The Research Of Construction Of Prokary Otic Secretory Expression Vector,Expression And Activeanalysis Of Human Soluble TRAIL

Cancer is a disease of serious harm to human life and health. Treatment of cancer to take the surgery, chemotherapy and radiotherapy and radiotherapy three traditional therapies, although able to remove most of the tumor cells, but there are still some resistance to cell residues, resulting in tumor recurrence and also have a greater toxic side effects on normal cells, the treatment of tumors into a major problem today, to find more effective anticancer drugs into the current study. Tumor necrosis factor-related apoptosis-inducing ligand (TNF-related of apoptosis inducing of ligand, referred to as TRAIL) is a normal expression of a protein of the human body, belonging to the new members of the TNF superfamily. The combination of TRAIL protein and the tumor cell suface death receptors induces the apoptosis of tumor cells but not normal tissues and cells.Based on the advantage, TRAIL is expected to become a new and effective drugs on treatment of cancer, and it is the research focus of current cancer therapy.The gene of human TRAIL molecule encodes281amino acids. We start from the first95or104amino acid residues cloned soluble molecules TRAIL (sTRAIL) to construct the recombinant expression vector and the expressed proteins are active. The active site is located in the114-281amino acids, and this fragment is now found in the soluble, non-toxic and biological activity of both the length of TRAIL. So we select this amino acid fragment as the object of study.In this paper, from HL-60cells (acute myeloid leukemia cell) to extract total RNA, amplification of human soluble TRAIL gene fragments of114to281primers were designed according to human TRAIL gene sequence by GeneBank providing. At the same time, the gene sequence of NcoI, BamHI, TEV enzyme restriction site and His tag were inserted. The target gene were subcloned into the expression vector phoA. The recombinant expression plasmid phoA-sTRAIL was identified by NcoI, BamHI restriction enzyme digestion and sequencing analysis, which was induced expression in E. coli MM294in Low-phosphorus medium. sTRAIL protein purified by Ni, reducing SDS-PAGE and non-reducing SDS-PAGE analysis.The results show that in this experiment purified sTRAIL protein in the form of dimmer, and by western blot analysis there was a specific band at27KD, indicating that the purified target protein is sTRAIL protein.Before the detection of the biological activity of sTRAIL protein,the His tag was cut by TEV enzyme. Selected three types of tumor cells were non-small cell lung carcinoma cell line A549, chronic myeloid leukemia cell line K562, the human hepatoma cell line HepG2to test. The results showed that sTRAIL protein can effectively inhibit the proliferation of non-small cell lung cancer cell strain A549and the human hepatoma cell line HepG2, the inhibition rates were46.7%and29.3%, inhibition of the proliferation of leukemia cell line K562was not obvious. It can be seen from the results, sTRAIL protein is expected to become a new clinical drugs on treatment of lung cancer, but also for future clinical study provide a scientific basis for the draw.