该研究选取中华草龟肉为原料，以其木瓜蛋白酶酶解所得多肽对人乳腺癌细胞MCF-7增殖的抑制功效为活性检测指标，采用MTT细胞毒性实验对具有抗癌作用的多肽组分进行追踪测定。先采用截留分子量10 ku的超滤膜对酶解液进行截留，再通过Sephadex G-75、G-50及LH-60等层析技术手段对截留所得活性成分进行分离纯化。RP-HPLC谱图的结果表明，最终分离纯化出了一个纯度较高的抗癌活性组分TP-1，其IC50值约为2.7 mg/mL，呈明显的抗癌剂量依赖性与时间依赖性，且对正常细胞毒性极小。随后通过紫外、红外、圆二色谱对TP-1进行理化性质表征，并采用质谱技术对该多肽主成分分子量进行鉴定。结果表明TP-1中含有β折叠，β转角和无规则卷曲结构，且主成分多肽分子量约为1410.7 u，本研究成果为进一步开展中华草龟活性肽的抗癌机理研究奠定了坚实基础。

The Chinemys reevesii was selected as the raw material, and the inhibitory effect of the polypeptide of papain enzymatic hydrolysate on the proliferation of human breast cancer cell MCF-7 was identified as the activity detection index in this study to evaluate the cytotoxicity effects of extracted peptides by using MTT assay. The enzymatic hydrolysate was retained by ultrafiltration membrane with a molecular weight of 10 Ku first, and then, the active components were purified by sephadex G-75, sephadex G-50, and sephadex LH-60. The results of RP-HPLC spectrum indicated that a high-purity anticancer component, here named TP-1 was isolated and purified with an IC50 value of approximately 2.7 mg/mL, showing a significant dose-dependent and time-dependent anticancer effect, and little toxicity to normal cells. In addition, the physicochemical properties of TP-1 were characterized by ultraviolet, infrared and circular dichroism, and the molecular weight of the main component of the polypeptide was identified by mass spectrometry. The results manifested the existence of secondary structure of β-sheet, β-turn and random coil in TP-1, and the molecular weight of the principal component in TP-1 was about 1410.7 u. The research laid a solid foundation for further studies on anti-tumor mechanism of bioactive peptides from C. reevesii.

安徽省水产产业技术体系项目（皖农科[2016]84号）