桑蚕丝素-RGD融合蛋白的固态结构及其细胞粘附性分析

利用基因工程方法把含有短肽RGD的氨基酸序列连接到桑蚕丝素蛋白的结晶序列GAGAGS上, 通过调节DNA的聚合度, 合成了具有TGRGDSPA(GVPGV)₂GG(GAGAGS)₃AS]_n一级结构、不同分子量大小的桑蚕丝素-RGD融合蛋白, 并且通过在M9培养基中添加3-¹³C]Ala的方法进行融合蛋白的稳定同位素标记. ¹³C CP/MAS NMR结果显示, 融合蛋白中的GAGAGS部分具有与天然桑蚕丝素结晶部分相同的分子结构, 即Silk I处理后为均一的分子结构, 而Silk II处理后为不均一的分子结构, 它包含了三种不同的结构成分. 另一方面, 通过对小鼠成纤维细胞BALB/3T3在不同蛋白材料载体上的粘附和增殖性能的测定结果显示, 融合蛋白对细胞的增殖性能与天然胶原蛋白相近, 但表现出了比胶原蛋白更好的细胞粘附性能. 该研究结果显示, 如果对该桑蚕丝素-RGD融合蛋白进行适当加工, 可能适合于组织工程支架材料的应用.

Solid-state Structure of Bombyx mori Silk Fibroin-RGD Fusion Proteins and Their Cell Adhesive Ability

An amino acid sequence containing the short peptide RGD was ligated with another sequence selected from the crystalline region of Bombyx mori silk fibroin by using the genetic engineering method. The Bombyx mori silk fibroin-RGD fusion protein with the primary structure of TGRGDSPA(GVP-GV)2- GG(GAGAGS)3AS]n was synthesized with the different molecular weights by regulating the polymerization degree of DNA. The 13C isotope labeling of fusion proteins were performed by adding 3-13C]Ala in a modi- fied M9 medium during cell cultivation. 13C CP/MAS NMR results showed that the GAGAGS region in the fusion proteins tended to take the similar molecular structure to the native silk fibroin protein, i.e., homoge- neous silk I structure after treatment or heterogeneous silk II structure after treatment including three struc- tural components. The evaluation of cell biocompatibility to the BALB/3T3 cell line showed that the fusion proteins had higher cell adhesive ability than that of native collagen, even though both have the similar abil- ity on the cell proliferation. These results suggested that the B. mori silk fibroin-RGD fusion proteins may be the candidate for the scaffolds in tissue engineering after suitable processing.