Interaction Between Potential Receptor Protein And Structural Protein Of Bombyx Mori Bidensovirus

Bombyx mori bidensovirus?BmBDV?is the only type species newly established within the new family Bidnaviridae by the International Classification Committee of viruses?ICTV?in 2012.The BmBDV infected silkworm midgut columnar epithelial cells,specifically shows symptoms such as the softening of sick silkworm,the nucleus of midgut columnar cells expanded and yellowed of midgut.The Bm BDV genome contains two linear ssDNA molecules?VD1 and VD2?,which are individually packaged in virus capsid.The major structural protein VP encoded by VD1 and the minor structural protein P133 encoded by VD2 play an important role in the process of BmBDV infect silkworm.Previous studies have shown that Bm BDV-sensitive silkworms express an amino acid transporter protein NSD-2 which consist of 12 transmembrane domains.The deletion of NSD-2 protein causes the silkworm to be non-sensitive to Bm BDV.Therefore,researchers speculate that the NSD-2 protein is a potential receptor for Bm BDV infection.The main objectives of this study include:?1?Construction of a silkworm ovarian cell line Bm N(+^nsd-2)stably express the complete NSD-2 protein;?2?Expression of the Bm BDV structural proteins VP and P133 in vitro and investigation of the interaction between viral structural protein VP and NSD-2protein in the Bm N(+^nsd-2)cell line;?3?Study the potential of Bm N(+^nsd-2)cell line as a sensitive cell line for BmBDV infection in vitro.This thesis can be divided into the following three parts:The first part is the construction of a silkworm ovarian cell line BmN(+^nsd-2)that stably expresses the complete NSD-2 protein.Firstly,we constructed pIZ-+ⁿsd-2sd-2 plasmid for expressing BmBDV sensitive gene+ⁿsd-2sd-2 and transfected into Bm N cells.The transfected Bm N cells were screened using the Zeocin-resistance selectable marker of the pIZ-V5/His vector.After 30 passages,a stable silkworm cell line Bm N(+^nsd-2)was obtained.The expression of NSD-2 protein and the transcription of+ⁿsd-2sd-2 gene were detected by western blotting and RT-PCR.The results showed that the expression of NSD-2 protein and the transcription of+^nsd-2gene could be detected in Bm N(+^nsd-2)cells;Immunofluorescence technique was used to detect the localization of NSD-2 protein in BmN(+^nsd-2)cells.The results showed that NSD-2 protein was mainly located on the cell membrane of BmN(+^nsd-2)cells.The second part is the expression of the Bm BDV structural proteins VP and P133 in vitro and investigation of the interaction between viral structural proteins and NSD-2 protein in BmN(+^nsd-2)cell lines.The expression vectors of the major structural protein VP and the minor structural protein P133 were constructed using the insect expression vector pIB/p IZ-V5/His and transfected into Bm N(+^nsd-2)cells respectively.We prepared the VP monoclonal antibody and constructed expression vector contained different lengths of the vp genes to determine the protein epitope based on VP?-barrel structure.The expression of VP and P133 protein was detected by western blotting.The results showed that the expression of VP protein,P133 protein and NSD-2 protein could be detected in Bm N(+^nsd-2)cells.In order to study the interaction between NSD-2 protein and the major structural protein VP of BmBDV,VP monoclonal antibody was used to detect co-precipitation of VP and NSD-2-V5 fusion protein;Secondly,V5-tag polyclonal antibody was used to detect the co-precipitation of NSD-2-V5 fusion protein with the VP protein;The results indicate that VP interacts with NSD-2 in Bm N(+^nsd-2)cells.Immunofluorescence was used to detect the co-localization of VP and NSD-2 proteins in Bm N(+^nsd-2)cells.These results show that there is an interaction between the NSD-2 protein and the major structural protein VP of Bm BDV.The third part is to study the potential of the BmN(+^nsd-2)cell line as a BmBDV-infective sensitive cell line in vitro.BmN(+^nsd-2)cells were infected with purified Bm BDV virus suspension?concentration:midgut dried 20 mg/m L?.Real-time quantitative PCR and immunofluorescence techniques were used to detect the transcription of Bm BDV genes and the expression of the major structural protein VP of the virus.The infectivity of Bm BDV to Bm N(+^nsd-2)cells was analyzed.However,immunofluorescence assays in infected Bm N(+^nsd-2)cells did not detect virus invasion and VP protein expression;Real-time quantitative PCR did not detect the transcription of vp and ns1 genes.The results of the study indicate that BmBDV may not invade Bm N(+^nsd-2)cells or cannot replicate in cells.For summarize,this study constructed a silkworm cell line Bm N(+^nsd-2)that stably expresses the Bm BDV sensitive gene+ⁿsd-2sd-2 successfully.This cell lines can be used to study the interaction between potential receptor protein NSD-2 and the major structural protein of BmBDV.The results showed that NSD-2 protein interacts with VP and co-localizes in the cell membrane.Bm BDV does not infect the Bm N(+^nsd-2)cell line,indicating that the NSD-2 protein is not the only receptor for the BmBDV.