肿瘤抑素抗肿瘤相关肽的克隆及生物活性

Cloning and Biological Activity of Anti-tumor Peptide of Tumstatin

为得到肿瘤抑素中具有直接抗肿瘤活性肽并检测其生物学活性,人工合成肿瘤抑素中185～2 0 3位氨基酸(19肽)所对应的核苷酸序列,将其连接到融合蛋白表达载体pTYB2中,酶切和测序鉴定后,转化到大肠杆菌BL 2 1(DE3)中诱导表达.表达的融合蛋白经几丁质亲和层析、二硫苏糖醇(DTT)的柱内还原,直接获得可溶性19肽.利用MTT法,细胞生长曲线,小鼠H2 2腹水型转移型肝癌实体瘤模型抑瘤实验并结合组织病理学切片,研究19肽的生物学活性.获得的19肽对B16小鼠黑色素瘤细胞、人SMMC 772 1肝癌细胞、人脐静脉内皮细胞的生长均具有抑制作用.小鼠H2 2腹水型肝癌抑瘤率达4 8 4 6 % .病理学切片显示,19肽可促使小鼠肿瘤组织坏死,血管数量减少.19肽具有较强的直接抗肿瘤活性,有可能成为肿瘤治疗的一种新的有前景的药物.

To obtain anti-tumor peptide of tumstatin and detect its biological activity, the nucleotide sequence encoding 185～203 amino acids (19 peptide) of tumstatin was synthesized and inserted into the fusion protein vector pTYB2.After identification by sequencing and restriction, the recombinant vector was transformed into ~E.coli BL-21 (DE3). Transformants were induced by IPTG, and then expressed. By DTT reduction, the soluble 19 peptide was obtained from chitin affinity chromatography. The biological activities of 19 peptide were determined by MTT assay, cell growth curve and effect of the ascitic fluid transfevent H22 hepatoma of mice and histopathological slice. The purified 19 peptide directly inhibited proliferation and migration of murine B16 melanoma cell, SMMC-7721 hepatoma carcinoma cell, human umbilical vein endothelial cell.The tumor inhibition rate of ascitic fluid transfevent H22 hepatoma in mice was 48.46%.Histopathological slice showed that it could promote tumor tissue necrosis and decrease the density of blood vessel. With higher anti-tumor activity, 19 peptide has potential to become a novel, potent anti-tumor agent.