Expression Analysis Of Serine Protease Inhibitor-20 In Silkworm, Bombyx Mori

Bombyx mori is a model organism of Lepidoptera,whose complete genome was already sequenced,and also as the uniqueness agricultural economy insects which was domesticated and reformed.Serine proteinase inhibitors are involved in some biological procession,such as cell differentiation,blood coagulation,inflammatory response,apoptosis,immune reaction,fibrinolysis,and tissue reconstruction.In this study BmSerpin-20 gene was inserted into the p ET-28a(+)vector.Recombinant plasmids were transfected into Escherichia coli Transetta(DE3),and the BmSerpin-20 protein was induced expression with IPTG.The recombinant BmSerpin-20 protein was used as antigen to immunize New Zealand rabbit and achieved the polyclonal antibody serum.The expression patterns of BmSerpin-20,immunostimulation,and antimicrobial peptide,were analyzed with real-time quantitative PCR and western blot.The BmSerpin-20 protein was successfully expressed in Escherichia coli cells and purified.The polyclonal antibody against BmSerpin-20 was prepared.To test the tisssue-specific expression of Bm Serpin-20 mRNA and protein,fat body,midgut,hemocyte,malpighian,integument,testis,ovary were sampled from fifth-instar larvae of B.mori.The experimental results show that BmSerpin-20 was specifically higher expressed in integument.Fifth-instar larvae of B.mori were injected with nuclear polyhedrosis viruses,Micrococcus luteus,Escherichia coli or Beauveria bassiana,the expression patterns of BmSerpin-20 gene in fat body was first down-regulated and then up-regulated.The expression level of BmSerpin-20 was different when injected with different microorganism or at different injection time.The expression of antimicrobial peptides cecropin D,gloverin 2 and moricin was significant decrease after pre-injection of BmSerpin-20 protein,respectively.In this study,the BmSerpin-20 protein and polyclonal antibody was successfully expressed.The expression pattern of BmSerpin-20 after induced by different microbe was well studied,and also was the expression pattern of antimicrobial peptides genes.This may be lay a foundation for the further research of its function in immune system of B.mori.