Functional Study And Analysis Of Bmserpin-15 In Domestic Silkworm, Bombyx Mori

Serine protease inhibitors(SPIs)play a key role in physiological responses by controlling protease activities.Serpin family is one important family of the SPIs.In the present,the serpin family has been studied in depth relatively,and the informed research indicated that serpin family play a key role in AMPs synthesis and hemolymph melanization in humoral immune.In this study,we studied the expression profiles and biochemical functions of serpin-15 in humoral immune of Bombyx mori,an serpin,from Bombyx mori(Bmserpin-15).We first constructed pET-28a-Bmserpin-15 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 45.4 kDa,which was consistent with prediction,indicating that the Bmserpin-15 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 Bmserpin-15 was achieved.The polyclonal antibody serum against Bmserpin-15 was used for subsequent functional study.QRT-PCR and Western blotting results showed that Bmserpin-15 was widely expressed in integument,silk gland,fat body,midgut,malpighian tubules,testis,ovary,hemolymph and hemocytes in larvae at the third day of the fifth instar stage,and particularly high expression in the silk gland and fat body,with the lowest levels in the hemocytes.Larvae from the third day of the fifth instar stage were respectivily injected the heat-inactivated Escherichia coli,Micrococcus luteus,Beauveria bassiana,B.mori nuclear polyhedrosis virus and PBS as the negative control.The transcript and protein expression analysis of Bmserpin-15 were performed by qRT-PCR or Westerrn Blot,the results suggested that the other three pathogenic microorganisms immune-challenged all can significantly upregulate or downregulate the expressionsof Bmserpin-15 mRNA and protein without E.coli,but the up-regulated or down-regulated expression degree and time of Bmserpin-15 were different.The purified Bmserpin-15 protein was incubated with screened hemolymph and M.luteus,and used to test whether Bmserpin-15 plays a role in proPO activation and PO activity,the results suggested that Bmserpin-15 inhibits proPO activation in the hemolymph,but does not affect PO activity.We injected larvae with Bmserpin-15 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 decrease in mRNA levels of the antimicrobial peptides cecropinD,gloverin2 and moricin,the results indicated that Bmserpin-15 protein could significantly inhibit the transcription of antimicrobial peptides.In conclusion,our results suggested that Bmserpin-15 may play an important role in the innate immunity of B.mori,further analyticed the relationship between serpin and immunity of B.mori at the molecular level,and also have an important referential meaning for studing innate immunity regulation mechanism of B.mori and other lepidoptera insects.