Cloning Of The Human Lung Cancer Associated MAGE-12 Gene And Research Its Expression In Eukaryotic Cell

BackgroundAlong with the advancement of immunology and molecular biology, human have deep comprehension of the molecule mechanism of tumorigenesis and evolution. People know that tumorigenesis is a gradually process with polygene and many stage. But people haven't yet found special mutation gene or defect gene of carcinoma. The key point of the specific immunity therapy of human tumor is finding tumor specific antigen.In 1991, van der Bruggen found the melanoma antigen-1 (MAGE-1). MAGE gene has a big family, above now, people has discovered at least six sub- family including MAGE-A,B,C,D,E,F.MAGE-12 gene was found in human melanoma cell line DM150 by Min D in 1994,which is belong to MAGE gene family.MAGE-12 gene has high homology with MAGE-2,MAGE-3. Many studies confirmed that MAGE-12 gene had high expression in lung carcinoma.MAGE-12 gene encode a species of tumor specific antigen, which has different degree expression in melanoma,lung cancer and any other kind of tumor, but never express in normal tissue except testis and placenta. The reports about using MAGE-12 gene vaccine to treat tumor are few. Green fluorescent protein (GFP) is a few reported molecules, which is developed from 1990s of last century. At first, as a kind of protein, it was found in Aequoreavictoria. GFP can be expressed and create fluorescent light in many creature, for example bacterium, myxomycetes, yeast, plant and mammal. GFP has small molecular weight, it is easy to form fusion gene with other purpose genes without any hazard to cells. Its chemical property is stable; we can observe its expression in living cell every now and then. So the vector pEGFP-C3 is widely used in molecular biology.ObjectiveRT-PCR was used for cDNA cloning of MAGE-12 gene from lung tumor tissue. Construct cloning vector pGEM-T easy-MAGE-12 and eucaryotic fluorescent expression vector pEGFP-C3 -MAGE-12. Then transfect the expression vector into eucaryotic cell (Eca109), Induce the vector express in eucaryotic cells and observe its expression in eucaryotic cell by fluorescence microscope. Make the foundation of contriving gene vaccine.Materials and methods1.mRNA was extracted from human lung cancer specimen. MAGE-12 gene was amplified by reverse transcriptase polymerase chain reaction(RT-PCR)and was identified by enzyme cutting with Pvuâ…¡.The PCR fragment was inserted into pGEM-T easy plasmid, and then was transformed into JM109.After the selection of blue/white screening, select the positive cloning and sent it to sequence analysis.2. After the base sequence was confirmed by sequence determination, the recombined cloning vector pGEM-MAGE-12 and expressing vector pEGFP-C3 were cutted by two enzymes. Construct the eucaryotic fluorescent expression vector pEGFP-C3 -MAGE-12.Then transformed the recombined expression vector into competence JM109.3.Select the positive cloning, after conformed by enzyme cutting identification, extract recombined expression vector, then transfect into eucaryotic cell. Observe its expression in eucaryotic cell by fluorescence microscope.4. MRNA was extracted from the eucaryotic cell, which contained the recombined expression vector pEGFP-C3 -MAGE-12.Then the expression of MAGE-12 gene was confirmed by RT-PCR. Result1.Taking the cDNA as template, take the special primer of MAGE-12 as the primer, amplified by RT-PCR. Electrophoreses the product with 1.5% agarose gel. The stripe we get is located at about 1000bp contrasting with DNA Marker; this is close to our anticipation. After the enzyme cutting, the result is consistent with the anticipation.2. Cloning vector pGEM-T-MAGE-12 has been constructed successfully. Select the positive cloning and send it to sequence analysis , the sequence of the target gene is consistent with the base sequence of MAGE-12 gene which is found in gene bank.3. Expression vector pEGFP-C3-MAGE-12 has been constructed successfully. After it was conformed by enzyme cutting identification, transfected it into eucaryotic cell. It could be expressed in eukaryotic cell stably.4.We conformed that MAGE-12 gene was expressed in eucaryotic cell by RT-PCR.Conclusion1. MAGE-12 gene was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) from human lung tumor tissue, and constructs the cloning vector pGEM-T-MAGE-12 successfully.2. The sequence of theMAGE-12 gene is consistent with the base sequence of MAGE-12 gene, which is found in GenBank.3. The eukaryotic green fluorescent protein expression vector pEGFP-C3-MAGE-12 was constructed successfully. After transfected it into eukaryotic cell, it could express stably. This made the foundation of immunotherapy for tumor.