Indentification and modification of two new bacteriocins from database that may have the anticancer potential.

• Main Author: 馮政凱
• Other Authors: 林志侯
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/52759588322145137534

碩士 === 國立清華大學 === 分子醫學研究所 === 101 === Recently, scientists pay attention to Antimicrobial peptides(AMPs) due to the fact that AMPs is not only antibacteria , but anticancer and can be used in drug as well. Not produced from artificial methods, the AMPs are made by an organism. Therefore, the AMPs do not have drug resistance. The AMPs made from bacteria that is called bacteriocin. we screen a potential bacteriocin and have a test about this bacteriocin in this study. Analyzing characteristics and structures of known bacteriocins, investigated by a recent study from the lab, we screen a potential bacteriocin from database like NCBI and PFAM. In summary, we find two genes are coding about bacteriocin. The genetic codes of potential bacteriocin are BD_21 and BD_22, which exist in bacteria like Lactobacillus casei. We produce BD21 and BD22 from E. coli by gene clonig system and test the activity . The result of activity shows that BD21 and BD22 have no activity after treated with bacteria. In addition, we do not find any activity of BD21 and BD22 after mixing BD21 and BD22 in the same proportion. Consequently, we decide to modify the peptides from amino acid sequence .Owing to the fact that bacteria and some cell membrane surface of cancer cells have negative charges, we change the amino acid which is negative of BD21 and BD22, Asp and Glu, to Ser which is no electricity, by a genetic technology. This modification can increase the interaction between BD21 and cell membrane or BD22 and cell membrane. After the modification, the antibacterial ability of BD21m and BD22m has no changes.(m means after modification) On the other hand, we test BD21, BD22, BD21m and BD22m by MTT assay in order to know an influence of cell with these four potential bacteriocins. According to MTT assay, we find that BD22 and BD22m, can inhibit growth of the human colon adenocarcinoma cell,SW480. In particular, BD22m has the great inhibition to SW480, however, BD21 and BD21m have no influence. Furthermore, we find that BD22 and BD22m can’t inhibit growth of human normal fibroblast,HFW. Analyzing stained PI and stained Annexin V、PI by flow cytometry in order to observe cell cyle and whether the cell death is apoptosis or necrosis, we discover that BD22 and BD22m are apoptosis and some part of necrosis for SW480. Hence, a future work will focus on apoptosis mechanism and improve the activity of BD22.