The Cloning And Purification Of Vitronection And Its Biological Activity Verification

Vitronectin(VTN)is a multifunctional glycoprotein present in blood and in the extracellular matrix with apparent molecular weight of 75 kDa,and it is present at high concentration in the peripheral vasculature,with the concentration of 200-400 mg/L.The VTN in human body is mainly synthesized in the liver,and then it is transfered to other organs and tissues.VTN contains a RGD integrin-binding motif,and thus it can mediate cells' adherence and spreading,and moreover,it can bind with many cytokines,and exert many physiological effects,playing an important role in angiogenesis,cancer's development,thromb,blood coagulation,humoral immunity and so on.VTN is always added to serum-free medium to promote cells' adherence and breeding in cell culture experiment in vitro,and it can promote many cells' growing and differentiation such as pluripotent stem cells.VTN from natural serum is mostly purified by means such as heparin affinity chromatograph,but the source is limited and the cost is expensive.The way to express recombinant VTN through culturing cells in vitro is the main pathway to obtain VTN for scientific research,and scientists have developed several cell strains expressing VTN with corresponding purification methods,and achieved to express VTN or VTN's functional fragments in E.coli,mammalian cells,tobacco and other cells and organisms,however,the papers of expressing complete recombinant VTN using HEK 293 T cells have not been published yet.In this research we tried to express recombinant VTN using HEK 293 T cells,CHO cells and E.coli,and mainly explored the conditions and procedures using 293 T cells.Via the way of calcium phosphate co-precipitating with DNA and gene's transient transfection,we transfected the recombinant plasmid comprising VTN's cDNA and pEXL-FTH-GFP(or pcDNA3.1-YFP)to 293 T cells,and harvested the medium 2-3 days after transfection.Then we centrifuged the medium,and purified VTN as the his-tag in it could bind specially with Ni2+ resin.After SDS-PAGE and Western Blot,we detected the expressed VTN,and we estimated its concentration using ultraviolet spectrophotometer,which gave a data of about 20.62 mg/L in the HEK 293 T cells' medium.After that,we dialyzed the product and lyophilized it using vacuum freeze drier.Finally we verified the function of purified VTN,and the results showed that our VTN had biological activity,which could promote cells' adherent growing and breeding.