Fathi Javad, Amani Jafar, Nazarian Shahram, Hadi Nahal, Mirhosseini Seyed Ali, Ranjbar Reza, Abianeh Hossein Samiei
Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Department of Biological Sciences, Faculty of Science, Imam Hossein University, Tehran, Iran.
Microb Pathog. 2023 May;178:106066. doi: 10.1016/j.micpath.2023.106066. Epub 2023 Mar 15.
Shigella spp. causes bloody diarrhea and leads to death, especially in children. Chimeric proteins containing virulence factors can prevent Shigella infection. The purpose of this study is to investigate the immunogenic and protective effect of trivalent chimeric protein containing IpaD-StxB-TolC antigens against shiga toxin, S. dysenteri and S. flexneri in vitro and in vivo conditions.
Recombinant vector was transferred to E. coli BL21. The expression of the chimeric protein was confirmed by SDS PAGE and purified using the Ni-NTA column. Mice were immunized with recombinant protein and antibody titer was evaluated by ELISA. 10, 25 and 50 LD of Shiga toxin neutralization was evaluated in vitro (Vero cell line) and in vivo conditions. Also, the challenge of immunized mice with 10, 25 and 50 LD of S. dysentery and S. flexneri was done.
The expression and purification of the recombinant protein with 60.6 kDa was done. ELISA showed increased antibody titer against the chimeric protein. MTT assay indicated that 1/8000 dilution of the sera had a 51% of cell viability against the toxin in Vero cell line. The challenge of mice immunized with toxin showed that the mice had complete protection against 10 and 25 LD of toxin and had 40% survival against 50 LD. Mice receiving 10 and 25 LD of S. dysenteri and S. flexneri had 100% protection and in 50 LD the survival rate was 60 and 50%, respectively. Organ burden showed that the amount of bacterial colonization in immunized mice was 1 × 10 CFU/mL, which was significantly different from the control group.
This study showed that chimeric proteins can create favorable immunogenicity in the host as vaccine candidates.
志贺氏菌属可引起血性腹泻并导致死亡，尤其是在儿童中。含有毒力因子的嵌合蛋白可预防志贺氏菌感染。本研究的目的是调查包含IpaD - StxB - TolC抗原的三价嵌合蛋白在体外和体内条件下对志贺毒素、痢疾志贺氏菌和福氏志贺氏菌的免疫原性和保护作用。
将重组载体转入大肠杆菌BL21。通过SDS - PAGE确认嵌合蛋白的表达，并使用镍 - 亚氨基二乙酸（Ni - NTA）柱进行纯化。用重组蛋白免疫小鼠，并通过酶联免疫吸附测定（ELISA）评估抗体效价。在体外（Vero细胞系）和体内条件下评估10、25和50个志贺毒素致死剂量（LD）的中和情况。此外，用10、25和50个LD的痢疾志贺氏菌和福氏志贺氏菌对免疫小鼠进行攻毒。
完成了60.6 kDa重组蛋白的表达和纯化。ELISA显示针对嵌合蛋白的抗体效价增加。噻唑蓝（MTT）测定表明，血清1/8000稀释度对Vero细胞系中的毒素具有51%的细胞活力。用毒素免疫的小鼠攻毒显示，小鼠对10和25个LD的毒素具有完全保护作用，对50个LD的毒素有40%的存活率。接受10和25个LD的痢疾志贺氏菌和福氏志贺氏菌的小鼠有100%的保护作用，在50个LD时存活率分别为60%和50%。器官负荷显示，免疫小鼠中的细菌定植量为1×10 CFU/mL，与对照组有显著差异。
本研究表明，嵌合蛋白作为候选疫苗可在宿主体内产生良好的免疫原性。
