Prokaryotic Expression And Purification Of L Protein Polymerase Activity Module In Rabv And Prepared Thepolyclonalantibody

Rabies is an acute zoonotic infectious disease with a high mortality rate, almost100%dead, bringing a horrible threat to people s life. Rabies virus has a single non-segmented negative strand RNA genome, coding five structural proteins, including the biggest L protein. The L protein is responsible for virus genome replication, transcription and post-translational processing. L protein is a highly conservative protein and its mutation could seriously affect the process of transcription and replication. Therefore, studying the structure and function of L protein s particularly important.In this study, the L protein polymerase gene was amplified by RT-PCR and cloned into pMD18-T vector, and then subsequent cloned into pET-32a to reconstructed an expression vector pET-L. The pET-L plasmid was transformed into Rosetta (DE3),and from which the L protein was induced to express by IPTG. Using Western blot, the L protein expression with fused His-tag label was detected The expression of fusion protein in E.coli was detected as a form of inclusion body by SDS-PAGE. The recombinant L protein was washed in5Mol urea for purification and then refolded by gradually dialysis method. The polyclonal antibody was acquired by using purified recombinant L protein as antigen to immunize mice.The anti-L polyclonal antibody and anti-His antibody specifically responsed to recombinant L protein by Western blot test. After1:8000dilution the polyclonal antibody also reacted with recombinant L protein quite well. This result indicated that the polyclonal antibody had reactivity and specificity with recombinant L protein.