Founctional Studies On Bombyx Mori Nucleopolyhedrovirus(BmNPV) Bm122 Gene

Bombyx mori baculovirus expression system has been used in the expression of foreign protein with complex modification after translation and the production of pesticides and vaccine and many other aspects. But the mechanism of how to express foreign protein efficiently using Bombyx mori baculovirus expression system is not clear. What’s more, the mechanism of DNA replication and gene expression regulation is not clarified, and the function of many protein factors involved in gene expression regulation is also unknown. Therefore, Bombyx mori Nucleopolyhedrovirus orf?122(Bm122) gene with function unknown was studied in this research.Gene knockout was recognized to be one kind of precise and effective methods to research the function of virus genes, especially in the research of genes in viral DNA replication and gene expression regulation during the infection cycles. In this study, red-recombination technology was conducted to delete Bm122 gene and research its function. Bm122 gene is located between 116323?nt and 116928?nt. Its orf is 606?bp in length. The protein it encoded is about 23?kD and located in the nucleus. Bm122 is present in the genomes of all lepidopteron NPV and GV, but is not present in those of hymenopteran or dipteran. Its homologous gene Ac146 is an essential gene of AcMNPV. Bm122 was predicted be related to viral DNA replication and late gene expression.To determine whether Bm122 was required for virus DNA replication, Bm122-ko-bacmid was generated by Red-recombination and then Bm122 was instead by chloramphenicol gene.?To eliminate the influence of Bm122 gene knock out to its upstream and downstream genes,?PCR was conducted to amplify the complete open reading frame of Bm122 gene.Fatherly,?Bm122-re-bacmid was constructed by Bac-to-Bac system in order to repair Bm122 gene.? After transfecting BmN cells with wtbacmid(wild type bacmid)、Bm122-ko-bacmid、Bm122-re-bacmid, the supernatant was harvested at particular time point and virus titer was determined by using TCID50 end point dilution assay. The results indicated that the Bm122 knockout virus is unable to produce infectious virus particles, while the repaired virus can restore infectivity, indicating that Bm122 is critical for the formation of infectious BV(budded virus). In addition, transmission electron microscopy samples were prepared to observe whether there was BV produced. There were only something like polyhedra observed.This result, goesone step further, it suggests that deletion of Bm122 significantly reduces the produce level of BV and affects BV morphogenesis. Harvest the cells at a particular time point after the transfection,?qPCR was performed.?At the same time,?purified wtbacmid was diluted and fluorescence quantitative PCR was conducted to get the linear relationship between the Ct value and the copy number index of virus DNA replication.?Then conversion of the Ct value obtained by fluorescence quantitative PCR into gene copy number index,?and compared them.? ?QPCR results show that in cells transfected with the Bm122 knockout bacmid, the level of viral DNA replication is evidently reduced compare to wtbacmid and Bm122-re-bacmid. More dates should be get to determine whether the deletion of Bm122 affects viral DNA replication.Bm122-ko-bacmid and wtbacmid were transfected into BmN cells,?the cells were collected for western blot experiment,?using the LEF-3 monoclonal antibody, the results showed that the knockout of Bm122 gene don’t affect the normal expression of lef-3 gene.