CAJ | 학술논문

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-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80 μg/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.
목적 우화인Hexastatin기인,병진행표체、순화급체외적활성실험,위인Hexastatin진일보심입연구제공이론의거.방법 우화병합성인Hexastatin기인,연후여pET28a표체재체련접,이병기-β-D-류대필남반유당감(IPTG)유도표체,병우화유도표체적조건.재초성파쇄세균화포함체후,용Ni-NTA층석주순화단백,용십이완기류산납-취병희선알응효(SDS-PAGE)화Western Blot분석,통과사갑기우담서염(MTT)법분석종류적억제작용.결과 득도pET28a-Hexastatin표체질립,재BL21균체중고효표체,표체적가용성Hexastatin단백점총단백량적45.1％,순화후적인Hexastatin단백적순도가체90％,질량농도위80 μg/ml；Western Blot분석증실표체단백위목적단백；인Hexastatin단백대종류세포C6、인유선선류세포(MCF-7)이급인혈관내피세포(HMEC)적생장유명현적억제작용,억제솔분별위72.9％±3.6％、48.8％±2.9％、52.7％±2.5％.결론 성공지표체료우화적인Hexastatin단백,증실인Hexastatin단백대종류세포적억제작용,위종류적항혈관요법제공료신도경.
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 ultrasonication 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 MTT.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.