人Hexastatin基因的优化、蛋白的表达纯化及初步应用  

Human Hexastatin genetic optimization, protein expression, purification and preliminary application

作　　者：唐晓[1] 宋娜玲[1] 贺欣[1] 王月英[1] 刘谦[2] 温镭[1] 王德芝[1] 韩英[1] 张恒[1] 

机构地区：[1]中国医学科学院北京协和医学院放射医学研究所,天津市分子核医学重点实验室,天津300192 [2]天津市第一中心医院泌尿外科,300192

出　　处：《国际生物医学工程杂志》2012年第2期103-107,I0006,共6页International Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目（30970851）;中国医学科学院放射医学研究所发展基金（SF1104、ST1113）

摘　　要：目的优化人Hexastatin基因，并进行表达、纯化及体外的活性实验，为人Hexastatin进一步深入研究提供理论依据。方法优化并合成人Hexastatin基因，然后与pET28a表达载体连接，异丙基-β—D-硫代吡喃半乳糖苷（IPTG）诱导表达，并优化诱导表达的条件。在超声破碎细菌和包涵体后，用Ni-NTA层析柱纯化蛋白，用十二烷基硫酸钠一聚丙烯酰胺凝胶（SDS—PAGE）和Western Blot分析，通过四甲基偶氮唑盐（MTT）法分析肿瘤的抑制作用。结果得到pET28a．Hexastatin表达质粒，在BL21菌体中高效表达，表达的可溶性Hexastatin蛋白占总蛋白量的45．1％，纯化后的人Hexastatin蛋白的纯度可达90％，质量浓度为80μg/ml；Western Blot分析证实表达蛋白为目的蛋白；人Hexastatin蛋白对肿瘤细胞C6、人乳腺腺瘤细胞（MCF．7）以及人血管内皮细胞（HMEC）的生长有明显的抑制作用，抑制率分别为72．9％±3．6％、48．8％±2．9％、52．7％±2．5％。结论成功地表达了优化的人Hexastatin蛋白，证实人Hexastatin蛋白对肿瘤细胞的抑制作用，为肿瘤的抗血管疗法提供了新途径。Objective TO optimize human Hexastatin gene, to express, purify protein and conduct activity experimental research, and to provide a theoretical basis for further study of Hexastatin. Methods Human Hexastatin gene was optimized and synthesized. It was connected to the pET28a expression vector, induced to express by isopropyl β-D-1-thiogalactopyranoside（IPTG）, and optimized induction conditions. After the uhrasonication of bacterial cells and inclusion bodies, the recombinant fusion protein was purified with Ni-NTA chromatographic column, analyzed and identified by SDS-PAGE and Western Blot, and conduct activity experimental research in vitro by MTF. Results Constructed production was pET28a-Hexastatin expression plasmid. The human Hexastatin protein was expressed in E. coli BL21 the high level and accounted for 45.1% of the total bacterial protein. The purification of recombinant protein purified with Ni-NTA chromatographic column reached 90%, and the concentration was 80 μg/ml. Human Hexastatin protein can restrain the growth of C6, MCF-7 and human vascular endothelial cell （HMEC） cells, and inhibition ratio reach to 72.9%±3.6%, 48.8%± 2.9%, 52.7%±2.5%, respectively through MTT test. Conclusion The optimized human Hexastatin protein was expressed successfully, which confirmed the inhibition to tumour cells. It is a new way for anti-angiogenesis therapy of tumour.

关 键 词：Hexastatin蛋白 表达纯化 MTT活性检测 

分 类 号：R730.2[医药卫生—肿瘤]