权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

魚類体表の非特異的防御因子-鰓及び腸管の抗菌ペプチドの網羅的解析

鱼体表非特异性保护因素——鳃及肠道抗菌肽综合分析

基本信息

批准号：
17658087
负责人：
飯島憲章
金额：
$ 2.24万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Exploratory Research
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

[目的]本研究では、マダイの鰓から構成的あるいは誘導的に合成・分泌される抗菌ペプチドを電荷・疎水性・分子量に基づく多次元クロマトと質量分析法を併用して網羅的に解析することを目的とした。[方法及び結果]未処理区及びLPS処理区(0.51mg LPS/100g魚体重)のマダイ未成魚(魚体重160g前後)よりそれぞれ鰓組織を採取し、液体窒素下で粉末状にした後、粗抽出液を調製した。次いで、Sep-PakC18カートリッジで脱塩した後、 SP-Sephadex C25により強酸性、弱塩基性、強塩基性の3分画を得た。この中で最も抗菌活性の高い強塩基性画分について、ゲル濾過HPLCにより分子量分画し、Fraction I〜IVを得た。そのうち、高い抗菌活性を示したFraction II、IIIついて2段階の逆相HPLCによりペプチド分画を行った。逆相HPLCの溶出画分から抗菌活性の高いピークを選択し、ESI-MSで分子量を推定した。なお、各精製段階における抗菌活性の測定には、B. sutilisを用いMBC(Mimimal Bactericidal Concentration)を求めた。ゲル濾過HPLC、逆相HPLCで得られた画分については、E. coliを用いたマイクロプレート法とB. sutilisを用いたコロニーカウント法を併用し、抗菌活性を測定した。未処理区及びLPS処理区の鰓粗抽出液から、SP-sephadex C-25、ゲル濾過HPLC、逆相HPLCにより抗菌ペプチドを精製した後、ESI-MSにより、平均分子量を解析した。その結果、未処理区のゲル濾過HPLC溶出画分Fraction. IIにおいて3種類(M. W.約3000〜7000)、またFraction.IIIに7種類の抗菌ベプチド(M. W.約1800〜4500)の存在を確認した。またLPS処理区のゲル濾過HPLC溶出画分Fraction.IIでは、未処理区で確認された抗菌ペプチドに加え、さらに2種類(M. W.約3000〜4000)が、またFraction.IIIからは5種類(M. W.約1800〜7000)の抗菌ペプチドの存在が確認された。これらは誘導型抗菌ペプチドである可能性が示唆される。現在、計14種類の抗菌ペプチドについてN末端配列を解析中である。

[objective] in this study, the synthesis and secretion induced by the molecular weight of water-borne molecular weight, molecular weight, molecular weight and molecular weight were analyzed. [methods and results] the untreated area and the LPS physiological area (0.51mg LPS/100g weight) were not successful (before and after the body weight was 160g). The tissue samples were collected from the tissue, the liquid asphyxiate was added to the powder, and the crude extract was extracted. The SP-Sephadex C25's strong acid, weak basic and strong basic properties were obtained after the secondary temperature, Sep-PakC18 temperature and temperature. The most potent antimicrobial activity was obtained by HPLC, molecular weight analysis and Fraction I~IV analysis. The anti-bacterial activity and high antibacterial activity of the two segments of Fraction II and III show that the inverse phase of HPLC is different from each other. Reverse phase HPLC stripping separation, antibacterial activity, high molecular weight, ESI-MS molecular weight, presumptive molecular weight. The antimicrobial activity of each segment was determined by MBC (Mimimal Bactericidal Concentration). B. the antibacterial activity of each segment was determined by MBC (Mimimal Bactericidal Concentration). By means of HPLC and reverse phase HPLC, the determination of antimicrobial activity was performed in E. coli was used to determine the antimicrobial activity. In the untreated and LPS regions, the crude extract was extracted, SP-sephadex C Mel 25, the crude extract was extracted by HPLC, the reverse phase of ESI-MS was determined by HPLC, the antimicrobial activity was purified, and the average molecular weight was analyzed. The results showed that the Fraction was extracted from the untreated area by HPLC. II is classified into three categories (M. W. About 3000,7000,000,000,000,000,000,000,000,000,7000,000,7000,000,7000,000,7000,7000,000,7000,7000,000,7000,7000,7000,7000,000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000,7000, About 1800 to 4500) there is a confirmation. Through HPLC, the LPS processing area can be extracted into several categories, such as Fraction.II, antimicrobial activity, and so on. About 3000-4000), and other Fraction.III species (M.W.) About 1800-7000) there is a confirmation that there is an antimicrobial agent. The possibility of antimicrobial activity in the form of antimicrobial agents has been indicated. At present, there are 14 kinds of antibacterials in the analysis of N-terminal antibacterials.