Construction And Expression Of Eukaryotic Coexpression Vector PEGFP-C3-B7.2-MAGE-1

Background and Objective:Melanoma antigen (MAGE) which were first found in melanoma cell is a teamof tumor-associated antigen(TAA). In fact, MAGE are a group of superfamily antigens, which contain four subfamilys: MAGE-A, MAGE-B, MAGE-C and MAGE-D, and among the total, MAGE-A contains 12 genes from MAGE-1 to MAGE-12. Recently, a large number of documents from domestic and abroad have reported occurring high MAGE-1 expression in many common tumors such as hepatoma, esophageal carcinoma, gastric carcinoma, carcinoma of tongue, leukemia, head and neck cancer, lung carcinoma, neuroblastoma and so on. In normal tissues, MAGE-1 only expresses in testiculus and placenta which are immune-exempt tissues and can escape from attacking from immune competent cells. Therefore the catholicity and relative specificity of expression of MAGE-1 in tumors make it possess a potential application prospect and consequently become a study hot spot. Up to now, we have discovered eight HLA-I-restricted CTL epi-positions which are encoded by MAGE-1 gene have been found. MAGE-1 can activate pre-thymocyte (pre-T) of immune system to produce specific CTL which can kill tumor cell specifically throught identifying MAGE-1 antigen expressing in surface of tumor cell. This result provide potent experiment evidence for application of MAGE-1. Research indicates that during tumor antigen is specific recognized, B7 as costimulatory signal can promote T cell proliferation, inhibit T cell apoptosis, enhance sensitivity of T cell, mediate adherence of T or B cell and enhance humoral immunoresponse (HI). General tumor cells are not able to express B7 molecule(B7.1 or B7.2)or only have low expression, which lead to the second signal road blocked and cann't induce specific CTL. However, tumor cells which were introduced cDNA of B7.2 molecule into can induce immune response of anti-tumor. Recently there are a lot of exploratory development about B7.2 in tumor immunotherapy, Vasilevko et al constructed coexpression plasmid of MUC1 and B7.2. They immunized mice by particle gun, then vaccinated breast cancer cell line which expresses MUC1 to mice, result shows which can prolong tumor- forming time and inhibit tumor growth. The finding of Disis indicated that hypodermic injection of plasmid which encodes neu and B7.2 can induce neu-specific cell and humoral immune reaction and consequently resist tumor. Up to now there is no a document have reported to construct B7.2 and MAGE-1 eucaryon coexpression plasmid, make B7.2 and MAGE-1 express in same a cell and carry out coe-effect of anti-tumor. In this topic we constructed pEGFP-C3-B7.2-MAGE-l eucaryon coexpression vector adopting MAGE-1 and B7.2 gene fragments as target gene and pEGFP-C3 plasmid as vector transfect; human esophageal carcinoma cell line EC9706 was transfected by the vector of pEGFP-C3-B7.2-MAGE-l through the technique of lipofectamine transfection and the expression of B7.2/MAGE-1 gene was observed in order to obtain green fluorescence eukaryotic coexpression vector pEGFP-C3-B7.2-MAGE-l, The study will provide experiment evidence for further approaching tumor activity immune therapy based on B7.2 and MAGE-1 targets.Methods:1. The construction of the eukaryotic coexpression vector pEGFP-C3-B7.2/ MAGE-1: Two pairs of primers of B7.2 and MAGE-1 gene were designed according to GenBank databases using Oligo 6.0 gene analysis software, respectively, and the primer of B7.2 and MAGE-1 will be added restriction enzyme site. The total RNA were extracted from mammary gland tissue and mammary cancer tissue with Trizol reagent, respectively. And also, interest of gene B7.2 and MAGE-1 were amplified by RT-PCR. The PCR product of B7.2 and MAGE-1 genes were purified with Gel extraction kit, and ligate with vector pGEM-T Easy, respectively. Extract the constructed pGEM-T-7.2 with plasmid purification kit. After digesting B7.2 gene with XhoI and SalI, ligated it to the vector pEGFP-C3, which was also digested by the two same enzymes. Ligated MAGE-1 gene that was cut form the recombinant plasmid pGEM-T-MAGE-1 by restriction enzymes KpnI SalI to the vector pEGFP-C3-B7.2 digested by KpnI andSalI to construct the eukaryotic expression vector, which was identified by PCR, enzyme cut and sequencing.2. Human B7-2/MAGE-1 transfect esophageal cancer cell EC9706: Use the method lipofectamine transfection to transfect Human esophageal cancer cell line EC9706 with the recombinant plasmid pEGFP-C3-B7.2/MAGE-l. 24 hours later, observe the expression of the fused gene under fluorescence inverted microscope. The transfected cells were screened with G418 for 20 days to obtain the EC9706 cell line which can consistently express fusion gene and detect the expression of B7.2/MAGE-1 gene in mRNA level by RT-PCR.Results :1. Amplify the gene of B7.2 through the method RT-PCR, the product was about 618bp with the enzyme recognition sits include. From the same way the MAGE-1 gene was also obtained, the expected product was about 545bp.2. We constructed successfully pGEM-T-B7.2 and pGEM-T-MAGE-1 prokaryotic recombinant plasmid, respectively. The sequencing to B7.2 and MAGE-1 gene is conformity with GenBank databases.3. We constructed successfully pEGFP-C3-B7.2-MAGE-l eukaryotic coexpression plasmid. The target fragment identified with PCR and XhoI,KpnI double-enzyme cutting system is conformity with expection4. We constructed EC9706 cell line which can express stably B7.2/MAGE-1. Transfected 24 hours later, there was fluorescence occurring in EC9706 cells under fluorescence inverted microscope. We detected the expression of B7.2/MAGE-1 gene in mRNA level by RT-PCR, The result showed that the sizes of which are conformity with what we expect.Conclusions:1. In this study we constructed successfully MAGE-1 and B7.2 eukaryotic coexpression plasmid first time. 2. We obtained EC9706 cell which can express stably MAGE-1 and B7.2.3. The study provided significant experiment evidence for further approaching tumor activity immune therapy based on B7.2 and MAGE-1 targets.