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Expression of serine proteinase P186 of Arthrobotrys oligospora and analysis of its nematode-degrading activity

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Abstract

The nematode-trapping fungi possess a unique capability of predating and invading nematodes. As a representative nematode-trapping fungus, Arthrobotrys oligospora has been widely used to study the interactions between nematode-trapping fungi and their hosts. Serine proteinase is one of the important virulence factors during process of invasion of the nematode-trapping fungi into nematodes. In this study, using reverse transcription polymerase chain reaction, we amplified the gene sequence of serine proteinase 186 from A. oligospora, cloned it into pPIC9K vector and expressed it in the yeast Pichia pastoris. The expressed recombinant serine proteinase186 (reP186) was purified via Ni-affinity chromatography. The in vitro nematode-degrading activity of reP186 was analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis revealed that reP186 with molecular weight of 33 kDa was successfully obtained. ReP186 was capable of degrading a series of protein substrates including casein, gelatin, bovine serum albumin, denatured collagen and nematode cortical layer. The reP186 exhibited the maximal activity at pH 8.0 and 55 °C and was highly sensitive to the inhibitor, phenylmethanesulfonylfluoride. Treatment of Caenorhabditis elegans and Haemonchus contortus with reP186 for 12, 24 and 36 h, respectively, resulted in 62, 88 and 100 % of killing rates for C. elegans, and 52, 65 and 84 % of killing rates for H. contortus, respectively, indicating a relatively strong nematode-degrading bioactivity of reP186.

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Acknowledgments

We thank the field staff who provided the samples for this study. This work was supported by the International Science and Technology Cooperation Program of China (No.2014DFR31310), National Natural Science Foundation of China (31260601, 31360596, 31460654).

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Authors and Affiliations

  1. Key Laboratory of Control and Prevention of Animal Disease of Xinjiang Production & Construction Corps, College of Animal Science and Technology, Shihezi University, North Street No.4, Shihezi, 832003, Xinjiang, China

    Hailong Zhao, Jun Qiao, Qingling Meng, Shasha Gong, Cheng Chen, Tianli Liu, Lulu Tian & Chuangfu Chen

  2. State Key Lab of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, Gansu, China

    Xuepeng Cai & Jianxun Luo

Authors
  1. Hailong Zhao
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  2. Jun Qiao
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  3. Qingling Meng
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  4. Shasha Gong
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  5. Cheng Chen
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  6. Tianli Liu
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  7. Lulu Tian
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  8. Xuepeng Cai
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  9. Jianxun Luo
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  10. Chuangfu Chen
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Correspondence to Qingling Meng.

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This manuscript has not been simultaneously submitted for publication in another journal and been approved by all co-authors. The authors declare that they do not have any conflict of interest.

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The experiments were carried out in accordance with the guidelines issued by the Ethical Committee of Shihezi University.

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Zhao, H., Qiao, J., Meng, Q. et al. Expression of serine proteinase P186 of Arthrobotrys oligospora and analysis of its nematode-degrading activity. Antonie van Leeuwenhoek 108, 1485–1494 (2015). https://doi.org/10.1007/s10482-015-0595-z

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