A Rapid Method For Titrating BmNPV With Reporter Gene Transformed BmN Cells

Determining of baculovirus titer plays a significant role in baculovirus studies. Due to thelimits in the traditional methods for determination of BmNPV titer, this study aimed atestablishing a rapid, convenient, and accurate method for titrating BmNPV based on the reportergene stably transformed BmN cells. The principal of this method is utilizing the expression ofreporter gene, which has been integrated into the genome of BmN cells, prompted by polyhedrinpromoter to indicate BmNPV infection. In titration assay, when the stably transformed cell linesare infected with BmNPV, the reporter gene was expressed as a result of the expression of earlyviral gene products which could activate polyhedrin promoter. The BmNPV titer was determinedthough detection of product of reporter gene.The first step was to construct two reporter gene stably transformed BmN cell lines, BmN-RFP and BmN-LacZ. Firstly, the construction of pBacPAK8-DsRed and pBacPAK8-LacZ wasperformed by inserting DsRed and lacZ genes into pBacPAK8, respectively. Secondly, theexpression cassette for corresponding reporter gene flanking by polyhedrin promoter andpolyadenylation sequence was amplified useing pBacPAK8-DsRed or pBacPAK8-LacZ astemplate, and then inserted in pIZT/V5-His vector to replace OpIE2promoter and OpIE2polyadenylation sequence. Therefore, two transform vectors, pIZT-DsRed and pIZT-LacZ wereproduced. Finally, stably transformed BmN cell lines BmN-RFP and BmN-LacZ were obtainedby screening with Zeocin on BmN cells transfected with pIZT-DsRed and pIZT-LacZ,respectively.To verify the applicability of the cells, BmNPV was used to infect BmN-RFP and BmN-LacZ. The expression products of DsRed or lacZ were successfully detected when BmN-RFP orBmN-LacZ cells were infected with BmNPV. While the expression products of DsRed or lacZcould not be detected if BmN-RFP or BmN-LacZ cells were not infected with BmNPV. Inaddition, Southern Blot assay revealed the reporter genes had successfully integrated into thegenome of BmN cells.Futher more, both BmN-RFP and BmN-LacZ cells were used to titrate deferent BmNPVson the basis of end-point dilution method. The assay results revealed that this method was quick,accurate, and reliable.