CDNA Cloning And Construction Of Eukaryotic And Pronucleus Expression Vector For Human Trail

So far the tumor has become the main reason which cause people to die. Genetherapy maybe become a new measure to therapy the tumor after surgery and chemotherapy. TRAIL(TNF-related apoptosis-inducing ligand, TRAIL) can specifically kill many tumor cells while have no toxicity to normal tissues and cells. So TRAIL is considered as a potential drug and ideal gene in tumor therapy. To investigate the mechanism of how TRAIL inducing apoptosis and the prospect of TRAIL as a clinic drug in cancer therapy, we do reseach about Cloning, Construction of eukaryotic and pronucleus expression vector for human TRAIL. The results of research is following: 1.　The whole human TRAIL cDNA sequence which consisted of 1 039bp was cloned by RT-PCR from human placenta tissue, and then subcloned into pGEM-T Easy vector. The fragment was sequenced and compared with human TRAIL cDNA sequence in GenBank. The results shows we cloned successfully the whole cDNA of TRAIL. 2.　T-TRAIL and pIRESneo3 plasmid was digested respectively with restrictive enzyme NotⅠ. And after the 5′end of digested pIRESneo3 product was phosphatased, it was connected with purified TRAIL fragment by agarose using T4 ligase.The result was verified by PCR and endonuclease digestion. So eukaryotic expressional vector of human TRAIL was constructed, which consisted in IRES and CMV promoter. 3.　we amplified 560 bp cDNA fragment of the soluble region of TRAIL by cloned whole cDNA as template with primer consisted of BamH I and Xho I site.The cloned sTRAIL cDNA was cloned into plasmid PET-28a.The result was verified by endonuclease digestion.So we construct pronucleus expressional vector of human sTRAIL. 4.　Recombined plasmid be transformed into Ecoli BL21,TRAIL was highly expressed after 4 hours' induction with IPTG.. After ultrasonic crash ,we collect deposition and supernatant. SDS-PAGE indicated that the expression of TRAIL is mainly in inclusion body form .