The Function Of Bmserpin32 In Silkworm (Bombyx Mori)

As the arthropods that do not possess acquired immune system,insects depend on their advanced innate immune system to resist the infection of various pathogens during the long process of evolution.Innate immunity consists of humoral immunity and cellular immunity.Humoral immunity mainly consists of Toll pathway and melanization,which can be induced to produce antimicrobial peptides,agglutination and melanotic encapsulation to achieve the purpose of eliminating pathogens.And the completion of humoral immunity requires the participation of extracellular serine protease cascade system.The important point in these immune defense process is that the precise regulation of serine protease inhibitor serpin to the extracellular serine protease cascade system to ensure the successful operation of the whole defense system.Serpin plays an important regulatory role in the immune process.At present,the researches on serpin in lepidopteran insects are mainly concentrated on the tobacco hornworm,Manduca sexta.However,the silkworm,Bombyx mori,as a model organism of lepidopteran insects,the reports on its serpin function are comparatively rare.In this study,Bmserpin32 was used as a target to investigate the function of serpin in the immune process of silkworm.Our result will lay the foundation for the further research on the activation and regulation of the innate immune pathway in the silkworm,B.mori.The main results are as follows:1.Basic information analysis,cloning,prokaryotic expression and purification and antibody preparation of Bmserpin32Firstly,we used the bioinformatics software to analyze the basic information of Bmserpin32.The Bmserpin32 protein consists of 395 amino acid residues with a molecular weight of about 43.9 kDa and an isoelectric point of 4.76,and 20 amino acids near the N-terminal as its signal peptides.A phylogenetic tree was constructed with the amino acid sequence of Bmserpin32 and other serine protease inhibitors from13 species of insects.Bmserpin32 and Msserpin7 were closely related and clustered together.Further,the results of multiple sequence alignment showed that the sequence homology of Bmserpin32 and Msserpin7 reached 64%,with the same P1 site?Arginine?.These results suggest that Bmserpin32,may have similar functions as Msserpin7,and be involved in regulation of the immune response in the silkworm.Subsequently,we cloned Bmserpin32,which was then linked with the His-tagged p28 vector and transformed into the expression strain E.coli BL21?DE3?.After induction by IPTG at 16°C for 20 h,the cells were harvested,centrifuged after ultrasonication,and the supernatant protein was purified by nickel column affinity chromatography and eluted with different gradient concentration of imidazole.Recombinant Bmserpin32 protein was mainly eluted at 50 mM,100 mM,and 200 mM imidazole concentration.The collected eluent was concentrated by ultrafiltration,and further purified using HiLoad 16/600 superdex 75 pg to obtain a higher purity protein.The recombinant Bmserpin32 protein was confirmed by His-tag antibody,and 8-10 mg recombinant protein was used for preparation of Bmserpin32 polyclonal antibodies.2.Activity analysis of recombinant Bmserpin32 proteinTo analyze whether the recombinant Bmserpin32 protein obtained from prokaryotic expression possesses inhibitory activity,we used FITC-casein as a substrate to determine the inhibitory activity of Bmserpin32 against six commercial proteases:trypsin,chymotrypsin,elastase,protease K,subtilisin and papain.The results showed that Bmserpin32 had a significant inhibitory effect on trypsin activity.After treatment of Bmserpin32 protein at different temperature and pH,Bmserpin32still has high inhibitory activity against trypsin when the treatment temperature is20-60°C and the treatment pH is 6-10.Next the incubated mixture of Bmserpin32incubated with trypsin was detected by SDS-PAGE and western blot.It was found that Bmserpin32 inhibits the trypsin by forming a stable covalent complex with trypsin.After incubation of Bmserpin32 with trypsin,peptide fragmentation was detected by MALDI-TOF/TOF mass spectrometry and the molecular weight of identified peptide was consistent with that of the amino acid residue fragments behind Arg³5959 of Bmserpin32.This result indicated that the peptide bond between Arg³⁵⁹ and Ile³⁶⁰ was broken during the Bmserpin32 protein inhibited trypsin activity,and then Arg³⁵⁹ was combined with trypsin to form a covalent complex.To further verify the correctness of the active cleavage site,we designed specific primers to mutate Arg³⁵⁹ to Ala³⁵⁹ of Bmserpin32,and obtained the mutant R359A protein by prokaryotic expression,then determined its inhibitory activity against trypsin.The results showed that the mutant R359A protein did lose its trypsin inhibitory activity compared to the wild-type Bmserpin32 protein.3.Functional analysis of Bmserpin32The expression levels of Bmserpin32 in fat body,integument and hemocyte were measured after injecting E.coli and M.luteus to day-3 fifth instar larvae.Quantitative PCR results showed that the expression level of Bmserpin32 in the integument was significantly upregulated at 12 h after injection,and the expression level of Bmserpin32 in hemocyte was significantly upregulated from 6 h to 24 h after injection.Western blot results also showed that the expression of Bmserpin32 protein in the hemolymph was induced to increase at 6 h and 12 h after injection.In order to explore whether Bmserpin32 is involved in the regulation of the expression of antibacterial peptide genes during the immunization process,Bmserpin32 protein was injected into the day-3 fifth instar larvae,and after 30 minutes,M.luteus was injected to activate the antimicrobial peptide pathway,and quantitative PCR was used to detect the expression of antimicrobial peptide genes.The results showed that after injection of Bmserpin32,the expression of antibacterial peptides Gloverin2,CecropinA,and Defensin2 in the immune tissues was not significantly different from with control groups.It was speculated that Bmserpin32 may not regulate the expression of antibacterial peptide genes in the immune response of silkworm.In order to study whether Bmserpin32 is involved in the inhibition of melanization in the silkworm,incubating recombinant Bmserpin32 protein with hemolymph from day-3 fifth instar larvae in vitro,lead to the hemolymph melanization became slower.When the mass of recombinant Bmserpin32 protein increased from 1?g to 10?g,the inhibition of hemolymph melanization became more and more obvious.These results indicated that Bmserpin32 played an important regulating role in the process of hemolymph melanization.In order to further elucidate the role of Bmserpin32 in the process of melanization,we examined the regulation of Bmserpin32on the activation pathway of prophenoloxidase?PPO?and the activity of PO.The activity of PO in hemolymph samples was measured using L-Dopa as substrate.Compared with the control group,the PO activity was significantly down-regulated by the addition of Bmserpin32 protein as well as the induction of M.luteus.However,if M.luteus was added to activate the PPO pathway and then Bmserpin32 protein was added,there was no inhibitory effect on PO activity.The above results indicated that Bmserpin32 did not directly inhibit the melanization process,but rather inhibited the activation of PPO to achieve the effect of inhibiting melanization.In insects,serpin generally plays a regulatory role by inhibiting its target protease.Therefore,in order to analyze the target proteases of Bmserpin32 in silkworm,we selected the clip domain serine protease CLIP18,which has the highest homology with M.sexta PAP3,for incubation experiments.MsPAP3 can activates PPO,and is a target protease of Msserpin7.The CLIP18 precursor was activated by factor Xa and then incubated with Bmserpin32.The binding band was detected by western blot between70-100 kDa with Bmserpin32 antibody.This experiment shows that Bmserpin32 may play a role in regulating melanization by inhibiting BmCLIP18 in silkworm.