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合成基序为LLLRRRDNEY*FY*VRRLL的短肽(pSP),其中含有两个可被JaK2蛋白激酶磷酸化的酪氨酸残基.将此短肽与壳聚糖(CS)相偶联,体外磷酸化及DNA释放实验检测哺乳动物细胞裂解液对短肽的磷酸化及pSP-CS/DNA复合物中DNA释放的影响.放射性标记DNA转移实验验证pSP-CS/DNA复合物的入胞能力后,将荷荧光素酶或GFP报告基因的质粒与pSP-CS制成pSP-CS/DNA复合物,转染体外培养的C2C12小鼠成肌细胞,观察GFP的分布及细胞裂解液中的荧光素酶活性以表征转染效率.继而进行多种细胞系的转染,衡量pSP偶联的壳聚糖对不同种属细胞的转染效率.结果表明,哺乳动物细胞裂解液可有效地使短肽发生磷酸化,并藉此促进DNA与壳聚糖载体的解离.以pSP修饰的壳聚糖进行转染时,细胞裂解液的荧光素酶活性可达普通壳聚糖转染的两倍,细胞中GFP的含量也明显增加.据此推论,短肽被磷酸化后产生电荷属性的改变,促进DNA与壳聚糖载体的解离从而显著提高壳聚糖的转染效率.
The synthetic motif is a short peptide (pSP) of LLLRRRDNEY * FY * VRRLL containing two tyrosine residues that can be phosphorylated by the JaK2 protein kinase.This short peptide is coupled to a chitosan (CS) Phosphorylation and DNA release assay were used to detect the phosphorylation of short peptide and the release of DNA from pSP-CS / DNA complex in mammalian cell lysate. The radioactivity labeled DNA transfer assay was used to verify the ability of pSP-CS / DNA complex to enter into cells The plasmids carrying luciferase or GFP reporter gene and pSP-CS were made into pSP-CS / DNA complex and transfected into C2C12 mouse myoblasts cultured in vitro. The distribution of GFP and the expression of fluorescein in cell lysate Enzyme activity to characterize the transfection efficiency, followed by a variety of cell lines transfected to measure pSP-coupled chitosan transfection efficiency of different species of cells.The results show that mammalian cell lysate can effectively make short peptides Phosphorylation and thus promote DNA and chitosan carrier dissociation.With pSP modified chitosan transfection, the luciferase activity of cell lysate up to twice the normal chitosan transfection, GFP content in cells also significantly increased.According to the corollary, the phosphorylation of short peptides generated charge properties change , Promoting vector DNA and chitosan dissociation which significantly improves the transfection efficiency of chitosan.