Studying On The Construction Of Eukaryotic Expression Vector Of Human HLA-G Gene And The Suppression Of Cytotoxic Action Of NK Cell

The aim of this study is to get a grip on the clone of HLA-G cDNA, the techniques of construction, appraisal and transfection of eukaryotic expression vector, and to have a preliminary discuss on the inhibitory action of HLA-G gene against the cytotoxicity of NK cell, which will provide an experimental basis for the comprehension of the tumorous immune escape phenomenon as well as the biohherapy of tumor, and a foundation for ulterior transgenic technology, organ transplant and reorganization engineering.Empirical method:Drawing assistance from PCR technology to amplify the cDNA of HLA-G and insert it into the eukaryotic expression vector of pCMV-HA2, and finally construct a eukaryotic expression vector of HLA-G. With the method of lipofection, the eukaryotic expression vector of HLA-G is transfected to carcinoma of bladder cell line of 5637(5637 cells) and carcinoma of prostate cell line of PC3(PC3 cells) to detect its proteinum expression by Western Blot. Through the co-culture with NK cell, an initial identification was found that HLA-G Gene, after transfected tumor cell, has a rejection capability against the cytotoxic action of NK cell.This study includes 3 experiments.Experiment 1 was about the clone of HLA-G Gene and the construction and appraisement of eukaryotic expression vector: to pick up general RNA from the chorionic villus of a pregnant woman, to obtain cDNA of human's chorionic villus by RT-PCR, which is taken as the template to amplify the ORF of HLA-G; After being double digested by EcoRâ… and Hind III, PCR product links to eukaryotic expression vector of PCMV-HA2 and transforms bacterium coli of Topo,10; to sieve the masculine clone bacterium by LB/Amp flat plate and...