Özet:

  Objective(s):  Infection by high-risk papillomavirus is regarded as the major risk factor in the development of cervical cancer. Recombinant DNA technology allows expression of the L1 major capsid protein of HPV in different expression systems, which has intrinsic capacity to self-assemble into viral-like particles (VLP). VLPS are non-infectious, highly immunogenic and can elicit neutralizing antibodies. VLP-based HPV vaccines can prevent persistent HPV infections and cervical cancer. In this study recombinant HPV-16 L1 protein was produced in Sf9 insect cells and VLP formation was confirmed. Materials and Methods: Complete HPV-16 L1 gene was inserted into pFast HTa plasmid and transformed into DH10BAC Escherichia coli containing bacmid and helper plasmid. The recombinant Bacmid colonies turned to white and non-recombinant colonies harboring L1 gene remained blue in the presence of X-gal and IPTG in colony selection strategy. To confirm the recombinant bacmid production, PCR was applied using specific L1 primers. To produce recombinant baculovirus, the recombinant bacmid DNA was extracted and transfected into Sf9 cells using Cellfectin. The expression of L1 in Sf9 cells was identified through SDS-PAGE and western blot analysis using specific L1 monoclonal antibody. Self-assembled HPV16L-VLPs in Sf9 cells was confirmed by electron microscopy. Results:The recombinant protein L1 was predominantly ~60 KD in SDS-PAGE with distinct immunoreactivity in western blot analysis and formed VLPS as confirmed by electron microscopy. Conclusion:Application of recombinant baculovirus containing HPV-16 L1 gene will certainly prove to be a constructive tool in production of VLPs for prophylactic vaccine development as well as diagnostic tests.

Anahtar Kelimeler: