Studies on the Antioxidative Potential of the Freeze-Dry Extract (Tsan-Ron-Bau-Yuan) of Fruits and Vegetables, DNA Vaccine Against Mycoplasma hyopneumoniae

• Main Authors: Hsiao-Ning Wang, 王筱寧
• Other Authors: 宣大衛
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/61166996234034868315

碩士 === 國立中山大學 === 生物科學系研究所 === 88 === The oxidative damage to DNA, protein and lipid, may be accumulated and play a role in the process of human cell aging. Oxidative stress may be due to the aerobic respiration and ozone-induced radiation which result in reactive oxygen species known as free radicals. Therefore, the antioxidant and free radical scanvenger which may reduce the oxidative damage, are of great interest, both in academic research and in the business world. The present study aims to evaluate the antioxidative potential of a very unique vegetable-fruit extract (Tsan-Ron-Bau-Yuan). The extract consists of over forty domestic vegetables and fruits, without using chemical fertilizers and pesticides during their entire growth period, is produced through a sophisticated freeze-dry technology. The anti-hydroxyl radical antioxidative potential were evaluated using the following four methods: (1) the plasmid DNA (2) the protein (3) the cell line and (4) the red blood cell . The results clearly demonstrat that the aqueous fraction of this extract can remarkably reduce the oxidative damage as evidenced by the DNA and protein model. In addition, the susceptibility of human red cells to oxidative stress can also be alleviated to some extents based on the RBC deformability studies. The efficacy of protection of the oxidative damage mediated by .OH generated by the Fenton’s reaction was in the order of : aquaeous extract >100% ethanol extract > ethanol/ethylacetatee extract > 50% ethanol extract. Conversely, no significant protection to the action of hydrogen peroxide was observed in the cell line. Lysozyme which is ubiquitous among various natural products has negligible contribution as an antioxidant as revealed in the RBC deformability tests. Swine enzootic pneumoniae （SEP）, is a disease caused by Mycoplasma hyopneumoniae infection, and usually lead to considerable economic loss. Though extensive research during the past years, the molecular mechanism about the infective pathway of M. hyopneumoniae was still elusive. The membrane proteins of this microorganism were considered as critical adhesion molecules and therefore are potential candidates for vaccine development. Through immunoscreening, we had isolated five recombinant phage clones expressing 10 kDa, 32 kDa, 36 kDa, 42 kDa and 60 kDa antigen proteins from the lEMBL3 library of Mycoplasma hyopneumoniae. The clone carrying the P42 gene was subcloned and further characterized as a heat shock protein gene. In the present study, the heat-shock protein gene encoding a 42 kDa/ 65 kDa protein（P42/P65）was cloned into the mammalian expression vector pcDNA3 and obtained plasmid pcDNA42. The immune response induced by pcDNA42 was evaluated in mice. The IgG titer was obviously elevated during the first eight weeks with the IgG1 titer slightly higher than IgG2a. However, the IgM titer was not changed signifcantly. Studies on the macrophage activity and T cell cytotoxity were still undergoning.