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Screening of the Genes Highly Expressed in Tumors by mRNA Fluorescent Differencial Display

Author: WenYan
Tutor: WangWenBing
School: Jiangsu University
Course: Biochemistry and Molecular Biology
Keywords: malignant tumor fluorescence differential display fluorescence quantitative PCR Calcium binding protein C3orf1 gene baculovirus
CLC: Q78
Type: Master's thesis
Year: 2006
Downloads: 35
Quote: 0
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Malignant tumor has at present become one of the most seriousdiseases that pose great theat to human’s health.Survey on death causesof Chinese population showed that malignant tumor is the most serviousdisease with one in five persons dying from malignant tumors. Commonmalignant tumors include; esophageal cancer, lung cancer, gastric cancer,leukemia and so on, all of which have become the focus of researchers inthe world.In this thesis, in order to investigate genes that express specificallyin malignant tumors, we studied gene expressed highly in esophagealcancer and gastric cancer as well the lung cancer cell line 95D with highmetastatic potential by fluorescence differential display (FDD). Thefollowing are what we have achieved and concluded:Thirty cDNA fragments were excised by performing FDD-PCRreactions with those cancer tissues and their respective normal tissuesadjacent to the tumor in esophageal cancer and gastric cancer, andtwenty-one differential bands were obtained by carrying out the secondPCR amplification. These fragments were cloned and sequenced, andnine cDNA fragments were obtained. Six of them come from esophagealcancer tissue, three from gastric cancer tissue. Seven cDNA fragmentsthat expressed more differentially than others were confirmed byfluorescence quantitative PCR, and the result showed that: five of theseven were significantly higher than that in the corresponding normaltissue adjacent to the tumor. The other two had no significantly difference.The result showed that it has highest homology (88.7%) with the calciumbinding protein of house mouse. Fluorescence quantitative PCR was used to detect the relative expression amount of it in six kinds of tumor cells,and the result showed that expression level of it in esophageal carcinomacell line TE1 was significantly higher than that of it in the other fivetumor cells(p<0.05) . The results above indicate that carcinogenesis oftumors may be associated with the specific expression of certain genes.Seven cDNA fragments were excised by FDD-PCR reactions withfrom lung cancer cell line with high metastatic potential (95D) and thelung cancer line(LTEP-a-2) . These fragments were cloned and sequenced,and three cDNA fragments were obtained. All of them come from the cellline 95D. Two cDNA fragments that expressed more highly than otherswere confirmed by fluorescence quantitative PCR in the cell line 95D.One gene highly homologous with the gene C3orf1 attracted our attention.The experimente get its span. The ORF of the gene was 858 bp, coding285aa. Expression level of genes of interest C3orf1 in some differenttumor cells was investigated by employing fluorescence quantitative PCR(p<0.05) .Both prokaryotic expression plasmid pET-28a-C3orf1 and baculovirusexpression transfer plasmid ETB-C3orf1 were successfully constructed,and the stable expression of C3orf1 in prokaryotic and baculovirusexpression systems was proved by SDS-PAGE.

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