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Cloning of the Human Lung Cancer Associated MAGE-12 Gene and Research Its Expression in Eukaryotic Cell

Author: WangHuaQi
Tutor: ZhangGuoJun
School: Zhengzhou University
Course: Internal Medicine
Keywords: MAGE-12 Eukaryotic expression Green Fluorescent Protein Clone
CLC: R734.2
Type: Master's thesis
Year: 2007
Downloads: 47
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Abstract

Background with the progress of the study of molecular immunology and molecular biology of tumor occurred, the development of the molecular mechanism of an insight into the understanding of the occurrence of the tumor is more than one gene, the gradual process of multi-stage, multi-step, but not yet find tumor specific mutations or defective gene, an important material basis of human tumor-specific immunotherapy is the presence of tumor-specific antigen. 1991, van der Bruggen et al found since the melanoma antigen (melanoma antigen, MAGE) MAGE-1, MAGE family has been found that at least include the MAGE-A, B, C, D, E, F six sub family. MAGE-12 gene belongs to a family of melanoma antigen gene (MAGE), MAGE-12 the 1994 Ding M, was found in human melanoma cell lines DM150 and MAGE-12 and MAGE-2, MAGE-3 The high degree of homology, the study found that the MAGE-12 gene in lung cancer cells have a higher expression rate. MAGE-12 gene encodes a tumor-specific antigen, melanoma, lung cancer and a variety of other tumors have different levels of expression in normal tissues was not expressed in the testis and placenta the MAGE genes found immediately become hot treatment of tumor biology. Green fluorescent protein (GFP) is a report on the mid-1990s developed a new molecule, initially discovered from the multi-tube the jellyfish (Aequoreavictoria) in a protein, GFP in a variety of organisms, such as bacteria, sticky expressed in bacteria, yeast, plants and mammals and generates fluorescent GFP is a small molecular weight, easy to other target gene to form a fusion gene and the toxic side effects on cells, but also the nature of its chemical stability, ease of use, can regularly observe in living cells, so the connection the GFP vector pEGFP-C3 been widely applied in molecular biology. The purpose of using RT-PCR technology, MAGE-12 gene was amplified from a human lung cancer tissue; build MAGE-12 gene cloning vector pGEM-T easy-MAGE-12 and green fluorescent protein expression vector pEGFP-C3-MAGE-12 and transfected into eukaryotic cells (Eca109 cells), to observe its expression in eukaryotic cells, for the preparation of the MAGE-12 gene vaccine, and lay the foundation for tumor immunotherapy. Materials and methods. Extraction of total RNA in lung tissue by RT-PCR method, from which reverse transcriptase amplified MAGE-12 cDNA gene fragment, and then PCR amplified MAGE-12 gene fragments after restriction enzyme digestion and cloned into the vector pGEM-T easy, for the MAGE gene cloning vector pGEM-T easy-MAGE-12, was transformed into competent bacteria JM109 white selection, selection of positive clones Shanghai Bioengineering profiles line sequence analysis. 2. Sequencing confirmed that the base sequence is correct, the two enzymes Qieke Long vector pGEM-MAGE-12 and the eukaryotic green fluorescent protein expression vector pEGFP-C3 build the MAGE12 green fluorescent protein eukaryotic expression vector pEGFP-C3-MAGE-12 and converted into competent JM109 Bacteria. 3. The positive colonies recombinant expression vector pEGFP-C3-MAGE-12 is correct by restriction endonuclease digestion, extraction method of application of liposome transfected into eukaryotic cells (Eca109 cells) in eukaryotic cells was observed under a fluorescence microscope expression. 4. Extract transfected with the recombinant expression vector pEGFP-C3-MAGE-12 eukaryotic intracellular total mRNA, application of the RT-PCR technique, identification of MAGE-12 gene expression in eukaryotic cells. Results 1. CDNA as a template, and MAGE-12, two pairs of specific primers for RT-PCR amplification, the amplified product obtained using 1.5% agarose gel electrophoresis, the obtained strip at nearly 1000bp bands located in the control DNA marker, at the close of the target gene size expected 944bp. By restriction endonuclease, the theoretical and experimental results. 2. Build a MAGE gene cloning vector pGEM-T Easy-MAGE-12. Selection of positive clones, send bio-engineering company sequencing target gene sequences obtained by computer analysis confirmed, with the gene pool MAGE-12 2960-3904bp fragment of the gene base sequence exactly. 3. Construct the expression vector pEGFP-C3-MAGE-12, MAGE-12 gene eukaryotic green fluorescent protein after double digestion, and the recombinant expression vector, transfection of eukaryotic cells, it is possible to be widely expressed in eukaryotic cells. 4. The technologies by RT-PCR confirmed MAGE-12 gene was successfully expressed in eukaryotic cells. Conclusion 1. MAGE-12 gene was successfully amplified from human lung cancer tissue and build MAGE-12 gene cloning plasmid pGEM-T-MAGE-12. 2. Sequencing results through computer software, the cloning of MAGE-12 gene MAGE-12 gene sequences in GenBank exactly the same. 3. MAGE-12 gene was successfully constructed green fluorescent protein eukaryotic expression vector pEGFP-C3-MAGE-12, confirmed that the recombinant transfection of eukaryotic cells by RT-PCR technology can be widely expressed MAGE-12 gene for tumor lay the foundation for immunotherapy.

Cloning of the Human Lung Cancer Associated MAGE-12 Gene and Research Its Expression in Eukaryotic Cell

Abstract

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