河蚬中活性蛋白CFp-a的分离纯化及其活性

Isolation, purification and property study of active protein CFp-a in Corbicula fluminea

采用硫酸铵分级沉淀 ,BlueSepharose 6FastFlow、SPSepharoseFastFlow和HPLC柱层析 ,从河蚬 (Corbiculafluminea)中分离纯化蛋白CFp a。经SDS PAGE测定 ,其分子量为 2 4 .0kD ;经苯酚硫酸法测定 ,其含糖量为 2 .3%。 10～ 16 0 μg/mLCFp a分别处理肝癌细胞BEL74 0 4 72h后 ,采用四氮唑还原法 (MTT)检测癌细胞的生长活性 ,结果显示 ,CFp a对BEL74 0 4癌细胞生长的抑制率在 2 4 .6 %～ 79.2 %。采用质量浓度为 2 0 μg/mL和 4 0 μg/mL的CFp a分别处理BEL74 0 4 4 8h后 ,用流式细胞仪 (FCM)检测细胞凋亡 ,可以在DNA直方图上观察到典型的亚“G1”峰 ,凋亡率分别为 10 .99%和 15 .5 1%。

Marine organisms have attracted much attention as potential sources for drugs over recent years. Mollusks, with a number of phylogenetic diverse, hold potential sources for anti-cancer drugs. A few kinds of proteins and glycoprotein isolated from mollusks showed high in vitro or in vivo anti-tumor activity. Corbicular fluminea, a kind of freshwater bivalve mollusks, is assisted in the treatment of some diseases such as hepatitis, measles and fever in Chinese traditional medicine. However, few studies on the components and biologically functional effects of C. fluminea were carried out. That provides a major interest to study the active components of C. fluminea for better utilization. In this study, an active protein, named CFp-a, was isolated and purified from the fresh C. fluminea with the methods of ammonium sulfate precipitation, Blue Sepharose 6 Fast Flow, SP Sephorose Fast Flow column chromatography and HPLC. Among them, Blue Sepharose 6 Fast Flow is a kind of affinity chromatography with Cibacron Blue F3GA as a ligand. The blue dye binds many proteins, such as albumin, interferon and lipoproteins. It is also ideal for separation of broad range of nucleotide-requiring enzymes, nucleic acid binding proteins. With the affinity chromatography, CFp-a was isolated and concentrated. HPLC analysis proved the purity of the protein. The purified CFp-a was a monomeric protein with 24.0 kD in relative molecular mass, estimated by SDS-PAGE. It was a glycoprotein in containing 2.3% sugar by phenol-sulfuric acid reaction. In its amino acid composition, Phe had the highest content, followed by Cys and Tyr, but there is a lack of Pro. The in vitro antitumor activity of CFp-a was determined by using BEL7404 human hepatoma cell line. BEL7404 cancer cells were treated with 10,20,40,80,160 μg/mL CFp-a for 72 h respectively. The growth inhibition rates of cancer cells were measured by MTT method. BEL7404 cancer cells were treated with 20 μg/mL and 40 μg/mL CFp-a for 48 h and Cell apoptosis was observed by flow cytometry (FCM). The results showed that the growth inhibition rates of cancer cells treated with CFp-a ranged from 24.6% to 79.2%.However, when treated with 100 μg/mL 5-Fluororacil, it was 71.8%. So CFp-a showed high activity at low concentration. A typical subdiploid peak sub G_1 appeared on DNA histogram in FCM. The apoptotic rate was detected in 10.99% and 15.51%. The effects of CFp-a on the growth inhibition and the induction of apoptosis of BEL7404 cancer cells were dependent on CFp-a's concentration. Above results revealed that the CFp-a inhibited the proliferation and induced the apoptosis of BEL7404 cells in vitro.