Screening,Cloning,and Characterization Of A Novel Gene Associated With Malignant Glioma

BackgroundGlioma is the most common intracranial malignancy and the leading cause ofintracranial tumor-related deaths. Despite the tremendous efforts have been made to improve the early detection of this disease and to design new treatment modalities, few of patients can alive more than three years as soon as it is diognosed. There is still a practically significant need to find new markers and therapeutic targets for the management of glioma patients. Likewise, the recent identification of critical biochemical pathways including apoptosis, cell adhesion, extracellular matrix, angiogenesis, and signal transduction have offered promising targets for new therapeutic approaches of glioma, but no one subject as a target to treat glioma shows the definite effect. The identification and characterization of new glioma-specific antigens or genes could provide new markers for the design of new drugs or vaccine and could be instrumental significance for the development of new treatment modalities. Objective1. To Screen malignant glioma associated novel genes with microarrays or gene chips. 2. To validate the results of microarray hybridization and the tissue expression specificity of candidate novel genes. 3. To cloning the full length sequences of the novel gene associated to the malignant glioma and to characteristic of this gene with bioinformatics. 4. To expression of the gene in E.coli and to purification of the protein; To get poly-clonal antibodies of the protein with immunize mice and to detect the expression of the gene in gliomaMethods1. Liquid nitrogen frozen stored clinic samples of glioma and matching para-cancer tissues were obtained from Neurological Department of ZhuJiang Hospital. The tissues were verified with pathology. 6 glioblastoma, 4 anaplastic glioma and 3 astrocytoma. The other organ tissues come from voluntary contributor.2. cDNA microarray hybridization and analysiscDNA microarrays were made by Max-Plank Guest laboratory of ShangHai Institute of Cell Biology. mRNA was extracted from clinical samples with Qiagen mRNA isolation kit cDNA probes for hybridization to the microarrays were made by reverse transcription of the purified mRNA in the presence of 33P dCTP.The labeled cDNA pools were hybridized to the genefiher arrays in a roller hybridization oven (Hybaid, America). The filters were then exposed to a Pachard phosphoscreen for 8h, and scanned in a Fujifilm ImageReader instrument (Fuji Photo Film Co. LTD). The signals resulting from the phosphorimaging of ImageReader were directly imported into the image analysis system of ArrayGauge (FUJi Photo Film CO. LTD). The image analysis system automatically locate, calculate, and store the intensity of cDNA spot from each array and simultaneously compare the two values of the same cDNA blot on the two different filters.3. Northern blot analysismRNA isolated from different tissues were electrophoresed on a standard 1.2%formamide agarose gel and transferred to Nylon membrane. The probes pick out the previously differentially expressed cDNA clones were labeled with DNA labeling kit (promega) in the presence of 32P dCTP. Blots were hybridized and washed as previously described in genefilter hybridization. All autoradiographs are scanned into computer and the signal density and signal area were analyzed.4. The full length sequence cloning of the gene specifically associated with glioblastoma was carried out followed the SMART?RACE cDNA Amplification kit protocol (Clontech Laboratories, Inc.). Two gene-specific primers for 3' and 5'-RACE reactions were designed according to the known partial sequences (364bp) of the target gene named HTB(clone ID HTB). The products of 3' and 5'-RACE reactions were enzyme digested and inserted into pt-Adv plastnid vector, and then transformed into E.Coli and sequenced.5. The bioinformatics analysis of the sequence were performed in the data bases of NCBI, PROSITE, SWISS-PROT, EBI, PDB, ALLALL through internet6.Expression and purification of the protein in E.coliThe open...