Laboratório de Imunologia Viral, Instituto Butantan, Avenida Vital Brasil, 1500, São Paulo Brazil.
Cell Line Development and Molecular Biotechnology Laboratory, iBET - Instituto de Biologia Experimental e Tecnológica, Av. República, Qta. do Marquês, Oeiras Portugal.
J Biotechnol. 2018 May 20;274:33-39. doi: 10.1016/j.jbiotec.2018.03.010. Epub 2018 Mar 22.
Viral hepatitis caused by the hepatitis C virus (HCV) affects millions of people worldwide. The non-structural protein 3 (NS3), one of the most conserved proteins in HCV, is the target of many therapeutic studies. The NS3 protease domain (NS3p) has a range of cytotoxic T lymphocyte (CTL) epitopes, and synthesizing the protein inside the cells is the most appropriate way to present it to the immune system. We developed a tool to study this kind of presentation, using two vectored particle (VP) systems, one based on the Semliki Forest virus (SFV) and the other on HCV pseudoparticles (HCVpp), both carrying the protease domain of the NS3 gene. In addition to producing the particles, we developed a method to quantify these VPs using qRT-PCR. We produced batches of approximately 2.4 × 10 SFV-NS3p/μL and 4.0 × 10 HCVpp-NS3p/μL. BHK-21 and HuH-7 cells treated with the VPs expressed the NS3 protein, thus showing the functionality of this system.
丙型肝炎病毒（HCV）引起的病毒性肝炎影响着全球数百万人。非结构蛋白 3（NS3）是 HCV 中最保守的蛋白之一，是许多治疗研究的目标。NS3 蛋白酶结构域（NS3p）具有多种细胞毒性 T 淋巴细胞（CTL）表位，在细胞内合成蛋白质是将其递呈给免疫系统的最佳方式。我们开发了一种工具来研究这种呈递方式，使用了两种载体粒子（VP）系统，一种基于 Semliki Forest 病毒（SFV），另一种基于 HCV 假病毒（HCVpp），两者都携带 NS3 基因的蛋白酶结构域。除了产生粒子外，我们还开发了一种使用 qRT-PCR 定量这些 VP 的方法。我们生产了大约 2.4×10 SFV-NS3p/μL 和 4.0×10 HCVpp-NS3p/μL 的批次。用 VP 处理的 BHK-21 和 HuH-7 细胞表达了 NS3 蛋白，从而显示了该系统的功能。
