Regulation Of Virus Like Particles Expressed In A Baculovirus System

Baculovirus expression vector system (BEVS) can be used to express structural proteins of some virus. These structural proteins can form virus like particles(VLPs) which have good antigenicity and immunogenicity. VLPs are safe and effective subunit vaccine candidate. BEVS is a powerful tool for research and production of this vaccine. Porcine circovirus type 2 and canine distemper virus are pathogen of post-weaning pigs multisystemic wasting syndrome(PMWS) and canine distemper. It is important to develop safe, efficient and inexpensive new vaccine for these pathogens. In this study, we regulated PCV2-LPs expression in a baculovirus system through modifying the baculovirus vector by muti-copies genes, two burst sequence modifying promoter and adding chiken insulator HS4 at the downstream of polyA. Furthermore, we studied CDV-VLPs expression and secretion in a baculovirus system.Firstly, we constructed a series of recombinant baculovirus expressing EGFP and Cap protein which were modified by muti-copies genes, two burst sequence modifying promoter and adding chiken insulator HS4 at the downstream of polyA. Sf9 cells infected with the recombinant baculovirus rBac-Cap were treated by negative staining using 3% PTA. We could observe some virus like particles under the electron microscopy. In order to improve the expression of PCV2 Cap protein in the baculovirus system, we used EGFP as a report protein to verify the effects of different vector constructed strategies. Then, we explored the reasons by quantitative RT-PCR. In order to research PCV2 Cap protein expression we established a quentitative ELISA detection method of PCV2 Cap protein. The results showed that the expression of protein in three copies gene and adding forward insulator HS4 at the downstream of polyA in the positive direction were increased about 1.24times compared to rBac-Cap (control). The protein expression level improved in similar degrees in recombant virus modified by two burst sequences and two copied genes.In order to study the expression and secretion of CDV-VLPs, we amplified four structu-ral protein genes(M,H,N and F)of canine distemper virus by reverse transcription, and compared and analyzed amino acid sequence homology of H, N, F of our CDV with the amino acid sequence published in NCBI. We found that N protein was relative conservation, H and F protein had higher mutation. Then, we constructed recombinant virus which could express M, H, N, F proteins. Sf9 cells were infected by these recombinant baculovirus virus in alone or co-infected. We could observe some virus like particles under the electron microscopy when Sf9 cells were infected by rBac-M. The results showed that CDV-VLPs could form virus like particles when M protein of CDV was expressed in insect alone. Sf9 cells infected with different combinations of recombinant virus were analyzed by immunoblotting, the results showed that N, H and F proteins couldn't to be secreted into the supernatant with M protein.