Characterization Of Bomyx Mori Serine Protease142(BmSP142) Against Viral Infection

Insect innate immune system include cellular immunity and humoral immunity. Cellular immunity is to engulf the invading microbes mainly by the blood plasma cells in insects. Insect humoral immunity involves complicated interactions between host proteins and microgram proteins. Previous researches showed that serine proteases and its homologue play important roles in food digestion, embryonic development and innate immunity responses against pathogens. The silkworm genome contains 143 serine protease coding genes, including 51 serine protease coding genes and 92 serine protease homologue coding genes. However, the specific functions of these proteins in silkworm and their role mechanism are still unclear so far.The sequence and expression pattern analysis of BmSP142 was carried out in this study. BmSP142 was cloned into pGEX-5X-3 to produce pGEX-5X-3-BmSP142, which was introduced BL21 for the expression of recombinant protein GST-BmSP142. Furthermore, the soluble protein was purified from the lysates of BL21 Ecoli harboring pGEX-5X-3-BmSP142. and the serine protease activity of BmSP142 was detected in vitro. Using the recombinant expression protein BmSP142, we maked mouse polyclonal antibodies. Additionally, analysis of BmSP142 transcription phase and expression phase were performed by qPCR and Western blot analysis. Finally, some experiments were designed to study the specific role of BmSP142 against viral infection and propagation, which will lay the foundation for further understanding of silkworm innate immune system.The main results are as follows:(11) Bioinformatics analysis revealed that, the first 1-22 amino acid of BmSP142 is a signal peptide sequence. Moreover, BmSP142 contains Trypsin-like serine protease domain, which contains a unique serine protease triplet catalytic center H(His)-Asp(D)-Ser(S). qPCR results revealed that, BmSP142 was expressed exclusively in the silkworm midgut, and it was strongly expressed in the middle part of the midgut, compared with the low level in the anterior and posterior part.(12) We constructed the recombinant plasmid for the expression of BmSP142 fusion with GST tag, and soluble recombinant target protein was obtained by purification using ProteinIsoTM GST Resin. The purified protein was identified by mass spectrometry, and the protease activity of BmSP142 was examined using ELISA kit of serine protease in vitro. The result confirmed the serine protease activity of BmSP142 in vitro.(13) The transcripts of BmSP142 were significantly up-regulated at 24 h p.i. in BmBDV-resistant strains(798) inoculated with BmBDV and Bm NPV-resistant strains(NB) inoculated with BmNPV, but not in BmBDV-susceptible strains(306). Surprisingly, the transcripts of BmSP142 were significantly down-regulated at 12 h p.i. in BmNPV-susceptible strains(HUABA35) inoculated with Bm NPV compared with that of healthy silkworm. Interestingly, we found that the transcripts of BmSP142 were significantly increased both in BmBDV-resistant strains(798) silkworm and BmNPV-resistant strains(NB) silkworm when they were suffered to viral infection, respectively. Therefore, it was inferred that BmSP142 may be involved with defence response against viral infection, and it was likely to be an important cause of resistant strains silkworm resisting the attack of pathogen.(14) Purified BmSP142 was incubated with BmBDV and BmNPV respectively,which was used to subcutaneously inject into the larvae. 30 larvae in each group were cultivated at 25 ℃ for 48 h with fresh mulberry. Then the total genome of silkworm midgut was extracted and viral DNA was detected by qPCR to examine replication levels of viral DNA. The silkworms infected with virus untreated with purified BmSP142 were used as control. Compared with the control group, the viral DNA levels of incubation with BmSP142 were significantly lower than that of the control group. The results indicated that BmSP142 impared the infectious ability of BmBDV and BmNPV. Additionally, recombinant BmNPV treated with different concentrations of purified BmSP142 were used to infect BmN cells. After 24 hpi, fluorescence microscopy results showed that, the number of fluoresce signal obviously decreased in BmN cells as more purified BmSP142 was incubated with recombinant BmNPV. the overexpression of BmSP142 in BmN cells also inhibited viral infection and viral propagation.These above results indicated that BmSP142 was a serine protease, and it was immediately involved with impairment effect on vial infection and propagation in silkworm. These results are very helpful to disclose the roles and molecular mechanism of BmSP142 in silkworm innate immune response.