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Cloning and functional identification of pmKPI cDNA in Poecilobdella manillensis

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Abstract

Background

Kazal-type serine protease inhibitors play a role in physiological processes such as blood coagulation and fibrinolysis. The amino acid residues at the P1 site are different, and they inhibit different types of proteases. The inhibitory mechanism of the protease in the salivary glands of Poecilobdella manillensis is still unclear.

Methods and results

Based on cloning, prokaryotic expression and bioinformatics analysis, we studied the role of Kazal-type serine protease inhibitors in P. manillensis and analyzed their expression by quantitative real-time PCR. The results suggested that the recombinant protein was successfully expressed in the supernatant when a prokaryotic expression vector was constructed and induced with 0.2 mmol/L IPTG at 37 °C for 4 h, and the enzymatic activity was determined. The mature protein encodes 91 amino acids and has a relative molecular weight of 9929.32 Da, and after removing the signal peptide, the theoretical isoelectric point was 8.79. It is an unstable protein without a transmembrane domain. The mature protein contains two Kazal-type domains, in which all P1 residues are Lys, consisting of an α helix and three antiparallel β sheets. The upregulated expression of the mRNA was induced after a meal was provided, and the results showed an increasing and then decreasing trend.

Conclusions

Taken together, the results indicate that mature proteins from P. manillensis inhibit thrombin activity, laying the foundation for the subsequent in-depth study of the function of genes encoding Kazal-type serine protease inhibitors.

Highlights

  • The pmKPI cDNA from salivary glands of Poecilobdella manillensis was cloned and expressed.

  • pmKPI proteins can inhibit thrombin activity.

  • pmKPI mRNA expression was upregulated after a meal was provided, showing an increasing and then decreasing trend.

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Abbreviations

p. manillensis :

Poecilobdella manillensis

pmKPI :

Poecilobdella manillensis Kazal-type serine protease inhibitors

KPI :

Kazal-type serine protease inhibitors

SPIs :

Serine protease inhibitors

Qpcr :

Quantitative Real-time-PCR

H. medicinalis :

Hirudo medicinalis

H. nipponica :

Hirudo nipponica

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Authors and Affiliations

  1. Department of Marine Science and Technology, School of Marine and Bioengineering, Yancheng Institute of Technology, 224051, Yancheng, Jiangsu, PR China

    Gui-Yan Shao, Qing-Qing Tian, Wen-Bo Li & Fei Liu

  2. School of Biological Sciences, Guizhou Education University, 550018, Guiyang, Guizhou, PR China

    Gui-Yan Shao, Qing-Qing Tian, Su-Yan Wang & Bo-Xing Cheng

  3. Institute of Animal Husbandry and Veterinary Science, Guizhou Academy of Agricultural Sciences, Guiyang, Guizhou, China

    Yu-Xi Lu

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Contributions

All authors contributed to the study’s conception and design. The research invested, conceived, and designed was studied by Fei Liu, Boxing Cheng, and Yuxi Lu, Material preparation, data collection, and analysis were performed by Guiyan Shao, Qingqing Tian, Wen-Bo Li, and Suyan Wang. The first draft of the manuscript was written by Guiyan Shao, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Fei Liu or Bo-Xing Cheng.

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This work was supported by the National Natural Science Foundation of China (Grant nos. 82073968), Guizhou Provincial Education Department Young Science and Technology Talents Development Project (KY [2021] 233), the Yancheng Institute of Technology Talent Introduction Project (XJR2021023), Major Program of Natural Science Research of Higher Education Institutions of Jiangsu Province (22KJA360011), Doctoral program of Guizhou Normal University (2020BS004) and youth foud of academy of agricultural sciences in Guizhou Province (2018(46)).

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Shao, GY., Tian, QQ., Li, WB. et al. Cloning and functional identification of pmKPI cDNA in Poecilobdella manillensis. Mol Biol Rep 50, 299–308 (2023). https://doi.org/10.1007/s11033-022-07944-7

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