摘要
获得可溶性表达的鲨肝再生因子类似物，初步研究性质与活性。构建含有鲨肝再生因子类似物片段的原核表达载体pET32a-S；转化BL21，IPTG诱导表达融合蛋白，表达产物经过分离纯化-FXa酶切-分离纯化，得到鲨肝再生因子类似物；利用MTT比色法研究该重组产物对肝癌细胞株SMMC-7721的增殖活性。结果 原核表达的融合蛋白在大肠杆菌中以可溶的形式存在，表达量为40％，分子质量为30ku，去除融合子后的鲨肝再生因子类似物分子质量为12ku，PI=4．7。活性研究显示在6．25-50μg／ml浓度范围内，类似物与天然产物均有相似的刺激SMMC-7721细胞株增殖的活性，但是在高浓度下却表现出了抑制活性。
To explore the fusion protein of hepatic regeneration factor analog from shark liver（SHRF） and to study its effects on SMMC7721. SHRF analog＇s cDNA was inserted in the downstream of TrxA in a pET32a expression vector. Recombinant TrxA-SHRF analog fusion protein was expressed under 0. 2 mmol/L/IPTG induction in BL21（DE3）. After having been purified with His Tag affinity chromatography, TrxA-SHRF analog was cleaved with FXa. Then it purified the SHRSF analog by FPLC Resource Q. Its activity on human hepatoma cell lines SMMC7721 was studied by MTT assay. Results： the fusion protein of TrxA-SHRF analog was expressed as a soluble form in bacterial cytosol and the quantity of the fusion protein was above 40% of the total soluble protein of BL21. The molecular weight of the fusion protein was 30 KD. After having been digested by FXa, the recombinant protein of SHRSF analog was detected by SDS-PAGE. The activity analyzed with MTT showed that the purified product could stimulate the proliferation of SMMC7721 cell under the concentration of 50μg/ml, but would inhibit the cells＇ proliferation up to 200μg/ml.
出处
《药物生物技术》
CAS
CSCD
2006年第4期244-249,共6页
Pharmaceutical Biotechnology
基金
国家海洋"863"(2005AA624090)
国家自然科学基金(30171103)
