The Cloning, Fusion Expression And Biological Characterization Of KininogenD5 And TRAIL

Folkman presented firstly the hypothesis that the proliferation and metastasis of tumor are dependent on angiogenesis as early in 1970s, which has been accepted widely now. The development of new blood vessels from pre-existing ones is generally referred to as angiogenesis. In the adult, new blood vessels arise via angiogenesis, a process critical for normal physiological events such as embryonic development, endometrium remodeling, and wound repair. In physiological state, there is a balance between angiogenesis and antiangiogenesis, uncontrolled neovascularization is associated with a number of pathological disorders including tumor growth and metastasis. Tumor growth and metastasis require angiogenesis, If the formation of new blood vessels are inhibited, tumor cells will undergo the process of apoptosis or necrosis. It provides the basis for anti-angiogenetic therapy for malignant tumors.Currently, a number of angiogenesis-inhibiting drugs have been developed for the treatment of tumors clinically. High molecular weight kininogen (HK) is a 120 kDa single-chain glycoprotein composes 6 domains. Its heavy chain consists domain 1-3 and light chain consists domain 5-6. Cleavage of HK by plasma kallikrein results in the release of the nonapeptide bradykinin (BK) from HK domain 4 and the generation of two-chain kininogen (HKa). HKa and HK have the similar structure endows with histidine-rich region in domain 5, and it is the Zn2 + binding region. HKa undergoes major conformational changes and shares the ability to bind to anionic surfaces and endothelial cell receptors including urokinase plasminogen activator receptor (uPAR). The weaken of adhesion induces apoptosis of endothelial cells, so new blood vessels'formation is inhibited. At present, the mechanism of Hka-induced endothelial cell apoptosis to inhibit angiogenesis has been relatively clear. The functional activity of the fragments has been identified. While there are still various disadvantages concerning the clinical application of HKa for antitumor treatment. Animal experiments showed that using HKa merely on anti-tumor treatment need large drug dose, and the effective time was to short. A fusing protein of an antiagiogenic inhibitor (KD5) and a cytotoxic factor (TNF-related apoptosis-inducing ligand TRAIL), KininogenD5-TRAIL was constructed in order to improve its antitumor effect.The encoding genes for Kininogen D560-148 and TRAIL114-281 were obtained by PCR, respectively, and the fusiing genes for Kininogen D560-148- TRAIL 114-281 (KT) and TR AIL114-281-Kininogen D560-148 (TK) were also obtained using the amino acid linker by SOE PCR. After the identification of the two fusion proteins, we found that KT elicited a significant effect both on anti-tumor and anti-angiogenesis, while TK had no evident effect. On this basis, the mechanisms of KT-induced apoptosis and its anti-tumor spectrum were also elucidated.Part One: Cloning, expression and bioassay of Kininogen D560-148 and TRAIL 114-281The Kininogen D560-148 and TRAIL114-281 were obtained by polymerase chain reaction (PCR), respectively. The PCR products were cloned into pMAL expression vector. pMAL-Kininogen D560-148 (PKD5) and pMAL-TRAIL 114-281 (PT) genes were transformed into E. coli BL21 and were efficiently expressed after IPTG induction. The expressed products accounted for 25% and 23% of the total bacterial proteins and existed mainly as soluble form, respectivly, as estimated by densitometry. The purified PKD5 and PT proteins were obtained by Amylose Resin affinity purification column and the purity of them reached up 95% and 93%, respectively. The two proteins showed the molecular weight of 52 and 62 kDa on SDS-PAGE, respectively, this was further confirmed by Western blotting. The biological activity of the two proteins were detected on their proliferation inhibition, cell apoptosis, as well as in vitro tube formation inhibition on endothelial cells and human pancreatic ductal epithelial cells (SW1990). The results showed PKD5 exhibited significant inhibitory effect on endothelial cell proliferation (ED50 was 10μg/ml), PKD5 also inhibited the tube formation in vitro. PT plays a significantly cytotoxicity against SW-1990 cells (ED50 was 25 ng/ml ). The results suggested that PKD5 and PT endowed with an inhibition effect on angiogenesis as well as an anti-proliferative effect on cancer cells, respectively.Part Two: Two way expression of the fusion gene of Kininogen D560-148–TRAIL 114-281 by SOE PCR (gene splicing by overlap extension PCR)The aim of this study is to fusing expression of Kininogen D560-148 with TRAIL 114-281 to yield a novel fusion protein, Kininogen D560-148-TRAIL 114-281, in order to improve its anti-tumor activity. The idea was originated from the fact of the specific interaction between Kininogen D560-148 and its receptors uPAR, thus it is possible to directly target the fusion protein to the endothelial cells in the malignant tumor tissues that have previously been shown to contain high levels of uPAR. The fusion protein is also targeted to tumor cells for TRAIL 114-281 specific interacts with the death recepter on the membrane of tumor cells. A bacterial expression system was used to produce Kininogen D560-148-TRAIL 114-281 fusion protein. First, we obtain two-way fusion genes TK and KT by SOE PCR, The TK and KT fusion genes were cloned into pMAL expression vector. pMAL-KT and pMAL-TK genes were transformed into E. coli BL21 and were efficiently expressed after IPTG induction. The expressed products accounted for 25% of the total bacterial proteins and existed mainly as soluble form, as estimated by densitometry. The purified MBP/KT and MBP/TK proteins were obtained by Amylose Resin affinity purification column and the purity of them reached up 95%. The recombinant products were tested for their cytotoxic or cytostatic activities against SW1990 and ECV304 cells. The results showed that MBP/KT played a significantly cytotoxicity against SW-1990 cells (ED50 was 5 ng/ml). Meanwhile, it also exhibited a growth inhibitory effect on proliferation of ECV304 cells (ED50 was 50 ng/ml). However, MBP/TK had no evident effect on these aspects. MBP/KT also obviously induced the apoptosis of SW1990 cells as determined by Flow cytometry, electron microscope and DNA ladder. So we further cloned KT fusion gene to pBV 220 expression vector and were efficiently expressed after heat induction. The main forms of expression of PBV 220-KT were inclusion body. We eventually got a little expression in the supernatant through improving fermentation conditions. This research provided the foundations of the target therapy in cancer treatment.