Infectivity Analysis Of AcMNPV Deleted Pk-1 Gene

Baculovirus is a kind of double-stranded circle large DNA virus, which has been widely developed as biopesticides and a powerful vector of foreign protein expressions. Revealing these applications will lead the better understanding of molecular mechanism of intacation between baculovirus and their hosts. Since the complete genome of AcMNPV was determined, extensive studies have been focused on the function of individual gene. The pk-1 gene of Autographa californica multinucleocapsid nuclear polyhedrosis virus encodes a protein kinase, but its function remains unclear.In this article, a series of experiments focusing on the function of pk-1 were carried on. The thesis encompasses four chapters.In chapter one a brief introduction of baculovirus was presented, including the classification status, replication, characteristics of the baculovirus genome, application and the background of pk-1, finally give aim to this study.Chapter two describes the materials and methods.The major results are presented in chapter three which contains three parts:Part one: Using theλ-RED recombinase system, the pk-1 gene was deleted from AcMNPV bacmid in Escherichia coli.Part two: Three recombinant baculoviral bacmids, AcMNPVΔpk-1, AcMNPVΔpk-1 repaired pk-1 and wild type AcMNPV, which all includes a gfp cassette, were constructed using the bac-to-bac system.Part three: The three recombinant baculoviral DNA were transfected into Sf9 cells respectively. GFP+ cells were observed in all samples at 96h post-trasfection. However, the health SF9 cells were infected with the supernants collected from the cells culture transfected with AcMNPVΔpk-1 were not observed GFP expression , while the others did. This means that pk-1 is an essential gene for productive AcMNPV replication and plays an important role in viral infection.In chapter four, we summarize and discuss the results of this experiment, making a view of further studying.