Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (8): 1687-1693.doi: 10.3864/j.issn.0578-1752.2013.08.019

• ANIMAL SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

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

 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   

  1. 1.Institute of Animal Science, Chinese Academy of Agricultural Sciences/State Key Laboratory of Animal Nutrition, Beijing 100193
    2.College of Animal Science and Technology, Inner Mongolia University for the Nationalities, Tongliao 028000, Inner Mongolia
  • Received:2012-08-22 Online:2013-04-15 Published:2012-11-28

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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

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