摘要
自行设计了抗肿瘤转移多肽-三聚β肽(β3),人工合成了β3的基因片段,构建了β3的表达质粒pET_His_β3,在大肠杆菌BL21(DE3)plysS中表达。在用IPTG诱导1·5h后可见明显的His_β3融合蛋白的表达,表达产物约占细胞总蛋白的4%,占细胞总不溶性蛋白的10%。每升pET_His_β3/BL21(DE3)plysS细菌培养液用金属螯合琼脂糖凝胶6BFF分离后可回收纯度为92·2%的β3产物约20mg。所表达出的β3肽对人肝癌细胞株SMMC_7721细胞及人肝癌高转移细胞株HCCLM6细胞与纤连蛋白(fibronectin,FN)粘附具有特异的抑制作用,呈现剂量效应相关关系和时间效应相关关系,抑制作用强于β肽(β1)、3倍浓度的β1(3×β1)和GRGDS。研究结果表明:pET_His_β3/BL21(DE3)plysS是β3适合的表达系统;表达的β3肽具有特异的抗肿瘤细胞粘附作用。
To block tumor cell adhesion, inhibit tumor metastasis and recurrence, the anti-adhesion peptide-trimeric β peptide (DLYYLMDLSYSMKGGDLYYLMDLSYSMKGGDLYYLMDLSYSMK, β3) was designed. The DNA fragment of β3 was cloned into expression vector pET-His and the fusion protein His-β3 was expressed in E.coli. BL21(DE3)plysS. After 1.5 hours' induction with IPTG, His-β3 peptide was expressed significantly amounting to 10% of the insoluble proteins and 4% of the total proteins. 20mg of β3 peptide was obtained from one litter culture medium after purification by using metal-chelating sepharose 6B FF. The purity of β3 is 92.2% according to Gel-Pro analysis. The anti-adhesion effects of β3 peptide, β1 peptide (DLYYLMDLSYSMK) and GRGDS on the hepatocellular carcinoma cell line SMMC-7721 and the high metastasis hepatocellular carcinoma cell line HCCLM6 were studied. The result showed the β3 blocked the adhesion of HCCLM6 cells and SMMC-7721 cells to fibronectin(FN) specifically. The inhibition effect was dose-dependent and time-dependent and the inhibition rate of β3 was higher than three times concentration of β1 and GRGDS. This suggested that pET-His-β3/BL21(DE3)plysS was a suitable expression system for β3, and the expressed β3 specially inhibited the adhesion of cancer cells.
出处
《生物工程学报》
CAS
CSCD
北大核心
2005年第4期558-562,共5页
Chinese Journal of Biotechnology
基金
国家高技术研究发展计划(863)项目(No.2001AA215411
No.2004AA215201)
上海市重点科技项目(No.024319212)~~
关键词
基因工程
抗粘附
多肽
表达
肝癌
molecular engeneering, anti-adhesion, peptide, express, hepatocellular carcinoma
