苦瓜MAP30蛋白基因克隆、表达及其抗肿瘤活性研究

Cloning, Expression and the Anti-tumor Activity of Recombinant MAP30 in Escherichia coli

通过PCR技术,从苦瓜总DNA中扩增出编码MAP30成熟蛋白的基因,经测序鉴定后亚克隆到原核表达载体pET 30a中,构建成带有N端6His tag的融合表达载体。表达载体用CaCl2 介导的化学转化法转化E .coliBL2 1 (DE3) ,然后利用PCR筛选阳性克隆。工程菌经1mmol/LIPTG诱导4h实现高效表达,而且在30℃时融合蛋白表达量最高,约占菌体总蛋白5 6%。可溶性分析表明,该融合蛋白在大肠杆菌中主要以可溶的形式存在。重组蛋白通过Ni2 +鏊合亲和层析进行纯化,纯化蛋白占上清总蛋白37. 2 % ,发酵液产率为2 5 0mg/L。Westernblot分析表明,重组蛋白可与兔抗his tag多克隆抗体发生特异性反应。利用MTT法分析重组MAP30的细胞毒性,结果表明其对小鼠3T3和S 1 80肿瘤细胞株具有明显的抑制作用,ID50 分别约为5 0 μg/ml和3 0mg/ml,而对人正常胚肺二倍体WI 38细胞株的毒性极小。

MAP30(Momordica anti-HIV protein of 30kDa)is a 30kDa, single-stranded, type-I ribosome inactivating protein purified from Momordica charantia(bitter melon). The gene, which encodes the mature MAP30 protein, was amplified by PCR from the total DNA of bitter melon. After sequencing analysis, it was subcloned into the vector pET-30a to construct the expression vector pET-30a-MAP30 with the N-terminal 6his-tag. The vector was transformed into E. coli BL21(DE3)by calcium chloride transformation method, and the recombinant was identified by PCR analysis. The recombinant MAP30 was expressed mainly in soluble form with 1mmol/L IPTG induction in E. coli, and the amount reached highest at 30℃. According to the N-terminal fusion 6his-tag, the recombinant protein was purified by affinity chromatography. Western blot analysis indicated that the recombinant MAP30 had the antigenicity to rabbit anti-his-tag polyclonal antibody. The cytotoxicity assay of recombinant MAP30 also showed that it exhibited dose-dependent inhibition to 3T3 and S-180 lines, and the dose required for 50% inhibition (ID 50) were 50μg/ml and 3 0mg/ml, respectively. However, little cytotoxic effect on WI-38 was found under the same assay condition. The results demonstrate that the recombinant MAP30 expressed in E. coli is active to tumor cells, it provides an abundant source of homogeneous material for the study of its other biological property and further development of genetic engineering products.