奶牛瘤胃微生物元基因组文库中二肽基肽酶Ⅳ的筛选与分析

doi:10.3864/j.issn.0578-1752.2013.08.019

摘要/Abstract

摘要： 【目的】研究奶牛瘤胃内参与蛋白质降解过程中二肽基肽酶Ⅳ（dipeptidyl peptidases Ⅳ，DPP-Ⅳ）的序列特征和酶学性质。【方法】从奶牛瘤胃微生物Fosmid文库中挑选17 664个克隆，利用dpp-Ⅳ简并引物筛选含dpp-Ⅳ的阳性克隆。提取阳性克隆的质粒，用限制性内切酶Hind Ⅲ进行酶切。PCR扩增含dpp-Ⅳ目的片段，并克隆测序。对阳性克隆的Fosmid末端进行测序。提取阳性克隆粗酶液，利用底物Gly-Pro-pNA检测肽酶活性。【结果】筛选后获得10个含有dpp-Ⅳ的阳性克隆（命名为DP1—DP10）。Fosmid末端序列经BLASTX比对后发现78%的序列可以与数据库的已知序列相匹配，但相似度变化较大（44%—94%）。DPP-Ⅳ序列经BioEdit比对后，发现均含有N端保守区域（DWVYEEE）和C端催化区域（GWSYGG），经BLASTP比对发现阳性克隆的DPP-Ⅳ分别与Cyclobacterium marinum（43%）、Capnocytophaga sp.（63%）、Prevotella ruminicola 23（66%）和Solitalea canadensis（50%）的DPP-Ⅳ相似度高。酶活力检测发现DP7肽酶活力最高，为6.88 U•mg-1。【结论】从瘤胃微生物Fosmid文库中获得了10个含有dpp-Ⅳ的阳性克隆，它们具有不同的dpp-Ⅳ序列特征和肽酶活性。

关键词: 瘤胃微生物 , Fosmid文库 , 二肽基肽酶Ⅳ , 末端序列 , 酶活性

Abstract: 【Objective】The purpose of this study was to reveal the characteristics of dipeptidyl peptidase Ⅳ (DPP-Ⅳ) sequences and enzymatic activity during the process of protein degradation in the rumen of dairy cows.【Method】The dpp-Ⅳ degenerate primers were used to screen rumen microbial Fosmid library including 17664 clones. The plasmids were extracted from the positive clones and digested by Hind Ⅲ. The dpp-Ⅳ sequences were obtained by PCR, cloning and sequencing. The Fosmid end sequences and DPP-Ⅳ sequences of the positive clones were analyzed by BLASTX and BLASTP, respectively. The peptidase activity from the positive clones was measured using Gly-Pro-pNA as a substrate.【Result】Ten positive clones named DP1-DP10 containing dpp-Ⅳ fragment were obtained. 78% of the Fosmid end sequences could match with the known genes (similarity 44%-94%). DPP-Ⅳ sequences contained N-conservative region (DWVYEEE) and C-catalytic domain (GWSYGG). DPP-Ⅳ sequences matched to Cyclobacterium marinum (43%), Capnocytophaga sp. (63%), Prevotella ruminicola 23 (66%) and Solitalea canadensis (50%). The activity of DP7 peptidase is the highest (6.88 U•mg-1).【Conclusion】Ten positive clones obtained from rumen microbial Fosmid library had different sequence characteristics and peptidase activity. This study implies that the substitution of some DPP-Ⅳ AA may affect the enzyme activity.

Key words: ruminal microbes , fosmid library , dipeptidyl peptidases Ⅳ , end sequence , enzyme activity

赵静雯, 王加启, 赵圣国, 孙鹏, 卜登攀, 胡小丽, 卢玉飞, 王丹丹, 金迪. 奶牛瘤胃微生物元基因组文库中二肽基肽酶Ⅳ的筛选与分析[J]. 中国农业科学, 2013, 46(8): 1687-1693.

ZHAO Jing-Wen, WANG Jia-Qi, ZHAO Sheng-Guo, SUN Peng, BU Deng-Pan, HU Xiao-Li, LU Yu-Fei, WANG Dan-Dan, JIN Di. Screening and Analysis of Dipeptidyl Peptidase Ⅳ from Microbial Metagenomic Library in the Rumen of Dairy Cows[J]. Scientia Agricultura Sinica, 2013, 46(8): 1687-1693.

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