Zhang Li-li, Ding Qian, Zhan Jin-biao
Department of Biochemistry and Genetics, College of Medicine, Zhejiang University, Hangzhou 310058, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2010 May;39(3):264-71. doi: 10.3785/j.issn.1008-9292.2010.03.009.
To clone and produce ribosome inactivating protein MAP30 from the seeds of Momordica charantia L(bitter melon), and to evaluate the biological activity of the recombinant protein.
The DNA sequence encoding MAP30 was cloned from the fresh seeds of Momordica charantia by PCR, the target DNA fragments were sequenced after T-A cloning. The expression plasmid was constructed by inserting the MAP30 fragment into vector pET30a. MAP30 was expressed in E.coli by addition of IPTG into final concentration of 1.0 mmol/L. The recombinant MAP30 was identified by SDS-PAGE, and the biological activity of MAP30 protein was evaluated by using MTT assay in cancer cells and normal cells following fluid-phase endocytosis.
The nucleotide and amino acid sequences of the cloned MAP30 were identical with those of reported MAP30. The solubility of recombinant protein was analyzed by SDS-PAGE, and the MAP30 was mainly produced in soluble form. The recombinant MAP30 showed a greater cytotoxicity to cancer cells than that to normal cells.
The gene of MAP30 has been successfully cloned.The recombinant MAP30 protein expressed by E.coli is bioactive.
从苦瓜种子中克隆并制备核糖体失活蛋白MAP30，并评估重组蛋白的生物活性。
通过PCR从新鲜苦瓜种子中克隆编码MAP30的DNA序列，T-A克隆后对目标DNA片段进行测序。将MAP30片段插入载体pET30a构建表达质粒。加入终浓度为1.0 mmol/L的IPTG在大肠杆菌中表达MAP30。通过SDS-PAGE鉴定重组MAP30，并在液相内吞作用后使用MTT法在癌细胞和正常细胞中评估MAP30蛋白的生物活性。
克隆的MAP30的核苷酸和氨基酸序列与报道的MAP30相同。通过SDS-PAGE分析重组蛋白的溶解性，MAP30主要以可溶性形式产生。重组MAP30对癌细胞的细胞毒性大于对正常细胞的细胞毒性。
成功克隆了MAP30基因。大肠杆菌表达的重组MAP30蛋白具有生物活性。
