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In 1997, O’Reilly found endostatin from the supernatant of the cultivation in vitro of the mouse angioendothelioma cell, the molecular weight is 20kDa. The endostatin can specifically act on endothelial cell, particularly for microvascular endothelial cell, it can inhibit the migration of endothelial cell, thus induct the endothelial cell to apoptosis. Human endostatin and mouse endostatin have homology, human endostatin molecular weight is about 18kDa, 86% amino acids of them are completely identical.As early as 1971, Folkman proposed that the majority of tumor cell ischemia or hypoxia through inhibiting tumorous angiogenesis, and thus slow down the growth of advanced tumor, prolong survival in patients with tumor, inhibit the growth of tumor, and delay the recurrence. In the past researchs, we found the mechanisms of the tumorous angiogenesis. On the one hand, angiogenesis factor was delivered from tumor cell to activate vascular endothelial cell, to promote the proliferation and migration of the endothelial cell. One the other hand, some angiogenesis factors was paracrined to activate the growth of the tumor cell from endothelial cell. In other words, the process of angiogenesis of tumor included the interaction of tumor cell and endothelial cell. So, the discovery of endostatin to make the hypothesis of the Folkman is possible and to provide a new way to treat cancer. However, the serum half-life of endostatin is very short (about 2 hours), so it’s necessary to develop the long acting endostatin.Human Serum Albumin (HSA) is a single-chain and free-glycosylation protein, composed of 585 amino acids. The molecular weight is approximately 68kDa and is a single protein of the highest level in blood. HSA can maintain blood pressure in the body, transport nutrients and plays other important role of biological material, with non-immunogenicity, fine human compatibility, and longer serum half-life (nearly 19 days). It can be used as the biological activity carriers. The technique of the albumin fusion don’t need additional chemical modification, the production of technology is simple, the substrate is uniform, the quality control is relatively easy, the amount of the expressed protein can increase after fuse with albumin, and the effect of prolong the half-life of drug is well, so the researcher pay close attention to the technique.The HGS(Human Genome Sciences) have researched a series of protein drug of the HSA fusion, the result of publication indicated that the half-life of HSA-IFNα(Albuferon) is get to 145 hours. Now the company have some others productions into the study of I/II clinical trails, including HSA-GH (Albutropin), HSA-GCSF (Albugranin), HSA-IFN (Albuferon), and so on.In this study, we constructed the endostatin-human serum albumin by albumin fusion to extend the serum half-life of endostatin. This study will be helpful for the pre-clinical research of long acting endostatin. Pichia pastoris is a methanol yeast expressed system. It’s a widely used eukaryotic expression system and developed rapidly in recent years. We have chosen to use the secreted expression vector pPIC9k. Compared with pPIC9, it increased a Kan resistance gene in expression vector gene so that it will have G418 resistance. Screening the transformants through G418-resistant, we hope that we can get a higher expression amount of strain.This study is described in 5 parts:1. The construction of pPIC9k/ES-HSA expressed vectorWe obtained the ES-HSA fusion gene and the fragment of pPIC9k from the expression vector of pPIC9/ES-HSA and the pPIC9k vector which constructed previously in our lab by double digestion of SalⅠand BamHⅠ, and then they were transformated after connection, got to the recombination expressed vector pPIC9k/ES-HSA. By double digestion of SalⅠand BamHⅠ, single digestion of SalⅠ, PCR amplification and sequencing, the recombinant expression vector was demonstrated to be correct.2 The expression and identification of ES-HSA fusion protein The pPIC9k/ES-HSA was linearized and electrotransformed in host vector GS115. We identified 15 positive transformants after G418 resistance screening, in situ double filters screening, the screening of the phenotype and PCR amplification, and then the higher expression vector of positive transformant was selected after carrying out a small amount expression and using protein content assay of BCA method. The result of the Western Blot of expressed supernatant show that the protein is a endostatin and human serum albumin fusion protein. After the expression of strain in shake flask culture, by the optimal expressed time and medium, we ensured that we got good expressed result to use the medium of BMMY+ and the ninety-sixth hour is the peak of the expression. The amount of expression is about 300mg/L.3 The purification of ES-HSA fusion proteinTo get the isoelectric point of ES-HSA fusion protein, we imported the sequence of amino acids which were correspond to the corrected of gene sequencing to the correlative network station, we can see the pI is 6.20. After optimizing the purification conditions, we used salting out (ammonium sulfate precipitation), CM Sepharose Fast Flow weak cation chromatography, Q Sepharose Fast Flow strong anion chromatography and Blue Sepharose Fast Flow affinity chromatography to purify the ES-HSA fusion protein successively. The purity of the ES-HSA fusion protein was about 93% and the yield was 24% approximately.4 The bioassay of ES-HSA fusion proteinMTT assay was used to test the biological activity of the ES-HSA fusion protein in vitro. The results indicated that the fusion protein could efficiently inhibit the proliferation of the human umbilical vein endothelial cells ECV-304, and the effect was dose-dependent. The half-inhibition concentration was 8.805μg/mL, exponential curve fitting was 0.98668. CAM assay was applied to detect the biological activity of ES-HSA fusion protein in vivo. We found that ES-HSA fusion protein could inhibit the blood vessels growth of CAM, indicating that the fusion protein have significant inhibition effect on angiogenesis.5 The half-life determination of ES-HSA fusion proteinThe ES-HSA fusion protein was injected into the tail vein of rats according to the dose of 1mg/kg, and then we obtained the blood of the rats from the orbital venous plexus at different time points (before protein was injected, after protein was injected 10min, 1h, 4h, 12h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 216h, and 240h) and got the blood serum by centrifugalization. By the way of double-antibody sandwich ELISA detection of human endostatin of rat serum levels, we got that the half-life of the fusion protein is about 16.3 hours after the software analysis. Compared to endostatin, it has significantly extended.To sum up, we constructed expression vector pPIC9k/ES-HSA successfully and got the expression strain. After the expression, purification, biological activity identification and half-life determination of the ES-HSA fusion protein, the results showed that the ES-HSA fusion protein achieved higher expression level, with good biological activity and extended half-life of endostatin. It provides as a theoretical basis for a pre-clinical research of long-term drug.
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