Isolation Of Ferritin And Its Functions In Response To BmNPV In Silkworm,Bombyx Mori

Ferritin is a ubiquitous iron binding protein that plays an important role in iron metabolism and host defence against pathogen infections.Bombyx.mori nuclear polyhedrosis virus（BmNPV）,is a primary silkworm pathogen and annually causes serious economic losses.The invasion and replication of virus needs iron,therefore,the study of interaction between ferritin and BmNPV can help understand the further function of ferritin,and made a certain degree complement for the mechanism of BmNPV infection.In this study,native ferritin was isolated from the hemolymph of B.mori using native-polyacrylamide gel electrophoresis（Native-PAGE）.Reverse transcription quantitative PCR（RT-q PCR）,employed to investigate the different tissues and development phase of BmFerHCH and BmFerLCH and the relative expression in silkworm challenged with Fe³⁺,bacteria and NPV.Combined with SDS-PAGE,Western-blot and Far-western bolt was used to identify the native ferritin and two subunits of ferritin which had interaction with BmNPV in vitro.The results were listed as follows:1.Ferritin was isolated successfully and identified it is a complex which consists of two types of subunits.2.BmFerHCH and BmFerLCH were detected in all tissues and development stages,and the highest relative expression in the hemolymph and adults.3.The transcription of BmFerHCH and BmFerLCH in the hemolymph and midgut could be strongly induced by Fe³⁺,bacterial and viral infection.4.The relative expression levels of two subunits were down-regulated in resistant silkworm strain BC9 and there was no obvious change in susceptible silkworm strain P50 after BmNPV infection.5.The ferritin complex can interact with BmNPV,but two subunits of ferritin cannot interact with BmNPV.6.The recombinant protein of BmFerHCH were successfully expressed and the purified recombinant protein for raising rabbit antibody.BmFerHCH had relatively highest expression in the head and hemolymph.The recombinant BmFerHCH protein possessed tolerance to H2O2.Taking together,this is the first study to extract B.mori ferritin successfully and verify ferritin can interact with BmNPV.These achievements provided a reference to understand the further function of ferritin and silkworm immune response to BmNPV infection.