Expression And Purification Of E1 Structural Protein Of Rubella Virus In Baculovirus Expression System

Objective 1.The method of baculovirus expression system and establishment of protein expression optimization condition;2.The full length recombinant protein of rubella E1 was expressed by baculovirus system to obtain the relatively pure recombinant protein of rubella E1;3.It lays a foundation for the detection and screening of antigens by high sensitivity and high specificity serological menthods,and is used for the establishment of high sensitivity and high specificity serological detection methods.Methods The E1 protein of rubella virus was digested and linked with p Fast Bac HTA of donor plasmid with restriction enzyme and then transformed into DH10 Bac TME.colic,the recombinant bacmid plasmid containing RV-E1 was extracted and transfected into Sf9 cells.and the viral culture supernatant was harvested,which was the P1 generation recombinant baculovirus Ac MNPV-RV-E1,The P1 generation was used to inoculate Sf9 cells to amplify the virus library,and the cell supernatant was harvested,which was P2 generation Ac MNPV-RV-E1.Identification by CPE,IFA and PCR indicated that the RV-E1 gene was successfully transfected into cells and assembled,replicated and expressed in the cells.The cells were seeded in the same manner as above,and the Ac MNPV-RV-E1 virus library was harvested.The virus was inoculated into the cells by endpoint dilution method,and the cells were seeded according to different MOI values,and plaque assay was performed to measure the virus titer.The conditions for the amplification of recombinant baculovirus were optimized to find the optimal number of days for which the protein of interest was harvested.Further,the protein containing RV-E1 was purified by nickel column affinity chromatography,and the expression of the target protein was identified.Results Successfully obtained the target gene of Rubella E1 by PCR amplification technology.At the same time,it was ligated to the donor plasmid p Fast Bac HTA and transformed into DH10 Bac TME.coli competent cells for homologous recombination.The Bacmid recombinant shuttle plasmid containing RV-E1 was extracted and transfected into Sf9 cells.After expression by the baculovirus expression system,the target protein was expressed.After optimizing the expression conditions,the harvested protein was inoculated and purified.It was identified that the target protein could be recognized not only by rubella-specific rabbit polyclonal antibody but also by 6*His monoclonal mouse antibody.It can also bind to rubella-positive human serum,while the control-negative serum does not react,indicating that the protein is immunoreactive.Conclusion 1.The gene of RV-E1 was successfully obtained by PCR amplification,and ligated with p GEM-T vector.After identification,it was ligated with the donor plasmid p Fast Bac HTA,transformed into DH10 Bac TME.coli competent cell clone,and the recombinant shuttle plasmid was extracted.Infected Sf9 cells,which were identified to indicate that the recombinant baculovirus can be assembled and replicated in the cells,and the recombinant baculovirus containing the gene of interest was successfully obtained;2.After identification by WB and DB,it can react with antibodies(specific rabbit anti-antibody,6*His antibody)and RV-positive serum,indicating that RV-E1 can react with antibodies of different species,And it has good immunoreactivity.Successful expression of Rubella E1 protein after optimization of expression conditions,It lays the foundation for the establishment of serological detection methods and antibody screening,and can be used for serological applications.