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Toxins 2015, 7(8), 2888-2905; doi:10.3390/toxins7082888

Molecular Cloning and Functional Studies of Two Kazal-Type Serine Protease Inhibitors Specifically Expressed by Nasonia vitripennis Venom Apparatus

1,2
,
2
,
1,2
and
2,*
1
College of Life Science, Anhui Agricultural University, Hefei 230036, China
2
State Key Laboratory of Rice Biology, Key Laboratory of Agricultural Entomology, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China
*
Author to whom correspondence should be addressed.
Academic Editors: Sokcheon Pak and Ren Lai
Received: 13 May 2015 / Revised: 29 June 2015 / Accepted: 27 July 2015 / Published: 4 August 2015
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Abstract

Two cDNA sequences of Kazal-type serine protease inhibitors (KSPIs) in Nasonia vitripennis, NvKSPI-1 and NvKSPI-2, were characterized and their open reading frames (ORFs) were 198 and 264 bp, respectively. Both NvKSPI-1 and NvKSPI-2 contained a typical Kazal-type domain. Real-time quantitative PCR (RT-qPCR) results revealed that NvKSPI-1 and NvKSPI-2 mRNAs were mostly detected specifically in the venom apparatus, while they were expressed at lower levels in the ovary and much lower levels in other tissues tested. In the venom apparatus, both NvKSPI-1 and NvKSPI-2 transcripts were highly expressed on the fourth day post eclosion and then declined gradually. The NvKSPI-1 and NvKSPI-2 genes were recombinantly expressed utilizing a pGEX-4T-2 vector, and the recombinant products fused with glutathione S-transferase were purified. Inhibition of recombinant GST-NvKSPI-1 and GST-NvKSPI-2 to three serine protease inhibitors (trypsin, chymotrypsin, and proteinase K) were tested and results showed that only NvKSPI-1 could inhibit the activity of trypsin. Meanwhile, we evaluated the influence of the recombinant GST-NvKSPI-1 and GST-NvKSPI-2 on the phenoloxidase (PO) activity and prophenoloxidase (PPO) activation of hemolymph from a host pupa, Musca domestica. Results showed PPO activation in host hemolymph was inhibited by both recombinant proteins; however, there was no significant inhibition on the PO activity. Our results suggested that NvKSPI-1 and NvKSPI-2 could inhibit PPO activation in host hemolymph and trypsin activity in vitro. View Full-Text
Keywords: Nasonia vitripennis; Kazal-type; serine protease inhibitors; humoral immunity Nasonia vitripennis; Kazal-type; serine protease inhibitors; humoral immunity
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MDPI and ACS Style

Qian, C.; Fang, Q.; Wang, L.; Ye, G.-Y. Molecular Cloning and Functional Studies of Two Kazal-Type Serine Protease Inhibitors Specifically Expressed by Nasonia vitripennis Venom Apparatus. Toxins 2015, 7, 2888-2905.

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