Construction Of E2F-1 Gene Eukaryotic Expression Vector And Its Expression In Gastric Carcinoma Cell

The incidence rate of gastric carcinoma is also in the first position among manlignant tumors in our country,and the mortality is about one quarter of global ones.Most patients have been progressing to advanced stage at the time of diagnosis.The rate of radical resection is lower,treatments including radiation,chemotherapy or preoperative chemotherapy have little infuence,even exert great side effect to patients.And at present having no normative chemotherapy, therefore the gene therapy has been introduced and displayed its promising. Two important properties of cancers are able to deregulate cell proliferation and suppress cell death.The transcription factor E2F-1 is capable of inducing apoptosis in many cells ,and has the potential to function at an tumour suppressor. However its use in human gastric carcinoma has little been reported so far.In present study,we constructed the recombinant eukaryotic expression vector contaning E2F-1 cDNA,which would transfect into gastric carcinoma cell with liposome, and also we investigsted the transient expression of E2F-1 in transfected cell by RT-PCR and western blot. This eukaryotic expression vector will be a powerful tool in our future study of gastric carcinoma.1.Method1.1 Construction of eukaryotic expression vector containing the E2F-1geneWe designed the primers with the position of restriction enzyme-Hindâ…¢,EcoRâ… ,E2F-1's cDNA fragment with complete open reading frame were amplified by PCR from pESV-G6-E2F-1 plasmid.First the products were cloned into pGEM-Teasy, transfer and select the positive recombinant,and then cloned into eukaryotic expression vector pCMV-HA after introducd the sites of restrictive endonuclease enzyme Hindâ…¢,EcoRâ… .