摘要
构建了鲨肝活性蛋白的原核分泌表达载体pET22b-S,经过周质蛋白的提取、DEAE Sepharose FF、MonoQ/AKTAexplorer、Bio-gel P10的分离纯化,获得可溶性重组鲨肝活性蛋白(rSHAP)。初步研究了rSHAP对肝细胞增殖以及肝细胞葡萄糖激酶活性的影响。结果显示rSHAP能够被成功分泌至细胞间质中,分离纯化后可获得纯度约95%的可溶rSHAP,产量约3.8 mg/L。所获得的rSHAP具有刺激SMMC-7721细胞株增殖活性,体外活性检测显示15～45μg/ml的rSHAP能够显著提高正常人肝细胞HL-7702葡萄糖激酶活性。
The gene encoding shark hepatic active protein（SHAP） was inserted into the downstream of signal peptide PelB to construct the secretory plasmid pET22b-S. The recombinant SHAP was secreted into the periplasm and purified using DEAE Sepharose FF, MonoQ/AKTA explorer and Bio-gel P10. The result of the N-terminal amino acid sequence analysis was NH2-M-L-V-G-P I-G-A-A-K V, which revealed that the signal peptide was cut correctly. The results of MTT showed that the 5～45μg/ml rS-HAP could improve the proliferation of SMMC 7721 cell and HL 7702 cell. Assay of glycogen contents showed that 15μ45/μg/ml rSHAP could increase hepatocellular glycogen contents. Assay of glucokinase activity showed that 15～45μg/ml rSHAP could improve the GK activity in vitro.
出处
《药物生物技术》
CAS
CSCD
2008年第6期439-443,共5页
Pharmaceutical Biotechnology
基金
国家自然科学基金资助
项目编号(N030701036)
关键词
鲨肝活性蛋白
分泌性表达
葡萄糖激酶
Shark hepatic active protein, Secretory expression, Glucokinase
