Fusion Expression And Biological Characterization Of TRAIL And Polypeptide Originating From HKa D5

In the 1970s, it was Folkman who for the first time ever brought forward the viewpoint of"the proliferation and metastasis of tumor relying on the angiogenesis". The angiogenesis is a process in which new capillary vessels are formed in the peripherals of the already existing capillary vessels through the proliferation and migration of endothelial cells. The process may occur in different aspects such as the embryonic development, periodical changes of the endometrium, repairment after trauma as well as the growth of tumor. The angiogenesis is regulated by both angiogenesis factors and anti-angiogenesis factors. Once the balance between these regulatory factors is broken, abnormal angiogenesis might be caused so as to arouse the growth and invasion of tumor, myocardial infarction and diabetes. So far, it has been proven that: the growth of primary tumor and its invasion into the peripheral tissues and organs rely on the angiogenesis which provides nutritious support. The focus of tumor may not exceed a couple of millimeters without sufficient nutrition provided by the angiogenesis. Therefore, if the formation of the the angiogenesis is inhibited inside the tumor tissues, the tumor cells will undergo apoptosis and necrosis so that the proliferation and metastasis of tumor are inhibited. In this way, a new way of thoughts is provided for the treatment of tumor.Currently, among the endogenous anti-angiogenesis factors that have already been found, the single-chain high molecular weight kininogen (HK) is a multi-functional glucoprotein in the plasm, which is closely related to blood coagulation, reactions to complement and the occurrence of inflammation. Structurally, HK consists of six domains, among which domains 1, 2 and 3 form its heavy chain while domains 5 and 6 form its light chain. Linking between the heavy chain and the light chain, there is domain 4 which consists of a nine-peptide bradykinin. With the effects of plasma kallikrein, the single-chain HK releases the bradykinin consisting of domain 4 which results in double-chain HK that is cleaved high molecular weight kininogen, HKa.HKa is composed of heavy chains including domains 1, 2 and 3 as well as light chains including domains 5 and 6. Linking between the heavy chain and the light chain, there is an independent disulfide bond. While HK is cleaved into HKa, major conformational changes take place so that the exposure of domain 5 becomes more obvious. As has been found in relevant researches: the light chain of HKa is rich in histidine-rich domain,HRD, that is to say, domain 5 is the active region where HKa is anti-cell spreading as well as the active region for HKa to inhibit the angiogenesis. GUO found that domain 5 of HKa conjugates with urokinase plasminogen activator receptor (μPAR) on the endothelial cell membranes so that the endothelial cells malfunction to adhere while the apoptosis of the endothelial cells is induced to inhibit the angiogenesis. Therefore, the domain 5 of HKa (HKD5)possesses potential antineoplastic activity. In recent years, according to the researches on the core amino acid sequence where HKD5 inhibits the antineoplastic activity, Zhang synthesized 14 polypeptide segments in the 384-503 amino acid sequences of domain 5 while each polypeptide segment consists of 16 amino acids. As a result Zhang found that the polypeptides HKD5 _480-495 and HKD5 _488-503Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is also called apoptotin-2 ligand (Apo-2L) which is a member of the tumor necrosis factor (TNF) superfamily. The protein TRAIL that can form codes is composed of 281 amino acids. TRAIL can selectively induce the apoptosis of tumor cells, but it has no obvious impairment on normal tissue cells. The _114-281 amino acid segments are the core sequences which play the mojor functions of TRAIL and soluble protein can be formed. have the most conspicuous effects of inhibiting the proliferation of endothelial cells.The animal-based experiments have shown that there may occur such problems as the needs for large dosages of drugs if HKa or TRAIL is used independently as the treatment for tumor and besides also it takes a long period of time to take into effect. Therefore, this experiment aims to integrate the active polypeptide segments of HKD5 and the active segments of TRAIL so as to obtain the fusion protein with bifunctional effects of specifically killing tumor cells and inhibiting the angiogenesis inside the tumor tissues, and in this way the anti-tumor activity is promoted.By applying gene engineering technologies, this experiment integrates the polypeptide of HKD5_480-495 and HKD5_488-503 with TRAIL_114-281 respectively. Through cloning and expression, we obtain recombinant proteins of MBP-HKD5_480-495-TRAIL_114-281 and MBP-HKD5_488-503-TRAIL_114-281. And then through analyzing the biological characterization of the two fusion proteins, it is found that MBP-HKD5_480-495-TRAIL has stronger effects of killing tumor cells and inhibiting the proliferation and migration of endothelial cells. Base upon this experiment, we have taken initiative steps of probing into the molecular mechanism of inhibiting the tomor cells in the HKa domain 5. Part One: Cloning, expression and purification of HKD5_480-495 -TRAIL_114-281 and HKD5_488-503-TRAILWe have integrated the primers consisting of polypeptide coded sequences of HKD5_{114-281 480-495} and HKD5_488-503. By applying PCR, we integrated the polypeptides of HKD5_480-495 and HKD5_488-503 and the coded sequences of TRAIL_114-281 with glycin as the linker-primer. The PCR products were inserted into pMAL-c2 vector to obtain recombinant plasmids pMAL-HKD5_480-495-TRAIL and pMAL-HKD5_488-503-TRAILIn the above-mentioned process, when producing the fusion proteins through utilizing prokaryotic expression vectors, we often choose colon bacillus as expressional parasitifer since it has high efficacies of transforming and expressing and can be fermented and cultivated in a large scale, and besides it is easy to manipulate and it is fast to produce recombinant proteins. The expressional and purification system was adopted in the experiment. The expression plasmid pMAL-c2x of this system possessed the regulatable strong promoter of Ptac and the maltose-binding protein (MBP) which was the initiation signal. The expressional and purification system could express the fusion protein in a high level. The purification system has a MBP protein which is favorable for the correct folding of the recombinant proteins. Therefore, it could be effectively promote the solubility of the interest protein. respectively. These recombinant plasmids were transformed into E.coli BL21(DE3). Then after recombinant plasmids were expressed by IPTG induction, the purified fusion proteins were obtained by Amylose Resin affinity purification column, filtrated, and stored at the temperature of minus 80℃for future uses. The expressed products accounted for more than 20% of the total bacterial proteins. Effective expressions were obtained after the induction. The analysis of SDS-PAGE showed the two proteins with a molecular weight of 61 KDa. The molecular weight of these fusion proteins turned to be 61kDa, as has been proved by Western Blot.Part Two: An analysis of the biological characterization of the the fusion proteins of MBP-HKD5_480-495-TRAIL_114-281 and MBP-HKD5_488-503-TRAILActing on the human umbilical vein endothelial cells (HUVECs), the purified fusion proteins had the effects of inhibiting the proliferation of the endothelial cells.As has been detected through a Transwell model with polycarbonate membrane, these fusion proteins were able to effectively inhibit the migration of HUVECs while the inhibitory effects were in direct proportion to the density of these fusion proteins. Through the experiments on lung cancer NCI-H460 cells and pancreas cancer _114-281 SW1990 cells, we have found: Compared to the respective utilization of TRAIL, the polypeptide of HKD5_480-495 and the polypeptide of HKD5_488-503, MBP-HKD5_480-495-TRAIL and MBP-HKD5_488-503-TRAILhave more obvious effects of killing tumor cells. Through further flow cytometric analysis, we have proven that the fusion proteins could obviously induce the apoptosis of the NCI-H460 cells. Through analyzing the biological characterization of the fusion proteins, we have found that, compared to MBP-HKD5_488-503-TRAIL, MBP-HKD5_480-495-TRAILThis Prokaryotic Expression Vector has effectively expressed the fusion proteins of MBP-HKD5 has more obvious effects of inhibiting the proliferation and migration of endothelial cells as well as inducing the the apoptosis of the tumor cells. _480-495 - TRAIL and MBP-HKD5_488-503-TRAIL. Acting on the endothelial cells, the purified fusion proteins had the effects of inhibiting the proliferation and migration of the endothelial cells. These two fusion proteins could kill the lung cancer NCI-H460 cells and pancreas cancer SW1990 cells and also induce the apoptosis of the tumor cells. It was proven that these fusion proteins have veyy good bifunctional effects. These two fusion proteins have both anti-tumor activities of TRAIL and effects of specifically inhibiting the endothelial cells, thus laying a solid foundation for these two fusion proteins to become anti-tumor candidates in the future.