Myllymäki Henna, Niskanen Mirja, Oksanen Kaisa, Rämet Mika
BioMediTech Institute, Faculty of Medicine and Life Sciences, University of Tampere.
BioMediTech Institute, Faculty of Medicine and Life Sciences, University of Tampere; MedEngine Oy.
J Vis Exp. 2018 Oct 30(140). doi: 10.3791/58453.
The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.
在过去二十年中，基于DNA的疫苗接种的关注度有所提高。DNA疫苗接种是基于将选定抗原或抗原组合的序列克隆到质粒中，这使得能够进行定制且安全的设计。将DNA疫苗接种到宿主细胞中会导致抗原表达，从而刺激体液免疫和细胞介导的免疫反应。本报告描述了将抗原序列克隆到pCMV-EGFP质粒中的方案、通过肌肉内显微注射用候选疫苗对成年斑马鱼进行免疫接种以及随后进行电穿孔以提高摄取量的过程。疫苗抗原以绿色荧光蛋白（GFP）融合蛋白的形式表达，这使得能够在紫外线下从活鱼确认抗原表达，并用酶联免疫吸附测定法（ELISA）对融合蛋白的表达水平进行定量，以及通过蛋白质印迹分析对其进行检测。在接种疫苗五周后用海鱼分枝杆菌感染鱼，然后在四周后用实时定量聚合酶链反应（qPCR）对细菌进行定量，以此来测试候选疫苗的保护效果。与哺乳动物临床前筛选模型相比，该方法为针对分枝杆菌感染的新型基于DNA的候选疫苗的初步筛选提供了一种经济高效的方法。该方法可进一步应用于筛选针对各种细菌和病毒疾病的基于DNA的疫苗。
