Reverse Genetic Manipulation HP-PRRSV Strain With Fluorescent Protein And PRRSV N Protein Expression With Eukaryotic Expression System

Porcine reproductive and respiratory syndrome(PRRS)is a kind of infectious disease caused by viruses,the occurrence of the disease to the global pig industry have caused a huge impact.N protein one of the structural protein of PRRSV is the most abundant protein content of virus particles,its known effect including the influence of virus replication,constitute the skeleton structure of the virus and interferences with the immune cells of susceptible animals or susceptible,N protein of PRRSV infection mechanism for research has the vital role.Reverse genetic technique for RNA virus research provides a powerful platform,can make use of this platform in DNA molecular level,by means of gene recombination,gene mutation to study RNA virus replication and infection,RNA viral protein function,the relationship between the virus and the host cell,and marker vaccine and antiviral strategy development is inseparable from the reverse genetic operation.The enhanced Green Fluorescent Protein(enhanced Green Fluorescent Protein)will be insert to HP-PRRSV XHGD strains cDNA,build complete EGFP-XHGD recombinant strains in this study,the recombinant strains of transfection into BHK cells,can obtain stable expression of Green Fluorescent and can infect and PAM Marc-145 cells of PRRSV strains,and can produce consistent with the original strain cell lesions.The green fluorescence can be stable,and the amount of fluorescence in cells increases with the time of infection.The successful construction of the viral strain with fluorescent label provides the convenience for the followup of the tracer and antiviral research of the susceptible animals and cells of PRRSV infection.Baculovirus expression system has a high level and large genetic capacity loads etc,are widely used in all kinds of production of recombinant proteins and certain viral vector,in principle,baculovirus expression system can produce any foreign proteins,and because the insect cells could glycosylation,phosphorylation and acylation and protein folding and so on the many kinds of modification after translation,it is often used to produce some bioactive protein and/or immunogenicity.N protein of PRRSV is a key role of virus infection replicate ability.In this study,118 and 120 phosphorylation sites of the N protein genome were mutated,and the N protein genome with mutation was constructed.The mutant N protein was successfully obtained by using baculovirus expression vector system to express the mutated genome.It was found that the optimized protein expression condition was that the recombinant plasmid was transfected into Sf9 cells 120 h,and the expression quantity of large protein was obtained.In the purification of protein,the eluent of 500 mM imidazole was better than that of protein eluting.It lays the foundation for the function research of N protein.