Aghdasi Marjaneh, Ghaffarifar Fatemeh, Forooghi Fatemeh, Dalimi Asl Abdol Hossein, Sharifi Zohre, Maspi Nahid
Dept. of Parasitology, Faculty of Medical Sciences, University of Tarbiat Modarres, Tehran, Iran.
Research Center of Iranian Blood Transfusion Organization, Tehran, Iran.
Iran J Parasitol. 2018 Jul-Sep;13(3):392-398.
Toxoplasmosis is a common infection all around the world. During pregnancy; it may lead to congenital disorders or abortion in human and animals. Severe damage of toxoplasmosis indicates to require effective vaccine. One of dense granules antigen is GRA4 that secrete from tachyzoite and bradyzoite. GRA4 genome is unique without intron and is one of the major immunogenic proteins from
We confirmed the cloning of GRA4 gene into pcDNA3 by restriction enzyme and PCR of GRA4 gene with pcGRA4 plasmids as template. Then with using calcium-phosphate method we transfected the pcGRA4 into CHO (Chinesehamster ovary) cells. The yielded protein was separated by SDS-PAGE and moved by electroblotting to nitrocellulose paper.
Result of SDS-PAGE analysis showed the appearance of band approximately 42 kDa which was absent in the negative control, that was able to identify toxoplasmosis antibody IgM serum in western blot analysis.
pcGRA4 plasmid is able to synthesis of antigenic protein in CHO cells. The ability of pcGRA4 for induction of protective immune response against toxoplasmosis will be evaluated in mouse model.
弓形虫病是一种全球常见的感染病。在孕期，它可能导致人类和动物的先天性疾病或流产。弓形虫病的严重危害表明需要有效的疫苗。致密颗粒抗原之一是速殖子和缓殖子分泌的GRA4。GRA4基因组独特，无内含子，是主要的免疫原性蛋白之一。
我们以pcGRA4质粒为模板，通过限制性内切酶和GRA4基因的PCR，证实了GRA4基因克隆到pcDNA3中。然后采用磷酸钙法将pcGRA4转染到CHO（中国仓鼠卵巢）细胞中。所得蛋白质通过SDS-PAGE分离，并通过电印迹转移到硝酸纤维素纸上。
SDS-PAGE分析结果显示出现了一条约42 kDa的条带，阴性对照中没有，该条带能够在蛋白质印迹分析中识别弓形虫病抗体IgM血清。
pcGRA4质粒能够在CHO细胞中合成抗原性蛋白。将在小鼠模型中评估pcGRA4诱导针对弓形虫病的保护性免疫反应的能力。
