关键词: 苦瓜, 克隆, 原核表达, MAP30, 活性片段

Abstract: BACKGROUND ＆ AIM: MAP30 (Momordica anti_HIV protein of 30 kDa) is a 30 kDa, a single_stranded ribosome inactivating protein purified from the fruits and seeds of bitter melon (Momordica charantia). In order to fully understand the relationship between gene function and domain of MAP30, the active gene fragment of this protein must be cloned and expressed precedingly. MATERIALS AND METHODS: Two PCR primers, one for amplifying coding gene for T8_N263 amino acids of mature MAP30 (large fragment), the other for amplifying coding gene for T8_S194 amino acids of mature MAP30 (small fragment) were designed firstly. The objective genes were amplified by PCR from the DNA of bitter melon, and then cloned into the prokaryotic expression vector pET_28a to construct the expression vectors, containing 6 His_tag in C_terminal. After sequencing analysis, the vectors were transformed into E. coli RosettaTM(DE3)pLysS by calcium chloride transformation method to obtain the recombinants respectively. The recombinants were induced by IPTG to express the recombinant proteins, which were analyzed by Western blot. RESULTS: Western blot analysis indicated that the recombinant proteins demonstrated antigenicity to rabbit anti_His_tag polyclona antibody. CONCLUSION: The result demonstrated that both of the two constructed expression vectors could express the expected recombinant proteins in E. coli RosettaTM(DE3)pLysS.

Key words: bitter melon, cloning, prokaryotic expression, MAP30, active fragment