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Purification and characterization of Moschatin, a novel type Ⅰ ribosomeinactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin.against human melanoma cells

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Cell Research 2003年第5期13卷 369-374页

作者： HENG CHUAN XIA, FENG LI, ZHEN LI, Zu CHUAN ZHANG~* Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. State Key Laboratory of Reproductive Biology Institute of Zoology Chinese Academy of SciencesBeijing 100080 China Department of Veterinary Pathobiology University of Missouri-Columbia MO 65211 USA

A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin （Cucurbita moschata） has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of～29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC50 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin.Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

关键词： ribosome-inactivating protein (RIP) Moschatin immunotoxin.

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Construction, expression, purification and functional analysis of recombinant 6C6 immunotoxin.to human breast-tumor cells

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Science China(Life Sciences) 1999年第3期42卷 281-285页

作者：刘晖朱玉贤李■秋 College of Life Sciences Peking University 100871 Beijing China

The 28 ku membrane protein is usually over-expressl m human bnasl bmast cancer and other tumor cells. licould be a larget for tumor therapy . By using genetie engineermg teehmgues.a 606 immunotoxin.(sefv606-PE40) was construeted by joining the 606 single-chain antibdy (SeFv606) with the truncll Pseudonwnas exotoxin A (PE40), SeFv606 contains both the heavy and light-chnia variable domams of 606 monoelonal antibody. Which speeifieally ree-ognizes the 28 ku protein. The bacterial expression level og 606 imnmmotoxin is 3.3%. about 5.5 mg ml baeterial *** singlc-step llisTrap (Nr2 chelating) column chronnetogaphy, the reeombinant peptide was obtained with a purit of 33.2%.This baeterial espressed 606 immunotosin binuls to MDA-231 human breast-tumer ccll surfaee and kill these cells with a median lethal dosage of 92 ngnd.

关键词： breast cancer 6C6 monoclonal antibody single chain antibody PE40 immunotoxin.

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