Cloning And Functional Analysis Of Serine Protease Inhibitor 6 Gene(apserpin-6)from Chinese Oak Silkworm,antheraea Pernyi

Protease inhibitors are a kind of peptides or proteins that inhibit the catalytic activity of proteolytic enzymes and are widely found in living organisms.They play an essential role in many life processes.Serpin is the most abundant and widely distributed superfamily of serine protease inhibitors.It worked by regulating the activity of serine proteases(SPs)and cysteine proteases(CPs).Insect serpin can regulate the body's immune defense response by inhibiting the activity of serine proteases.In order to elucidate the role of serpin in the immune defense response of Antheraea pernyi,the structure,evolution and expression characteristics of A.pernyi serpin gene were studied by gene cloning,which established foundation for the further research of A.pernyi immune denfense response to pathogenic microorganisms.The results are as follows:1.Cloning of A.pernyi serine protease inhibitor 6 gene(Apserpin-6)PCR primers were designed based on a serpin CDS sequence in the midgut transcriptome database of A.pernyi to clone a 1 239 bp sequence from the cDNA of A.pernyi larvae.According to sequence analysis,it was named A.pernyi serine protease inhibitor 6 gene(Apserpin-6)(GenBank accession No.MF944108).The gene encodes 412 amino acid residues.The predicted signal peptide sequence is located at N-terminal from the first to seventeenth amino acid residues.There is a reaction center loop from the 357th to the 391th amino acid residues at C-terminal,and the predicted protein cleavage site is between 374serine and 375 serine.The analysis above demonstrated that the candidate protein belonged to the serpin superfamily and was a typical inhibitory serpin.In addition,Apserpin-6 protein has a molecular mass(Mw)of 46.49 kDa and an isoelectric point(pI)of 7.15,and contains six phosphorylation sites.2.Phylogenetic analysis of A.pernyi serine protease inhibitor 6(Apserpin-6)Multiple alignment shows significant similarity among Apserpin-6 and serpin-6 from other insects.Phylogenetic analysis shows that Apserpin-6 was closely related to Manduca sexta Msserpin-6,Bombyx mori Bmserpin-6 and Bombyx mandarina Bmdserpin-6,and the homologous serpin-6 proteins from A.pernyi,B.mori and B.mandarina get togather on the same branch.3.Analysis of Expression Profile of A.pernyi serine protease inhibitor 6 gene(Apserpin-6)Apserpin-6 is mainly expressed in fat body,followed by hemolymph and head,and has the lower expression level in silk gland,midgut and malpighian tubule,which was detected by semi-quantitative RT-PCR.The results of real-time fluorescence quantitative RT-PCR(qRT-PCR)show that the mRNA level of Apserpin-6 in A.pernyi hemolymph increased significantly after induced by Enterococcus pernyi,Beauveria bassiana and A.pernyi nucleopolyhedrovirus.4.Prokaryotic expression and functional analysis of recombinant A.pernyi serine protease inhibitor 6(rApserpin-6)Recombinant Apserpin-6 protein was producted by E.coli BL21(DE3)transformed by pEASY~?-Blunt E1-Apserpin-6 prokaryotic expression plasmid,and purified by NTA-Ni affinity chromatography resin.Furthermore,recombinant Apserpin-6 was used to interact with hemolymph of A.pernyi larvae to detect its inhibitory effect on pro-phenoloxidase(proPO)activation and enzyme activity of phenoloxidase(PO)in vitro.The results show that recombinant Apserpin-6 inhibited proPO activation but did not affect PO activity in A.pernyi hemolymph.In summary,a inhibitory serpin gene from A.pernyi named Apserpin-6 whose expression was upregulated by pathogen induction was cloned in this study.Detection of recombinant Apserpin-6 protein activity in vitro suggests that Apserpin-6 acts as a negative regulator to the A.pernyi immune defense response to pathogens by inhibiting the proPO activation in hemolymph.