression strain of E. col i. M15 and the bacteria was induced to express ODC protein by IPTG.7. The protein was purified by Ni-NTA affinity chromatography and tested by SDS-PAGE.RESULTS AND CONCLUSION:1. Using the designed primers, the complete encoding sequence of ODC gene was amplified from human ODC total RNA.2. A expression plasmid contain ODC cDNA was constructed by ligating the expression vector pQE30 with ODC cDNA.3. The protein was purified by Ni-NTA affinity chromatographyPart Two: Preparation of Monoclonal Antibody Against Human ODC METHODS:1. The BALB/C female mouses were immunized with the prepared ODC protein direct admistrating into the spleen and were reimmunized every two weeks by intraperitoneally.2. The monoclonal antibodies against human ODC were prepared with hybridoma technique.3. The antibody in the supernatant of cell clones were tested by ELISA and the positive hybridoma cells were recloned four times in HAT medium by limiting dilution.4. The purified ODC protein were separated by standard SDS-PAGE and tested by Western blot.5. The chromosom of the cell strains were tested. RESULT AND CONCLUSION:1. 4 strains of hybridoma cells that could secret monoclonal antibodies stably were obtained.2. The chromosome number of hybridoma cells was between 100-110 that is about the total number of the two mother cells. There were metacentric and submetacentric chromosomes in hybridoma cell.3. ELISA showed that the subtype of the McAb was IgG2a with high specificity and affinity.Part Three: Using of The Obtained McAb METHOD:1. Test the ODC expression in colon carcinoma by Immunohistochemical staining using the prepared anti-ODC McAb.2. Test the ODC expression in colon carcinoma by Western blot using the prepared anti-ODC McAb.RESULT AND CONCLUTION:1. Immunohistochemical analysis shows that density staining in deep browncolor in most of the colon carcinoma tissues while few faint stainingin the normal tissue.2. The same result can be seen in Western blot.