人内皮抑素抗肿瘤相关肽基因的克隆表达及活性研究  

Gene Cloning,Expression and Activity of Anti-Tumor Peptide of Human Endostatin

作　　者：李丹[1] 刘兴汉[1] 赵炜明[1] 李冀宏[1] 马洪星[1] 张宇雯[1] 栗亚[1] 

机构地区：[1]哈尔滨医科大学生物化学与分子生物学教研室黑龙江省生物医药工程重点实验室--省部共建国家重点实验室培育基地,哈尔滨市150081

出　　处：《医学分子生物学杂志》2007年第6期475-479,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金(No.30400063)~~

摘　　要：目的 克隆并表达人内皮抑素抗肿瘤相关肽，检测其生物活性。方法 人工合成人内皮抑素1—30位氨基酸（30肽，序列25—31由RGIRGAD改为RGDRGD）所对应的核苷酸序列，连接到质粒pTYB2中，再转化至大肠埃希菌BL21（DE3）中表达，几丁质亲和层析树脂一步纯化30肽。通过MTT法、鸡胚绒毛尿囊膜（CAM）实验、小鼠体内抑瘤实验比较30肽和内皮抑素抗肿瘤活性。结果 MTT证实30肽体外对人脐静脉内皮细胞（HUVEC）、胃癌7901细胞（SGC-7901）半数抑制浓度IC50为36μg／ml、47μg／nl，显著低于内皮抑素IC50179μg／ml、202μg／ml。CAM实验中30肽对血管的抑制作用更强。30肽在小鼠体内抑瘤率47．8％，效果优于内皮抑素28．7％。结论 30肽具有更强抗肿瘤活性，有可能成为治疗肿瘤的一种新药物。Objective The anti-tumor peptide of human endostatin was cloned and expressed and its bioactivity was detected. Methods The nucleotide sequence encoding 1-30 amino acids （30 peptide, sequence RGIRGAD was changed to RGDRGD） of human endostatin was synthesized and inserted into plasmid pTYB2, and then the plasmid was transformed into E. coli BI221 （ DE3 ） for expression. At last 30 peptide was directly purified by using chitin affinity chromatography. MTT, chick embryo allantoid membrane （CAM） and tumor inhibition test were conducted in mice to compare the anti-tumor activity between 30 peptide and endostatin. Results MTT showed that 30 peptide had a lower dose of IC50 （HUVEC： 36 μm/mL, SGC-7901：47μg/mL） than endostatin （HUVEC： 179 μg/mL, SGC-7901 ： 202 μg/mL） . CAM revealed that 30 peptide could inhibit angiogenesis more effectively. Tumor inhibition rate of 30 peptide in mice was 47, 8 %, which was obviously higher than that of endostatin （28. 7 % ） . Conclusion The 30 peptide has stronger anti-tumor activity and can be produced more easily. It promises to be used as an agent for tumor treatment.

关 键 词：内皮抑素 RGD序列 克隆 基因表达 抗肿瘤活性 

分 类 号：Q78[生物学—分子生物学]