Choi Nak-Won, Estes Mary K, Langridge William H R
Center for Molecular Biology and Gene Therapy, Department of Biochemistry and Microbiology, School of Medicine, Loma Linda University, Loma Linda, Califronia 92354, USA.
Viral Immunol. 2006 Spring;19(1):54-63. doi: 10.1089/vim.2006.19.54.
A 90-amino acid peptide from the simian rotavirus SA-11 nonstructural protein, NSP4 was linked to the N-terminus of the Ricinus communis A-B toxin B subunit protein (RTB) and synthesized in Escherichia coli. Recombinant RTB and the NSP4(90)::RTB fusion protein bound artificial receptor glycoprotein asialofetuin in an in vitro enzyme-linked immunosorbent assay (ELISA), demonstrating biological activity of the recombinant protein. Mice co-inoculated with purified recombinant RTB plus NSP4(90) peptide proteins or heat denatured NSP4(90)::RTB fusion protein generated higher titers of serum anti-NSP4(90) IgG antibodies than mice immunized with NSP4(90) peptide alone, indicating the presence of adjuvant functions for N-terminal linked RTB. Serum anti-NSP4(90) IgG titers were highest in mice immunized with native recombinant NSP4(90)::RTB fusion protein, confirming the immunostimulatory function of RTB. Results of experiments described here demonstrate the feasibility of using RTB mediated adjuvant functions for stimulation of the antigenicity of a rotavirus nonstructural protein. The ability of recombinant NSP4(90)::RTB fusion protein synthesized in E. coli to bind glycoprotein receptor molecules effectively indicates that protein linkage to the RTB N-terminus and synthesis of the recombinant NSP4(90)::RTB fusion protein in bacteria do not interfere with the immunostimulatory properties of the RTB subunit.
来自猿猴轮状病毒SA - 11非结构蛋白的一种90个氨基酸的肽NSP4与蓖麻毒素A - B毒素B亚基蛋白（RTB）的N端相连，并在大肠杆菌中合成。在体外酶联免疫吸附测定（ELISA）中，重组RTB和NSP4(90)::RTB融合蛋白与人工受体糖蛋白去唾液酸胎球蛋白结合，证明了重组蛋白的生物学活性。与单独用NSP4(90)肽免疫的小鼠相比，同时接种纯化的重组RTB加NSP4(90)肽蛋白或热变性的NSP4(90)::RTB融合蛋白的小鼠产生了更高滴度的血清抗NSP4(90) IgG抗体，表明N端相连的RTB具有佐剂功能。用天然重组NSP4(90)::RTB融合蛋白免疫的小鼠血清抗NSP4(90) IgG滴度最高，证实了RTB的免疫刺激功能。此处所述实验结果证明了利用RTB介导的佐剂功能刺激轮状病毒非结构蛋白抗原性的可行性。在大肠杆菌中合成的重组NSP4(90)::RTB融合蛋白有效结合糖蛋白受体分子的能力表明，与RTB N端的蛋白连接以及在细菌中合成重组NSP4(90)::RTB融合蛋白不会干扰RTB亚基的免疫刺激特性。
