The Study On Eukaryotic Expression, Purification Of Recombinant Human Vasostatin And On Its Inhibition To Retinal Neovascularation

1.Objective:Ocular angiogenesis and a series of accompanying pathologicalalterations, such as exudation, bleeding and proliferation, willseverely damage the structure and function of eye and result in seriouscomplications, which is the main reason of blindness. How to controlangiogenesis effectively has long been a problem that ophthalmologistspay close attention to. At present, several angiogenesis inhibitorshave been identified, such as antibody of VEGF, VEGF soluble receptor,antibodies of integrinαvβ3, angiostatin, endostatin, Vasostatin,interferon γinducible protein-10,interleukin-12, fragment ofprolactin, platelet factor-4, thrombospondin and so on. They can takeeffects as follows: to prevent cells from secreting angiogenesisfactors, block the effects of angiogenesis factors, preventproliferation and migration of endothelial cells, interfere with theinteraction of endothelial cells and extracellular matrix, prevent theformation of vascular network and inhibit the growth of vascular smoothmuscle cells. Vasostatin, the NH2-terminal domain of calreticulin (amino acids1-180), was found and named by Pike, an American scientist, and thenwas purified from supernatant of an Epstein-Barr virus-immortalizedcell line in 1998.The gene to express Vasostatin has been localizedon human chromosome 19. Its protein product is composed of 180 animoacids, and its molecular weight is approximately 21KD. Vasostatin,which shows the functions of inhibiting the proliferation ofendothelial cells in vitro and suppressing angiogenesis in vivo, isa novel angiogenesis inhibitor. The molecule of Vasostatin is small,soluble, stable, effective and specific. The investigation onVasostatin will be significant to increase our understanding on thoseangiogenesis diseases such as rheumatic arthristis, Psoriasis,diabetic retinopathy, age related macular degeneration ans solidtumors.In order to get enough amount of Vasostatin and investigate intoits functions, scholars usually obtain this protein by the way ofprokaryotic expression system. In that way the recombinant proteinexisted as inclusion body, which makes the recombinant protein in anonatural folding. Subsequent elaborate denaturization andrenaturation alter the activity of recombinant protein. For thisreason, we express and Purify Vasostatin by eukaryotic yeast cell forthe first time all over the world. We investigate its function in vitroand in vivo.2.Content:We obtained human Vasostatin cDNA which includes 6 His tags fromhuman liver tissues by using RT-PCR techniques and cloned it into thePichia Multi-Copy Expression Vectors pPIC9K .we constructedeukaryotic expression plasmid:pPIC9K-Vasostatin. After DNAsequencing, eukaryotic expression plasmid:pPIC9K-Vasostatin andempty vecter pPIC9K were linearized with restriction enzyme SacI,then the exogenous genes were transformed into Pichia yeast competentcell KM71 by Lithium Chloride.After two times Screening by MDS plateand YPD+G418 plate , we have screened MutS multi-insertronTransformants clones. After Total DNA Isolation from Pichia and PCRAnalysis of yeast fungus, the desire was grown in BMGY/BMMY. Add 100%methanol to a final concentration of 0.5% methanol every 24 hours toinduce the expression of recombinant Vasostatin protein. The productof expression was analyzed by SDS-PAGE. The recombinant protein waspurified with immobilizedmetal affinity chromatography(IMAC). MTTassay was applied to measure the effect of recombinant human Vasostatinprotein on the proliferation of human umbilical vein endothelial cellsin vitro. Using Flow cytometric analysis we detected whether or notVasostatin induce apoptosis of ECV304 cells. Hyperxia induced retinalneovascularation model was applied to the study of Vasostatininhibiting angiogenesis in vivo.Our results showed that the cDNA fragment of Vasostatin we havegot is 540bp;After inserting it into pPIC9K vector the sequence ofpPIC9K-Vasostatin proved that the sequence we got was totallycoincident with that reported by Genebank NM004343. the eukaryoticexpression plasmid pPIC9K-Vasostatin we have constructed wassuccessful. We could transform the exogenous gene into Pichia yeastcompetent cell by Lithium Chloride method effectively. After two timesScreening by MDS plate and YPD+G418 plate, the desire KM71 couldexpress Vasostatin protein in 0.5% methanol. We got highly purifiedrecombinant human Vasostatin protein with IMAC. MTT assay proved thatrecombinant human Vasostatin protein inhibited the proliferation ofendothelial cells in vitro in a dose-dependent basis. Flow cytometricanalisis demonstrated that Vasostatin could induce apoptosis of ECV304cells. Recombinant human Vasostatin protein could also inhibitangiogenesis of Hyperxia induced retinal neovascularation in vivo.3. Main achievements:⑴ We have expressed the recombinant human Vasostatin protein with6-histimine tag using eukaryotic yeast expression systemeffectively for the first time all over the world. Meanwhile, we haveacquired the KM71 strain which can express Vasostatin proteinstably.⑵ We have obtained highly purified protein with IMAC.⑶ We amplified the application scope of Vasostatin protein to retinalneovascularation field for the first time all over the world. We provedthat Vasostatin expressed by eukaryotic yeast expression system couldinhibit the occurrence or development of retinal neovascularation invivo.4.The value of our achievements in ophthalmology:Since Vasostatin was found and named by Pike in 1998, which couldinhibit pathological neovascularation, scholars have done a lot ofwork on its expression and functions. All of the protein was obtainedeither extracted from supernatant of virus or expressed by Escherichiacoli .Our achievements filled the blank of expressing recombinant humanVasostatin protein effectively by eukaryotic yeast expression system.Our research work has provided the theoretical and methodologicalbases for others to express the recombinant human Vasostatin proteinrepeatly.The succese of applying Vasostatin to inhibit retinalneovascularation in whole animal contribute to exploit and convert therecombinant human Vasostatin protein to a gene engineering drugs. Thestudy on Vasostatin is not only significant to the prevention andtreatment of ocular angiogenesis diseases which lead to blindness, butalso has social significance to those diseases which severely threatenhuman health such as tumors, diabetes, rheumatoid arthritis, psoriasisand so on.