Expression Profiles And Function Analysis Of The Hemolymph Proteinase 6 From Antheraea Pernyi

Insect immune responses like prophenoloxidase(proPO)activation and Toll pathway initiation are mediated by serine proteinase and serpins.Hemolymph proteinases play important roles in physiological responses by controlling protease activities.In this study,we studied the biochemical functions of hemolymph proteinase 6 from Antheraea pernyi(ApHP6).We first constructed pET-28a-ApHP6 prokaryotic expression vector,then recombinant vector was expressed in Escherichia coli,SDS-PAGE and Western Blot analysis showed that the molecular mass of the mature protein was 39.6 kDa,which was consistent with prediction,indicating that the ApHP6 protein was successfully expressed.Soluble fusion proteins were purified under native conditions using an Ni-NTA column,and the interest protein with high purity was obtained.The acquired protein was employed as antigen to immunize a New Zealand white rabbit and ultimately the polyclonal antibody serum against ApHP6 was achieved.The polyclonal antibody serum against ApHP6 was used for subsequent functional study.QRT-PCR and Western blotting results showed that ApHP6 was widely expressed in epidermis,hemolymph,hemocytes,silk gland,fat body,midgut,and malpighian tubules,in larvae at the third day of the fifth instar stage,and particularly high expression in the hemolymph and hemocytes,with the lowest levels in the midgut.Larvae from the third day of the fifth instar stage were respectivily injected the heat-inactivated Escherichia coli,Micrococcus luteus,Beauveria bassiana,nuclear polyhedrosis virus and PBS as the negative control.The transcript and protein expression analysis of ApHP6 were performed by qRT-PCR or Westerrn Blot,the results suggested that the other three pathogenic microorganisms immune-challenged all can significantly effect the expression of ApHP6 mRNA and protein without E.coli,but the effect on the expression degree and time of ApHP6 were different.The purified ApHP6 protein was incubated with screened hemolymph and M.luteus,and used to test whether ApHP6 plays a role in proPO activation and POactivity,the results suggested that ApHP6 stimulates proPO activation in the hemolymph,but does not affect PO activity.We injected larvae with ApHP6 protein or BSA(as control)first,and then injected larvae with M.luteus 30 min later to stimulate the antimicrobial response.Quantitative real-time PCR analysis revealed a significant increase in mRNA levels of the antimicrobial peptides attactin,cecropin,gloverin,moricin,and proPO,the results indicated that ApHP6 protein could significantly promotion the transcription of antimicrobial peptides.After the injection of siRNA-ApHP6,expression levels of antibacterial peptide and proPO had significantly lower.By RNA interference method,injection of ApHP6 dsRNA was decreased the expression of AMPs and proPO in A.pernyi.In conclusion,our results suggested that ApHP6 may play an important role in the innate immunity of A.pernyi,further analyticed the relationship between hemolymph proteinases and immunity of A.pernyi at the molecular level,and also have an important referential meaning for studing innate immunity regulation mechanism of A.pernyi and other lepidoptera insects.