Study On Cloning And Expressing Of Two Genes From Blear Pear: MAP30 And α Momorcharin

The bitter gourd is the vegetables, and medicines too. MAP30 and alpha momorcharin both are the ribosome-inactivating proteins (RIPs) in the bitter gourd , which is a cytotoxin ,antitumor and resistant-disease of plant protein existing in the plant kingdom extensively . After MAP30 protein had isolated from bitter gourd, it is proved anti-HIV, anti-virus, anti-tumor activities and little cytotoxin to normal cells. Recombinant MAP30 has full biological actives as well as his counterpart from his original plant source. Alpha momorcharin has inhibitory effect on DNA, RNA and protein synthesis of NKM cell and also selectively injured choriocarcinoma and melanoma. Thereby, they have bright application future in agriculture and clinical medicine. So we cloned these two protein genes and then studied on their expressing in Ecoli. This research includes the following several respects mainly:First, A method by using PCR for rapid identification of positive clone was eveloped. Result showed this screening method could be used to detect positive colonie s from samples of bacterial, purified plasmid, liquid culture, and liquid culture treated with mixture of phenol/ Chloroform. The result from liquid-culture-treated-PCR (LCT-PCR) is very close to that of by plasmid-PCR. LCT-PCR is reliable and much easier to used than plasmid-PCR; therefore the LCT-PCR can be used for clone screening during the molecular cloning.Second, MAP30 and Alpha momorcharin both had successfully expressed in Ecoli in the way of solubility, though their genes have many rare usage coden of bacteria. Recombinant MAP30 has antimicrobial activity against Staphylococcus aureus, Enterobacter cloacae and Staphylococcus hominis.Last, constructed plant- expressing vectors of MAP30 and chitinase genes, which will be used for improving ability of peanut to resistant virus. These are the basis of construction of bivalent genes of plant -expressing vector.