Expression Of Rabies Virus M Protein In Bombyx Mori By BmNPV Bac-to-Bac System And Preparation Of Antibody

Baculovirus Expression Vector System(BEYS)is a recombinant baculovirus vector that transduces foreign target genes into baculovirus genome and then infects insect larvae or cultured cells.Bac-to-Bac expression system completes the recombination of baculovirus genome in E.coli through site-specific translocation.BmNPV Bac-to-Bac baculovirus expression system is an excellent choice for preparing bioactive protein because it can express a large number of exogenous proteins in silkworm vector and save time and effort.The ORF of Rabies Virus(RV)M gene is 609 nucleotides,encoding 202 amino acid residues of matrix protein with molecular weight of about 25 KD.It plays an important role in the assembly,germination and morphogenesis of progeny viruses.In this study,the recombinant RV M(rRV-M)protein was expressed by BmNPV Bac-to-Bac expression system.The purified rRV-M protein was used as an antigen to immunize mice.The indirect ELISA titer of the serum antibody was determined.The results of this study can provide antigen and antibody sources for exploring the function of RV M protein,and also provide reference for the development and utilization of silkworm bioreactor.The research contents and results are as follows:1.Recombinant donor plasmid pFastBacHTA-M was constructed by directional cloning of ORF of RV M gene onto donor plasmid pFastBacHTA,and then transformed into E.coli DH10BmBac containing BmNPV Bacmid.Under the function of transposase expressed by auxiliary vector,RVM gene was translocated and inserted into Bacmid DNA,and its transcription and expression were controlled by promoter of polyhedron gene.Finally,recombinant baculovirus rBmNPV-M was obtained by injecting recombinant rBacmid-M and liposome into the 5th instar silkworm.2.Indirect immunofluorescence assay was carried out on the blood lymphocytes of Silkworm Infected with rBmNPV-M virus.The results showed that the blood lymphocytes infected with rBmNPV-M emitted specific bright green fluorescence,and the fluorescence concentrated around the plasma membrane.Western-blot was used to identify the expression of rRV-M protein in the haemolymph and fat body of silkworm.The results showed that rRV-M protein was expressed in a large amount in the body of silkworm.The relative molecular weight of the recombinant protein was about 25 KD.3.rRV-M protein was purified from silkworm body by nickel affinity chromatography.The purified protein band was single and the purity was good.The purified rRV-M protein was mixed with Freund's adjuvant and used as antigen to immunize mice.Polyclonal antibodies were isolated from serum.The titers of serum antibodies were determined by indirect ELISA.The titers were 1:2560.