Clone And Expression Extra-cellular Fragment Of TRAIL And Preliminary Study On Its Apoptosis Effect

The TNF-related apoptosis-inducing ligand, TRAIL, is a type II membrane protein which belongs to TNF superfamily. It can induce apoptosis rapidly in susceptible cells including both malignant and some transformed cells.Although TRAIL shows high homology in sequence with TNF and FasL molecules, it is believed that TRAIL selectively induces tumor cell apoptosis that may be more important in biologic homeostasis.The animal model of malignant tumor-7402 hepatic carcinoma was established. The eukaryotic vector of human TRAIL cDN A has been constructed, then was used for malignant tumor antiangiogenesis gene therapy of the animal model.Using RNA extracted from the U937 cell as template,Human TRAIL cDNA was amplified by means of polymerase chain reaction(PCR), The PCR products were cloned into pGEX-6p-l vector routinely,and the recombinants were identified by endonuclease digestion and then sequenced.The recombinant expression vector pGEX-6p-l-TRAIL was induced expression by IPTG in E.coli expression system. The products of prokaryotic expression was injected abdomenly into BALB/c for anti-TRAIL polyclonal antibody. The human TRAIL cDNA was cloned into eukaryotic expression vector pcDNA3 after verified by sequencing. The CHO cells were transfected by the plasmid pcDN A3-TRAIL with liposome, monitoring the expression of TRAIL gene by means of indirect fluorescent assay(IFA). The apoptosis rate of carcinoma cells was measured by Flow Cytometry and MTT. Animal model of tumor was established by subcutaneouslytransplanting tumor cell of 7402 hepatic carcinoma into the oxter of nude mice .The plasmid pcDN A3-TRAIL was injected into muscle of nude mice, which were subcutaneously transplanted with 7402 hepatic carcinoma cell.The human TRAIL cDNA is identical to that reported previously in literature reference. The results of SDS-PAGE showed that the human TRAIL cDNA could be expressed highly in E.coli. Indirect fluorescent assay demonstrated that the CHO cells could express TRAIL in cytoplasm. TRAIL may inhibit the proliferation of 7402 liver carcinoma cell.the inhibitory rate was 31%.The growth of subcutaneous tumor was inhibited after gene therapy with the plasmid pcDNA3-TRAIL.The human TRAIL cDNA was cloned successfully and expressed in prokaryotic or eukaryotic expression system. The plasmid pcDNA3-TRAIL can be used for gene therapy against tumor.