| BmNPV is a kind of baculovirus,has produced a very harm in our country’s Sericulture.As the research on baculovirus is gradually thorough,the main role in the infection process is the membrane protein GP64 of the virus surface.As the advanced protein infected by BmNPV,when knocking or interfering,it can have a significant reduction in infestance of BmNPV,so GP64 is often used as an antivirus target.Studies have shown that virus invasion of the host is related to the cholesterol transport pathway.As a key protein in the intracellular cholesterol transport pathway,NPC1 plays an important role in virus invasion.In the process of Ebola virus(EBOV)infection,h NPC1 has been identified as an intracellular receptor for filovirus to invade the host.Preliminary laboratory studies have shown that BmNPC1 is structurally similar to h NPC1 and interacts with the envelope protein GP64 of BmNPV.When BmNPC1 is knocked out,BmNPV infectivity is greatly reduced.The above results indicate that BmNPC1 is the key to BmNPV infecting the host factor.Among the three main functional areas of BmNPC1,BmNPC1-C plays an important role in the interaction with GP64.Based on the results of previous studies,this study identified the key sites of the interaction between BmNPC1 and GP64,and evaluated the effect of the mutant protein in inhibiting virus proliferation.The research results obtained so far are as follows:1.Identification of BmNPC1 and GP64 interactionThrough analysis of the structure of BmNPC1,molecular docking and homology modeling,and alignment with the sequence of h NPC1,looking for flexible segments in BmNPC1-C that may form loop loops,and analyzing the possibility of interaction with GP64 in these segments After screening these predicted key positions by FRET and yeast two-hybrid,the scope of key positions was narrowed to three,namely V503,L510,D529 located in the functional region of BmNPC1-C.After mutating these three key sites on BmNPC1-C,the prokaryotic expression protein and purification were performed,and then the virus blocking experiment was performed.The interaction between the mutant protein and GP64 was analyzed by fluorescence observation and quantitative PCR.The experimental results showed that this After the three key sites are mutated,the ability to interact with GP64 is indeed reduced.2.Research on the anti-disease effect of mBmNPC1 at the cellular levelBy analyzing the structure of BmNPC1,trying to use molecular docking and homology modeling methods to align with the sequence of h NPC1,look for flexible segments that may form loop loops in the BmNPC1-C region,and analyze the interaction between these segments and GP64.These predicted potential key sites were screened by FRET and yeast two-hybrid technology,and finally the range of key sites was narrowed to three,namely V503 and L510 located in the functional region of BmNPC1-C.D529.Perform point mutations on these three key sites on BmNPC1-C,then perform prokaryotic expression and purification,carry out virus blocking experiments,and analyze the interaction between the mutant protein and GP64 through fluorescence observation and quantitative PCR.The results show that these three After the key site mutation,it does reduce the ability of the mutant protein to interact with GP64.3.Construction of mBmNPC1 transgenic expression vector and production of transgenic silkwormAfter functional verification of the selected key sites at the cellular level,the BmNPC1 promoter was cloned using silkworm genomic DNA as a template,and the p Bac-[PN-mBmNPC1-PA] expression vector was constructed after ligation with the full-length coding sequence of mBmNPC1.The mBmNPC1 expression cassette and two sg RNA expression cassettes targeting BmNPC1 were sequentially inserted into the p Bac-[A3-EGFP] vector,and the expression vector p Bac-[sg RNA1+mBmNPC1+sg RNA2] was successfully constructed to knock out the WT-BmNPC1 gene.And the gene knockout vector has been microinjected into the early embryos of silkworm,but the positive transgenic silkworm has not been screened yet.The above research shows that after the key sites of BmNPC1 and GP64 interaction are determined,the intensity of the interaction between the two can be reduced,and the virus infection can be reduced.Based on this result,the creation of resistant materials can be carried out to fight the virus for the silkworm.The creation of resistant materials in terms of aspects provides a practical strategy. |
