Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Iran J Basic Med Sci. 2013 Aug;16(8):891-5.
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.
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.
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.
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.
高危型人乳头瘤病毒（HPV）感染被认为是宫颈癌发展的主要危险因素。重组 DNA 技术允许 HPV 的 L1 主要衣壳蛋白在不同的表达系统中表达，该蛋白具有内在的自我组装成病毒样颗粒（VLP）的能力。VLPs 是非感染性的，具有高度的免疫原性，可以引发中和抗体。基于 VLP 的 HPV 疫苗可以预防持续性 HPV 感染和宫颈癌。在本研究中，HPV-16 L1 蛋白在 Sf9 昆虫细胞中进行了表达，并证实了 VLP 的形成。
完整的 HPV-16 L1 基因被插入 pFast HTa 质粒中，并转化到含有 bacmid 和辅助质粒的 DH10BAC 大肠杆菌中。重组 Bacmid 菌落因 X-gal 和 IPTG 的存在而变为白色，而非重组菌落则因含有 L1 基因而保持蓝色，这是在菌落选择策略中实现的。为了确认重组 bacmid 的产生，使用特定的 L1 引物进行了 PCR。为了生产重组杆状病毒，使用 Cellfectin 将重组 bacmid DNA 转染到 Sf9 细胞中。通过 SDS-PAGE 和使用特异性 L1 单克隆抗体的 Western blot 分析，鉴定 Sf9 细胞中 L1 的表达。通过电子显微镜证实 Sf9 细胞中 HPV16L-VLPs 的自组装。
SDS-PAGE 中 L1 重组蛋白主要为~60KD，Western blot 分析中具有明显的免疫反应性，电子显微镜证实形成了 VLPs。
应用含有 HPV-16 L1 基因的重组杆状病毒肯定将被证明是生产预防性疫苗开发和诊断测试用 VLPs 的有建设性的工具。
