A Method Recombinant Baculovirus Based On PCR Product And Related Functional Protein Research

Baculovirus expression vector system is one of the most widely used eukaryotic expressionsystems. For BmNPV system, the traditional method for constructing recombinant baculovirushas been difficult to meet the demand of quick and high throughput expression of target proteinfor it is time-consuming and laborious. Even using Bac-to-Bac method still takes about twoweeks to express the gene of interest. This study aims to establish a method for rapidlyconstructing recombinant viruses without sub-cloning and screening.The method is based on a replication-defective shuttle vector RD-BmBacmid. First, a pair ofhomologous recombinant arms, RA-L and RA-R, is synthetized using PCR amplification onBmNPV genome, which contain50bp homogous sequence with RD-BmBacmid, respectively.Further by overlapping PCR, the target DNA flanked with RA-L and RA-R is amplified. Theresulting PCR product can be used to cotransfect BmN cells or silkworm larvae with linearizedRD-BmBacmid, and then generate recombinant virus by one step without screening.Using thismethod, the recombinant BmNPVs habouring AcGFP and DsRed are constructed and expresscorresponding fluorescent proteins in BmN cells and silkworm larvae, respectively. Therefore,the feasibility of this method is verified.For further exploring the mechanism of homologous recombination of short arms, it is foundout that baculovirus LEF-3and AN are homologus to phage Beta and Exo in Red homologousrecombination, respectively. So, both lef-3and an are cloned, expressed, and purified forfunction research.This study successfully established a method that can rapidly construct recombinantbaculoviruses. This new method bears some advantages such as simplified operation, saving time,effortlessness and efficient expression of foreign genes, providing a new approach to apply thebaculovirus expression vector system.