Study On The Function Of P24 In Bombyx Mori Nucleopolyhedrovirusiii

In the previous studies, The P24 was considered to be a nucleocapsid related protein. The role of p24 in baculoviral life cycle still unknown. In this study, we constructed a Bmp24 knockout virus by the λ Red recombination in Escherichia coli. We generated the target segment was amplified by PCR that containing homologous arm and cat gene.The replacement between the Bmp24 fragment and the target segment in DH10 Bac because of the recombination enzyme expressed by p KD46 plasmid. The Bmp24-ko-Bacmid was established sucessfully and named Bmp24-ko-Bacmid. Then we generated a recombinant plasmid(p Fast Bac HTB-p24) and identificated by PCR and digested with Bam HI/Eco RI.Then the Bmp24 repair Bacmid was constructed by inserting the ORF and promoter element of Bmp24 into the polh locus by Bac-to-Bac system.In order to study the role of Bmp24-ko-Bacmid in Bm N, we transfecting the DNA of Bmp24-ko-Bacmid, Bmp24-re-Bacmid and wt Bacmid into Bm N cells. The viral titer detection results showed that Bmp24 knockout Bacmid was still able to produce viral progeny but its viral tite lower than wt Bacmid and Bmp24 repaired Bacmid. Results of transmission electron microscope also confirmed that the virus can replicate and assemble normally after Bmp24 deletion, but only some small and slender rod structure in the Bmp24-ko- Bacmid infected cells,in contrast, a large number of packaging of mature virus particles appear in the wild type virus and Bmp24-re-Bacmid infected cells. In conclusion, Bmp24 is unessential for virus DNA replication and it has an important influence on the virus gene transcription and the virus assembly.We examined the level of virus DNA replication, Furthermore, the replication level of Bmgp41 in the absence of Bmp24 were also examined by q PCR. Our results showed that The Bmp24 deletion didn’t show obvious effect on viral genome replication; Moreover, the transcription levels of early gene Bmlef-3, late gene Bmvp39 and very late gene Bmp10 were much lower than wild-type virus and Bmp24 repair Bacmid.In order to further explore the chracterization of Bmp24, the Bmp24 was cloned into p ET-28a(+)expression vector and then the expression of 6x His-Bm P24 coding region in E.coli result in the induction of a 24.8KDa band in accordance with the predicted size. The fusion protein was purified by the Ni-agarose gel and quantitated by the BCA Protein Quantitation Kit then injected in New Zealland white rabbit to produce antibody against Bm P24, ELISA test to detect the antibody titer is more than 128,00 U.Recombined prokaryotic expression vector, the purified protein and prepared polyclonal antibody were the necessary materials for further study of Bm P24. Western-blot assay obtained that the Bmp24 is a late gene in Bm NPV.