Effects Of LEF-10 And Secretory Protein Maturation Region On Late Gene Expression And Secretion Of AcMNPV

As a eukaryotic expression system,baculovirus expression vector system（baculovirus expression vector system,BEVS）is widely used in the research and production of various recombinant proteins and vaccines,BEVS has the advantages of having the capacity for the insertion of large foreign gene fragment,which allows the expression of multiple foreign genes using a single vector,and it can also be used to produce proteins with mammalian-like post-translational modifications.Previous studies have found that the late expression factor LEF-10 of baculovirus has prion properties,and when infected with high MOI（multiplicity of infection,MOI）,it will lead to the conversion of LEF-10 to an aggregated state that inhibits the high expression of the late viral genes,which may be one of the limiting factors for the high level expression of foreign proteins in BEVS.Studies have shown that the mature region sequences of secreted proteins can affect the secretion of proteins.This thesis is divided into two parts,in order to overcome the restriction of LEF-10 aggregation on the expression of foreign proteins in BEVS,LEF-10 was studied in the first part of this thesis.In the second part of this thesis,in order to promote the secretory expression of foreign proteins in the BEVS,the secretion of exogenous proteins guided by different signal peptides and mature region sequences was investigated.1.Effects of LEF-10 truncation on aggregation and late virus gene expressionPrediction of LEF-10 secondary structure showed that the carboxyl terminal region of LEF-10 was disordered.In order to explore the role of the disordered region in LEF-10solubility and function,the carboxyl terminus was truncated to express the truncated proteins containing amino acids of 1-62,1-65,1-66 and 1-70,Meanwhile,eGFP regulated by the late p10 promoter was expressed as the reporter protein to reflect the expression level of late viral genes.Sf9 cells were infected with recombinant baculoviruses expressing truncated LEF-10protein.eGFP expression in the recombinant virus infected Sf9 cells was detected by fluorescence microscopy and flow cytometry.The results showed that,all the truncated mutant except LEF-10_1-65 resulted in decreased late genes expression,compared with the wild-type LEF-10.The fusion expression of LEF-10 with eGFP showed that compared with LEF-10-eGFP,LEF-10_1-65-eGFP fusion protein produced less aggregates after the deletion of the disordered C terminus,which was consistent with previous studies.Flow cytometry results showed that the expression level of late virus gene regulated by LEF-10-eGFP was significantly higher than that of wild-type LEF-10,indicating that the fusion with eGFP affected the function of LEF-10.2.Effects of overexpressing LEF-10 by different promoters on late virus gene expressionLEF-10 and LEF-10_1-65were overexpressed by different promoters,and reporter gene expression under the control of a viral late promoter was analyzed by flow cytometry and SDS-PAGE.The results showed that the reporter gene expressed in the cells infected with baculovirus overexpressing LEF-10_1-65 by p10 and polh promoters was significantly higher than that of wild-type viruses,Especially,the expression level of late virus gene was significantly enhanced by overexpressing LEF-10_1-65controlled by polh promoter.These results indicated that overexpression of soluble LEF-10_1-65 could effectively improve exogenous protein production in BEVS.3.Effects of overexpressing LEF-10_L21Amutant on late virus gene expressionPrevious studies have shown that LEF-10_L21A mutation is able to up-regulate the expression of viral late genes at high MOI infection.In order to further expand the advantage of LEF-10_L21A in late gene expression,recombinant viruses,expressing LEF-10_L21Aby plef10,p6.9 and polh promoter,were respectively constructed,and the virus titers were determined by end-point dilution method.The results showed that the titer of virus expressing LEF-10_L21Aby its own promoter nearly dropped an order of magnitude.However,the viral titers were comparable to the wild type LEF-10 when the mutant was overexpressed by p6.9 and polh promoters,suggesting that overexpression of the mutant could compensate for the downregulated function caused by the mutation.Then,Sf9 cells were infected with virus overexpressing LEF-10_L21Aat different MOI.The results showed that the late gene expression level was increased in different degrees when the cells were infected by the viruses overexpressing LEF-10_L21A at different MOI.The expression level of late virus gene can be significantly improved by the virus expressing LEF-10_L21A under the control of polh promoter at low MOI.4.Effects of mature region sequence of secreted protein on protein secretionSome proximal sequences with signal peptide from chitinase,melittin and fibroin light chain were respectively fused with eGFP to guide the secretion of the reporter protein,using the signal peptide of baculovirus GP64 as the control,and Western blot detection showed that the reporter protein expression and secretion could be effectively improved by retaining some mature region sequences of N-terminal chitinase,meanwhile,long mature region sequences seriously affected the protein secretion.Similarly,retaining short fragment of the N-terminal mature region of melittin increased the expression level of the eGFP protein.5.Secretory expression of coronavirus IBV and BwCoV S2 proteinsPrevious studies in our lab have revealed that S2 proteins from Avian infectious bronchitis virus（Avian infectious bronchitis virus,IBV）and Beluga whale coronavirus（Beluga whale coronavirus,BwCoV）can be expressed at high expression levels in the BEVS,but they are not able to secrete out of insect cells.In this thesis,we successfully achieved the high secretory expression of two S2 proteins using melittin secretion tag containing part of the mature region.In summary,this study has proved that overexpression of LEF-10_L21A and LEF-10_1-65,which have reduced aggregation tendency,is an effective way to break through the restriction of late baculovirus genes expression by aggregated LEF-10 in BEVS,which sheds a new light on the improvement of proteins expression in BEVS.In addition,the influence of the mature region sequences of secreted proteins on protein secretion has been demonstrated in this thesis.The S2 proteins of IBV and BwCoV,which are difficult to secrete in BEVS,have been successfully expressed and secreted by retaining part of the mature region sequences with a signal peptide,providing a new idea for promoting the secretion of recombinant proteins.