Fusion Protein PTD-hA20 Protect Endothelial Cells Against High Glucose Injury

In the recent years, the incidence of cardiovascular disease(CVD)has been increasing. There are many factors can cause CVD , Diabetes mellitus(DM) is one of the most important reasons. DM is a kind of metabolic diseases characterized by chronic hyperglycemia, it is caused by little insulin secretion and(or) disturbance of the insulin utilization. According to reports, there is systematic small vessels diseases and microangiopathy in the 70%-80% diabetic.While for diabetic, the complications of capillary and great vessels can lead to death and disability. Why is it more often for the diabetic to have vessel complications than other people? It is because chronic hyperglycemia can lead the endothelial structure to change and disfunction. While the disfunction of endothelium is an important marker for vessel diseases.It is well known that only a few micromolecule can permeate the eukaryotic plasma membrane under normal physiological status. The biomacromolecule such as protein must form vesicles firstly then be transported into cells by endocytosis or exocytosis. However, plasma membrane can maintain cell physiological structure integrity, but inhibits many biomacromolecule medicine to permeate cell so as to treat many diseases. In the recent years, people found a kind of protein functional domain , they can guide the biomacromolecule to permeate the plasma membrane and accumulate in the cells, so they were called protein transduction domain(PTD). Those molecules carried into the cells hold their native activity. They can do their native work. So PTD is very charming because the character. It is possible for the macromolecule protein or polypeptide to permeate cytoplasmic membrane because of the PTD, meanwhile, another therapy platform that is to say treatment technic by protein becomes ture. PTD accelerates the protein engineering and the exploitation of protein medicine. At present, many kinds of protein have been carried into the different cells or the animals, they hold their native activity. PTD will be applicated to find new therapy method and new function of protein. A20 belongs to the zinc finger protein family, it is very helpful to anti-inflammatory, anti-apoptosis and anti-rejection reaction, meanwhile, it can protect endothelial cells from injury caused by many diseases. At present, A20 is considered as a protective gene for endothelial cells. Many scholars think A20 as an important measure to treat inflammation in the future. But in the past, the majority of the researches about A20 were finished by gene transfection. For example, A20 gene was conveyed into the cells or organism by liposome or viral vector. But there are many problems during the operative procedure. Such as poor feasibility, low efficiency and stability, low transfection rate. So at present, the researches about A20 are executed only in cells or animals. the application of the important gene-hA20 is inhibitted. If we can get quantity of active protein hA20(human A20) in vitro, meanwhile, if the protein can permeate plasma membrane spontaneously,the application range of zinc finger protein hA20 will be expanded greatly. But in vitro, does the fusion protein hA20 have good biologic activity? Can the exogenous zinc finger protein hA20 protect endothelial cells against injury and apoptosis induced by high glucose? Can it give some benefit to cure the cardiovascular disease caused by DM? These questions are very valuable for us, however, there is no report about that till to now.We hope to solve three problems in the study. The first is to get recombinant plasmid pPIC9k-PTD-hA20 through vector construction. In the second part of the study, deliver the recombinant plasmid pPIC9k-PTD-hA20 into the Pichia yeast GS115 through transformation. We can get excellent expression Pichia yeast monoclone by G418 selection, then induce the excellent expression Pichia yeast to express protein by methanol, after that we can get the yeast supernatant including our target protein. After depuration, we get purified target protein. In the third part, we tested the fusion protein hA20 biological activity though culturing endothelial cells and establishing cells model in high glucose surroundings.Main results and the conclusions are as followed:Partâ… Construction and identification of the recombinant plasmid pPIC9k- PTD-hA20.In the experiment, we got PTD-hA20 gene by means of PCR and constructed the eukaryotic expression vector pPIC9k-PTD-hA20 through enzyme digestion and ligation. At last the appreciation result digested by enzyme and sequencing result all indicated that the recombinant plasmid vector is identical with the one we designed previously.Partâ…¡Transform the yeast, screen the excellent expression yeast strain, induce the yeast to express the fusion protein and depurate the target protein.The recombination plasmid was transformed into the yeast GS115 successfully by means of electrotransformation. The excellent yeast strain was got by G418 screening. We achived the yeast supernatant including target protein induced by methanol. After the yeast supernatant passed the Ni-NTA resin, the majority hybridprotein was removed, then the target protein is purified.Partâ…¢The cultivation and assessment of human umbilical vein endothelial cells(HUVECs), the establishment of cell model cultured in high glucose medium and the protection of exogenous zinc finger protein A20.In the third part of experiment, The HUVECs was cultured successfully and the cell model cultured in high glucose was established. The results indicate that high glucose can induce the endothelial cells damage and apoptosis,the expression level of P-selectin and Caspase-3 are increased significantly. The recombinant protein PTD-hA20 can not only permeate the cellular membrane but also protect endothelial cells against damage and apoptosis induced by high glucose. It is very possible that hA20 is helpful to the cardiovascular diseases caused by DM.In the experiment, we constructed the recombinant plasmid pPIC9k-PTD-hA20 successfully and got the purified target protein. The recombinant protein has the ability to permeate the cellular membrane and good biological activity. All those provide a more security and convenient pathway for the application of A20 to clinic, mainwhile, it maybe give some help to treat the complications of capillary caused by DM.