截短型细胞毒素Gelonin的分子构象和生物活性

Molecular Conformation and Biological Activity of Truncated Cell Toxin, Gelonin

为了探索免疫络合物中具杀伤靶细胞的毒素,gelonin的结构与功能的关系,根据化学合成的gelonin基因序列和3维分子构象设计了N端区Gly,Leu,Asp和或C端区Asp,Lys,Asp,Pro,Lys缺失的gelonin.以重组质粒pEgel为模板,在相应引物存在下,用PCR法获得5′端区和或3′端区碱基序列缺失的gelonin基因片段.经克隆、表达和纯化得到3种截短型gelonin(GN3、GC5、GN3C5).CD谱和荧光谱表明,完整型gelonin(GO)与截短型gelonin的分子构象有明显的差异.它们的构象变化与类DNase活性和抑制肿瘤细胞生长的能力均为GO≥GN3>GC5>GN3C5.结果再一次证明了具有α+β型结构蛋白,gelonin的构象与生物活性的一致性.

To explore the relationship between the structure and function of cell toxin gelonin as the bullet of immunoconjugate to specially kill and wound target cells, truncated gelonins deleted at N-terminal region Gly, Leu, Asp and/or C-terminal Asp, Lys, Asp, Pro, Lys were designed on the basis of its gene sequence and three dimentional molecular conformation. DNA fragments of recombinant gelonin and its truncated products were amplified by PCR with plasmid pE-gel as the template in the presence of different primers, and in turn, the relevant proteins, e.g. intact gelonin （G-O） and truncated gelonins （G-N3, G-C5, G-N3C5） were obtained by processes of cloning, expression and purification. CD and fluorescence spectra evaluating the conformational change of protein show that truncated gelonins are obviously different from the intact molecule, showing that the α-helix relative content in truncated form decreased. Furthermore, DNase-like activity in vitro and the ability of inhibiting tumour cell growth in vivo are as following G-O≥G-N3〉G-C5〉G-N3C5. In conclusion, results in this study demonstrate the consistency of the structure and bioactivity of gelonin with the protein of α ＋ β conformation.