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  

Screening and Analysis of Dipeptidyl Peptidase Ⅳ from Microbial Metagenomic Library in the Rumen of Dairy Cows

作　　者：赵静雯[1,2] 王加启[1] 赵圣国[1] 孙鹏[1] 卜登攀[1] 胡小丽[1] 卢玉飞[1] 王丹丹[1] 金迪[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所/动物营养学国家重点实验室,北京100193 [2]内蒙古民族大学动物科技学院,内蒙古通辽028000

出　　处：《中国农业科学》2013年第8期1687-1693,共7页Scientia Agricultura Sinica

基　　金：国家"973"计划项目(2011CB100804)

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 peptidasesⅣ,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HindⅢ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。[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-lV 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.mgl）. [ 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.

关 键 词：瘤胃微生物 FOSMID文库 二肽基肽酶Ⅳ 末端序列 酶活性 

分 类 号：S852.6[农业科学—基础兽医学]