23 Jul Treatment effect of tissue plasminogen activator kringle-2 on stroke and preparation method for tissue plasminogen activator kringle-2
Abstract
The invention relates to the technical field of biomedical engineering, and discloses preparation and application of tissue plasminogen activator kringle-2 polypeptide. The invention discloses two methods for preparing the tissue plasminogen activator kringle-2. One method comprises that: a human tissue plasminogen activator variant reteplase is taken as a raw material and is cut by plasminogen to obtain the polypeptide consisting of the disulfide bond-connected kringle-2 and carbon-terminal serine protease; beta-mercaptoethanol is used for restrictive reduction to break the disulfide bond and H2O2 is used for restrictive oxidization; the kringle-2 polypeptide is further purified by lysine-Sepharose 4B affinity chromatography; and the purity of proteins is detected by SDS-PAGE electrophoresis. The other method comprises that: a kringle-2 gene is obtained by the PCR technology, and is cloned into an expression plasmid pET29a provided with a T7 promotor; the recombinant plasmid is converted into Escherichia coli BL21 and is subjected to IPTG induction expression; and the product exists in an inclusion body form, and is subjected to in vitro refolding to form the soluble kringle-2. The kringle-2 is offered after cerebral ischemia, can reduce the death rate of rats with the stroke, reduce the volume of the cerebral infarction, reduce the expression of endogenous tPA and adjust the BBB opening mode, makes the BBB permeability reduced after 24 hours of the cerebral ischemia, effectively prevents encephaledema, and reduces cerebral neuronal apoptosis. The tissue plasminogen activator kringle-2 polypeptide is mainly used for treating the stroke and can also be used for neuroprotection of cerebral injury and neurodegeneration.
Year: 2010
Country: CN
Doc No: 101886066