Davod Jafari, Fatemeh Dehghan Nayeri, Honari Hossein, Hosseini Ramin
Medical Biotechnology Department, School of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.
Biotechnology Department, Faculty of Agricultural and Natural Sciences, Imam Khomeini International University (IKIU), Qazvin, Iran.
Mol Biol Rep. 2018 Dec;45(6):2237-2246. doi: 10.1007/s11033-018-4385-3. Epub 2018 Sep 22.
Shigella dysenteriae causing shigellosis is one of the diseases that threaten the health of human society in the developing countries. In Shigella, IpaD gene is one of the key pathogenic genes causing strong mucosal immune system reactions. Anthrax disease is caused by Bacillus anthracis. PA protective antigen is one of the subunits in anthrax toxin complex responsible for the transfer of other subunits into the cytosol of host cells. The 20 kDa subunit of PA (PA20) has the property of immunogenicity. CTxB or B subunit of Vibrio cholerae toxin (CT) is a non-toxic protein and has the function to transfer toxic subunit into cytosol of the host cells by binding to GM1 receptor. The aim of this study was to fuse PA20, ipaD and CTxB and transform tomato plants by this cassette in order to produce an oral vaccine against shigellosis, anthrax and cholera. CTxB was used for these two antigens as an immune adjuvant. IpaD and PA20 genes were cloned in pBI121 containing the CTxB gene and Extensin signal peptide. In order to evaluate the transient expression of Shigellosis, Anthrax and Cholera antigens, agro-infiltrated tomato tissues were inoculated with Agrobacterium tumefaciens containing the gene cassette. Cloning was confirmed by PCR, enzymatic digestion and sequencing techniques. Expression of the antigens was examined by SDS-PAGE, dot blot and ELISA. Maturate green fruits demonstrated the highest expression of the recombinant proteins. The first phase of this study was carried out for cloning and expressing of CtxB, ipaD and PA20 antigens in tomato. In the next phase, we aim to analyze the immunogenicity of this vaccine candidate in laboratory animals.
引起志贺氏菌病的痢疾志贺氏菌是发展中国家威胁人类社会健康的疾病之一。在志贺氏菌中，IpaD基因是引发强烈黏膜免疫系统反应的关键致病基因之一。炭疽病由炭疽芽孢杆菌引起。PA保护性抗原是炭疽毒素复合物中的亚基之一，负责将其他亚基转运到宿主细胞的胞质溶胶中。PA的20 kDa亚基（PA20）具有免疫原性。霍乱弧菌毒素（CT）的CTxB或B亚基是一种无毒蛋白质，具有通过与GM1受体结合将毒性亚基转运到宿主细胞胞质溶胶中的功能。本研究的目的是将PA20、ipaD和CTxB融合，并通过该盒式结构转化番茄植株，以生产针对志贺氏菌病、炭疽和霍乱的口服疫苗。CTxB用作这两种抗原的免疫佐剂。IpaD和PA20基因被克隆到含有CTxB基因和伸展蛋白信号肽的pBI121中。为了评估志贺氏菌病、炭疽和霍乱抗原的瞬时表达，用含有基因盒的根癌农杆菌接种经农杆菌浸润的番茄组织。通过PCR、酶切和测序技术确认克隆。通过SDS-PAGE、斑点印迹和ELISA检测抗原的表达。成熟绿色果实中重组蛋白的表达最高。本研究的第一阶段是在番茄中克隆和表达CtxB、ipaD和PA20抗原。在下一阶段，我们旨在分析这种候选疫苗在实验动物中的免疫原性。
