Study Of Endoplasmic Reticulum Molecular Chaperones Involved In The Expression And Secretion Of Proteins Led By BmNPV GP64 Signal Peptide

The baculovirus expression system is a eukaryotic protein expression system expressed in insect cells,which has long been deployed for recombinant protein expression.However,the expression level of membrane protein and secretory protein in this system is very low,the reason is not clear.Glycoprotein GP64 is the key membrane fusion protein on Budded virus(BV)particles,and its signal peptides are often used to express secretory proteins and membrane proteins,and the most widely used baculovirus expression systems are Autographa californica multiple nucleopolyhedrovirus(Ac MNPV)expression system and Bombyx mori nucleopolyhedrovirus(Bm NPV)expression system,however,there is a difference on GP64 signal peptide between these two viruses.In this paper,transcriptome sequencing was used to analyze host differentially expressed genes caused by the transient expression of foreign proteins in Bm N cells guided by intact signal peptide(SP)and n-free region signal peptide(SPII),and the data analysis showed that SP significantly up-regulated the expression of Bi P and other genes related to the protein secretion pathway.Firstly,the transient expression plasmid of Bm NPV GP64 SP fusion luciferase gene was constructed for quantitative analysis of protein secretion.The results showed that the total expression quantity significantly increased with the increasing dose plasmid,but the secretion rate was decreased.Further studies showed that the genes involved in the Endoplasmic Reticulum Associated Degradation(ERAD),including protein disulfide isomerase(PDI),ubiquitin fusion-degradation protein gene 1(UFD1),sphingosine-1phosphate(S1P),and apoptosis signal-regulating kinase1(ASK1)were significantly upregulated,and the expression level was positively correlated with the transfection dose of plasmids.while the knock-down of PDI,UFD1,S1 P,and ASK1 significantly increased the secretion rate of foreign proteins.Furthermore,these results were further analyzed by the recombinant virus.Bm NPV carrying SP or SPII GP64 was constructed then infected Bm N cells.The infection of virus-containing SP GP64 significantly activates the expression of ERAD related genes,and ERAD-related genes' knock-down enhanced virus infectivity significantly.In summary,we preliminarily revealed the mechanism of endoplasmic reticulum molecular chaperones involved in the expression and secretion of proteins led by Bm NPV GP64 signal peptide.This study provided a reference for the study of the mechanism of signal peptides involved in protein secretion and provided new insights into the application of using the proteins of ERAD pathway as a molecular target to increase membrane protein expression.