Study On Co-expression And Application Of Multiple Antigenic Proteins By MultiBac Efficient Display System

Baculovirus surface display(BSD)system can not only be used to express foreign proteins,but also widely be used in gene presentation,gene therapy and vaccine development and other fields.The construction of traditional BSD is the use of "GP64 + exogenous protein" fusion expression,showing to the membrane surface.Early in the baculovirus replication,the wild-type GP64 protein has begun a large number of expression.Therefore,there is a strong competitive inhibition between wild-type GP64 protein and GP64 fusion protein,resulting in a low display efficiency of the system.At present,our group is committed to solving the problem of low display efficiency of BSD.We have started to develop a silkworm-baculovirus multigene display system(SBMDS),which can improve the efficiency of multiple target antigens and use this system to develop a new animal vaccine carrier.PCV2,CSFV and PRRSV are three major pathogens of swine disease,and the subunit vaccines for the three viruses have been developed independently or the combination of the two vaccines in the three vaccines has been developed.However,the case of three kinds of antigen protein combined into one vaccine hasn't been found.One reason is that the interaction between the three antigens is not clear,can not be mixed by physical methods to prepare a combined vaccine.The second reason is that the traditional BSD failed to express three or more foreign proteins,restricting the use of the technology research and development of the combined vaccine application value.In this study,we used the SBMDS to express the C-terminal truncated fragment of Invasin protein and the main antigen protein Cap protein,E2(179)truncated fragment and GP5 protein of PCV2,CSFV and PRRSV.We enhanced the immunological level of PCV2,CSFV and PRRSV by the immunostimulatory function that may exist in the C-terminal truncated fragment of Invasin protein,and detected the expression and immunological effect of the four proteins at the protein level.By analyzing the codon bias of pcv2,invasinC,e2(179)and gp5 genes,we explained the factors influencing the codon preference and codon bias and gene expression level,determined the strategy and plan of improving expression of four gene amount in SBMDS.The results of the innovations were as follows:The pF-p64spFP64-p64spSI64-phG and pU-p64spCE64-p64spHG64-phM transfer vector were successfully transferred into AcMultiBac by Tn7 transposon and Cre-Lox P homologous recombination,and the recombinant strain Ac-p64spFP64-p64spSI64-p64spCE64-P64spHG64-phG-phM was used to obtain AcBV-p64spFP64-p64spSI64-p64spCE64-p64spHG64-phG-phM recombinant baculovirus by infecting sf9 cells.Cap-GP64,InvasinC-GP64,E2(179)-GP64 and GP5-GP64 fusion proteins were successfully assembled on the surface of recombinant baculovirus which could be detected by SDS-PAGE and Western-blot.And it was proved by indirect ELISA that the PCV2-Cap and PRRSV-GP5 proteins had a stronger antigenicity than CSFV-E2(179)protein.The codon bias of four foreign genes,such as pcv2,invasinC,e2(179)and gp5 were analyzed to be related to the expression of the protein,and the expression level could be improved by optimizing the codon.The SBMDS which was constructed by our group has overcome the problems such as limited number of BSD display targets and low display efficiency.In this study,many foreign genes such as pcv2,invasinC,e2(179)and gp5 genes can be displayed efficiently.It can not only greatly play the advantages of its multi-gene display,but also can meet the demand of production by a high level of expression and conducive to the development of combined vaccine.