Du Lanying, Zhang Xiujuan, Liu Jixiang, Jiang Shibo
Lindsley F. Kimball Research Institute, New York Blood Center, USA.
J Vis Exp. 2011 May 2(51):2444. doi: 10.3791/2444.
Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity. Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method with some modifications. Compared with the lipid transfection method, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory. The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.
基于其安全性以及诱导针对感染的有效免疫反应的能力，亚单位疫苗已被用作多种病原体的候选疫苗。由于哺乳动物细胞系统能够进行翻译后修饰，从而形成正确折叠和糖基化的蛋白质，在哺乳动物细胞中表达的重组蛋白显示出维持高抗原性和免疫原性的最大潜力。自2004年以来，虽然没有新的SARS病例报告，但未来的疫情爆发始终是一个威胁；因此，研发针对SARS-CoV的疫苗是一项审慎的预防措施，应该开展。SARS-CoV S蛋白的受体结合域（RBD）在受体结合以及诱导针对病毒感染的特异性中和抗体方面发挥着重要作用。因此，在本方案中，我们描述了开发一种基于RBD的SARS亚单位疫苗的新方法。简要来说，重组RBD蛋白（rRBD）在哺乳动物293T细胞的培养上清液中表达，以获得具有正确构象和高免疫原性的正确折叠蛋白。然后使用经过一些改进的磷酸钙转染方法将编码RBD的重组质粒转染到细胞中。与脂质转染方法相比，这种改进的磷酸钙转染方法更便宜、更易于操作，并且一旦形成具有合适大小和形状的转染复合物，就有可能达到高效。最后，本方案引入了一种SARS假病毒中和试验，并用于检测用rRBD蛋白免疫的小鼠血清的中和活性。该试验相对安全，不涉及传染性SARS-CoV，并且无需在生物安全3级实验室中即可进行。这里描述的方案也可用于设计和研究针对其他具有I类融合蛋白的病毒的重组亚单位疫苗，例如，HIV、呼吸道合胞病毒（RSV）、埃博拉病毒、流感病毒以及尼帕病毒和亨德拉病毒。此外，产生假病毒并随后建立假病毒中和试验的方法可应用于所有这些病毒。
