苦瓜MAP30蛋白的原核表达及其生物活性研究

以天然苦瓜基因组为模板PCR扩增去前导肽后成熟的MAP30蛋白基因，克隆至可诱导表达载体pET28a中。将含MAP30基因的表达载体pET28a-MAP30转化至E. coli Rostta（DE3）中并通过IPTG诱导表达。经聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白杂交（Western blot）以及液相色谱-质谱（LC-MS）对表达的重组MAP30蛋白进行鉴定，并通过镍柱亲和层析纯化。将pUC19质粒与不同浓度的纯化后的重组MAP30蛋白孵育，分析其切割DNA的活性。同时将纯化后的重组MAP30蛋白体外作用于人乳腺癌细胞（MCF-7），采用MTT、AO/PI双染等方法进行抗肿瘤活性分析。实验结果表明纯化后的蛋白经质谱鉴定和Western blot分析，目的蛋白成功地与His-tag融合表达。首次发现大肠杆菌异源表达的重组MAP30蛋白同天然蛋白一样可以切割超螺旋DNA活性。MTT、AO/PI双染结果证实重组MAP30体外可诱导MCF-7细胞发生凋亡。通过基因工程技术大量制备MAP30蛋白，进一步研究其体外生物学活性，为以后的临床应用奠定基础。

Prokaryotic Expression of MAP30 from Momordica charantia and Its Biological Activity

Mature map30(signal peptide sequence excluded) was amplified from the genome of bitter melon and inserted on the expression vector pET28a. The final expression vector pET28a-map30 was transformed into E. coli Rostta (DE3) for MAP30 expression. The recombinant MAP30 was identified by SDS-PAGE, Western blotting and LC-MS. After purification by nickel-affinity chromatography, The DNA-cleaving activity was analyzed by electrophoresis after supercoiled plasmid pUC19 as substrate treated with MAP30 of various concentrations. Meanwhile MAP30 was used to treat MCF-7 human breast tumor cells, MTT, DNA Ladder, AO/PI double-stained is used to detect the anti-tumor activity. Results show that map30 gene was successfully expressed in E. coli Rostta (DE3)and been identified by LC-MS and Western blot using His-tag mAb. First discovered through Escherichia coli heterologous expression of recombinant MAP30 like natural MAP30 protein also exert the activity conversion supercoiled plasmid pUC19 to relaxed or linear forms. The cytotoxicity assay of recombinant MAP30 showed that it exhibited dose-dependent and time-dependent inhibition to MCF-7, AO/PI double-stained displayed typical of apoptosis. It through genetic engineering technology preparation MAP30 protein to further study of its biological activities in vitro and laid important foundation for the future clinical application.