Functional Study Of The Length Of N Region In BmNPV GP64 Signal Peptide On Protein Localization And Secretion

Baculovirus is a type of enveloped DNA virus that mainly infects lepidopteran insects.During its infection cycle,it produces two types of virion: occlusion-derived virus(ODV)and budding virus particles(Budded virus,BV).Bombyx mori nucleopolyhedrovirus(Bm NPV)BV's envelope protein GP64 has a total length of529 amino acids whiles Autographa californica nucleopolyhedrovirus(Ac MNPV)BV envelope protein GP64 has a full length of 512 amino acids.Signal peptide prediction analysis shows that Bm NPV GP64 is 18 amino acids longerth(1-18 aa at N terminal,referred to as SPI)than Ac MNPV GP64 signal peptide(SP).Studies have shown that constructing a transient vector of 1-18 amino acids truncated Bm NPV GP64,transfected Bm N cells,immunofluorescence analysis found that the deletion of 1-18 amino acids will result in the position of GP64 protein from the ectopic cell membrane to the cytoplasm.This article introduces the baculovirus infection mechanism,research status and application;envelope protein and its research progress,the application and research progress of signal peptides and signal peptidases;and introduces domestic and foreign research on different signal peptides to guide protein secretion pathways progress.We deeply studied of the role of the N-terminal sequence length of the Bm NPV GP64 signal peptide in mediating the transmembrane transport of foreign proteins.1.In this study,we constructed Bm NPV GP64 signal peptide of different truncated length to guide e GFP transient expression vector,Bm N was transfected,cell membrane was labeled with R18,cell nucleus was labeled with Hochest,and the location of e GFP was observed using laser confocal microscope.The results showed that the truncated signal peptides of 3,6,9,12,15,16,17 amino acids at the N region of Bm NPV GP64,respectively,Can mediate e GFP protein localization on cell membrane.while SPII guides the transient expression vector of e GFP,e GFP protein is located in the cytoplasm.It shows that the deletion of 1-17 amino acids does not affect the position change of e GFP.Only when the first 18 amino acids at the N-terminus are truncated will the position of e GFP change.2.Quantitative analysis of truncating Bm NPV GP64 signal peptides of different lengths on the expression and secretion of foreign proteins.GP64 signal peptide-directed Luciferase(Luciferase)transient expression vectors of differentlengths were constructed and transfected into Bm N cells.The Luciferase activity outside and inside the cells was quantitatively analyzed by a luciferase reporter system.The results showed that after truncating 3,6,9,12,15,16,17 amino acids of GP64 N-terminus,The ratio of luciferase activity secreted out of the cell to the total luciferase activity were 18.97%,19.30%,18.92%,16.91%,19.87%,18.25%,17.89%.There is no significant difference in the secretion rate of Luciferase guided by the full-length signal peptide.However,the ratio of Luciferase activity outside SPII cells to total cellular luciferase activity was 2.30%.The analysis of differences showed that the ratio of Luciferase guided by signal peptides of different lengths was significantly different from that of SPII guided Luciferase.3.Analyzes the host differences of signal peptidase expression using q PCR.The Bm NPV GP64 full-length signal peptide(SP,1-36 amino acids),1-18 amino acids(SPI),and 19-36 amino acids(SPII)e GFP transient expression vectors were transfected into Bm N and Sf9 cells.The transient expression of e GFP without signal peptide was used as a control and q PCR was used to quantitatively analyze the expression of signal peptidase in different cells.The results showed that in Sf9 and Bm N cells,the relative expression levels of the control group,SP,and SPI were not significantly different;However,the relative expression levels of SPII in Sf9 cells were 11.479,11.1,19.806,and 11.68,and the relative expression levels in Bm N cells were 1.493,1.171,1.172,and 1.118,respectively,and there were significant differences.In summary,the research in this paper shows that the Bm NPV GP64 signal peptide guide protein secretion depends on the length of its signal peptide N region.When the N-terminal 18 amino acids are truncated(the n region was removed),the guide protein secretion rate is significantly reduced.