摘要
目的原核表达旋毛虫抗肿瘤基因A200711,并纯化该蛋白,CCK-8检测其对H7402人肝癌细胞的生长抑制作用。方法 PCR获得A200711基因片段,构建重组表达载体pET-28a(+)-A200711,转入大肠杆菌Rosetta(DE3)。SDS-PAGE及Western blot检测IPTG诱导后重组菌,采用Ni-NTA亲合层析柱纯化并柱上复性目的蛋白。细胞计数试剂盒(Cell Counting Kit-8,CCK-8)检测A200711蛋白对H7402细胞的生长抑制作用。结果成功构建了原核表达载体pET-28a(+)-A200711,目的蛋白以包涵体形式在大肠杆菌中高效表达;SDS-PAGE及Western blot分析显示,相对分子量约22KD处出现目的蛋白条带,与理论值相符;经Ni-NTA亲合层析柱获得了高纯度可溶性的A200711蛋白;H7402细胞增殖抑制率与A200711蛋白浓度存在量效关系,同时随作用时间的延长增殖抑制率也明显增加。结论成功克隆、表达并纯化了旋毛虫A200711蛋白。A200711蛋白可抑制H7402细胞的增殖,且呈现时效和量效关系,本研究表明旋毛虫A200711蛋白作为潜在的抗肝癌生物制剂有进一步研究的价值。
Objective A200711 protein from trichinella spiralis was expressed and purified in E.coli Rosetta（DE3）.Growth inhibition of H7402 human liver cancer cells co-cultured with A200711 protein was detected by CCK-8.Methods In order to express A200711 gene in E.coli,the PCR product of 457bp A200711 fragment was cloned into prokaryotic expression vector pET-28a（＋） with BamHⅠand XhoⅠ,The recombinant pET-28a（＋）-A200711 then was transferred into E.coli Rosetta（DE3）,induced by IPTG,purified and renatured by Ni-NTA affinity column chromatography,detected by SDS-PAGE and Western blot.Growth inhibition of H7402 cells treated with the purified A200711 protein was analysised by Cell Counting Kit-8.Results Prokaryotic expression vector of pET-28a（＋）-A200711 was constructed successfully,the protein were expressed as inclusion bodies in E.coli after induction with IPTG;SDS-PAGE and Western blot analysis detected the protein at molecular weight of about 22 kD.High-purity soluble A200711 protein were harvested by Ni-NTA affinity chromatography;CCK-8 assay have revealed that A200711 had the inhibitory effect on cell proliferation（P〈0.05）.Conclusion Trichinella A200711 protein was expressed and purified successfully.These results demonstrate that A200711 had the inhibitory effect on H7402 cells proliferation and valuable biologics in againsting hepatoma.
出处
《中国实验诊断学》
2012年第2期194-197,共4页
Chinese Journal of Laboratory Diagnosis
基金
国家自然科学基金资助(30972177)
