可磷酸化短肽偶联壳聚糖促进CS/DNA转染效率

Phosphorylatable Short Peptide Coupled Chitosan Facilitates Transfection Efficiency of the CS /DNA Complex

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.

We have previously demonstrated that the degradation of chitosan promoted by the enhanced intracellular unpacking of the exogene from chitosan carrier could markedly improve the transfection efficiency of CS /DNA complex. Here we tested whether chitosan modification by phosphorylatable short peptide facilitated the intracellular DNA unpacking to further enhance CS /DNA transfection. A synthesized phosphorylatable short peptide （ pSP） with the sequence of LLLRRRDNEY FY VRRLL contained two potential phosphorylatable tyrosine residues by constitutively expressed cytoplasmic protein kinase Jak2. The pSP-CS /DNA complex was prepared using a GFP or Luciferase reporter plasmid and chitosan conjugated to pSP. In vitro phosphorylation and DNA releasing assays showed that mammalian cell lysate phosphorylated SP effectively and promoted the DNA unpacking from the carrier. High cell membrane permeability of the pSP-CS /DNA complex was observed by 32P labeled plasmid transforming assays. C2C12 myoblast cells were transfected with pSP-CS /DNA of the GFP or luciferase plasmid and the transfection efficiency determined by the expression levels of the reporter genes. Various cell lines were transfected by the complex with a Luciferase reporter and the results showed that the transfection efficiency was near to that of lipofectamine 2000.