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为探讨Ⅱ型抗癌晶体蛋白(Parasporin-2)上与抗肝癌作用相关的关键氨基酸,利用5-BU对Parasporin-2活性区编码DNA(P2Y)进行PCR诱变,之后在大肠杆菌中表达,产物纯化后经MTT法检测其对肝癌细胞系和正常肝细胞系的作用。获得的9个突变体其抗肝癌活性差异极大,其中P2M1和P2M8对两种肝癌细胞系SMMC7721和Bel7402均有较强的细胞毒杀作用而不影响正常肝细胞系Chang-liver。比较了P2M1、P2M8和P2Y的二级结构与三级结构,发现二级结构上的变化如β折叠变长或α螺旋增加影响着Ⅱ型抗癌晶体蛋白的抗肝癌活性。基于突变体间氨基酸序列比对、突变体与受体间分子对接以及模拟突变等研究的结果表明,位点52、56、58和208上的氨基酸残基特别是芳香族氨基酸在Parasporin-2与受体间的互作中可能起着重要作用。
To investigate the key amino acids associated with anti-hepatocarcinoma on type II anti-cancer crystal protein (Parasporin-2), PCR was performed on the DNA encoding P2 domain of Parasporin-2 by 5-BU and then expressed in E. coli. The purified product was assayed for its effect on hepatocellular carcinoma cell lines and normal liver cell lines by MTT assay. The nine mutants showed great difference in anti-hepatocarcinoma activity. Among them, P2M1 and P2M8 had strong cytotoxicity on both hepatoma cell lines SMMC7721 and Bel7402 without affecting the normal liver cell line Chang-liver. The secondary and tertiary structures of P2M1, P2M8 and P2Y were compared and it was found that secondary structural changes such as β-sheet elongation or α-helix increase affect the anti-hepatoma activity of type II anticancer crystal protein. Based on amino acid sequence alignment among mutants, molecular docking between mutants and receptors as well as mock mutations, the results showed that the amino acid residues at positions 52, 56, 58 and 208, especially the aromatic amino acids, are closely related to Parasporin-2 and Inter-receptor interactions may play an important role.