Studies On The Relationship Between The Number Of Gene Copies And Protein Expression Efficiency In The Baculovirus Expression System

Most of cellular proteins work as the subunits of protein complexes which are the basic functional unit in cell. Baculovirus expression system has been a popular tool for expressing eukaryotic multiprotein assemblies have become a method of choice in many laboratories, because it is safer and has higher expression level.MultiBac system is a useful method for expression of eukaryotic multiprotein complexes and introducing multiple copies of the same gene insect cells. The MultiBac system is consisted of two such modular transfer plasmids pFBDM and pUCDM. pFBDM is derived from pFastBacDUAL and has Tn7 transposition sequences (Tn7R, Tn7L),contains two expression cassettes in a head-to-head arrangement with multiple cloning sites MCS1 and MCS2 flanked by polh or p10 promoters and SV40 or HSVtk polyA signal sequences, respectively. Vector pUCDM is flanked by a Loxp inverted repeat sequences and contains a chloramphenicol resistance marker and a conditional R6Kγ.Recombinant baculovirus DNA by accessing the viral genome via two specific sites(Tn7 transposition transposon site and Loxp inverted repeat sequences). The resulting vectors are introduced into MultiBac baculoviral DNA in DH10MultiBacCre E. coli cells which contain the factors for Tn7 transposition (for pFBDM derivatives) and cre-loxp site-specific recombination (for pUCDM derivatives).The relationship of gene copies number and the protein production are followed gene dosages in theory. Related findings indicate that increasing the number of integrated copies of the expression cassette generally has the effect increasing the amount of protein expression .Other studies have found that there is no direct linear relationship between gene copies number and protein expression level. There is no research about the relationship between protein production in baculovirus expression system and the number of gene numbers.In this study, we choice gfp gene and try to found the relationship of gene copies number and the protein production in the baculovirus expression system The green fluorescent protein GFP from the jellyfish Aequorea ictoria has become an excellent marker, it has been successfully expressed in insect host cells and has the advantages of easy to detect for emiting bright green light when simply exposed to blue light. The reconstruct of seven virus is accomplished :rAcMultiBac-p10-gfp,rAcMultiBac-ph-gfp,rAcMultiBac-p10-ph-2gfp,rAcMultiBac-p10-2ph-3gfp,rAcMultiBac-2p10-ph-3gfp,rAcMultiBac -2p10-2ph-4gfpå'ŒrAcMultiBac-3p10-3ph-6gfp.After analyzed by Western blot with homemade GFP antibody,laser scanning confocal microscope and fluorescence spectrophotometer analysis. First we compares the activity of two very late promoter (ph and p10), we found the fluorescent protein expression of recombinant baculoviruses under polh promoter are 1-fold than p10 promoter. Second, we find protein expression is increase when the gene dosage was increase in the expression of recombinant baculoviruses embrace gfp from one to four copies .we find a 10-fold increase in activity when four copies gfp gene than three copies of the gfp gene and 20-fold than single copy gene .The third ,an increase in the expression level of GFP protein was observed when the gene copies number increase from two to four are 2-fold growth.,but the GFP protein expression of the gene copies number of six are declined.So, the target protein expression are improved as the increasing the number of gene copies significantly in baculovirus expression system .we only need to construct to four copies can obtain the highest amount of protein expression.It provides a theoretical basis and technical route for high-efficient expression in baculovirus mutigene expression system .