School of Environmental Science and Engineering, Kochi University of Technology, 185 Miyanokuchi, Tosayamada, Kami, Kochi, 782-8502, Japan.
Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi, 144 Xuan Thuy St., Cau Giay Dist., Hanoi, Vietnam.
Appl Microbiol Biotechnol. 2018 Jan;102(1):261-268. doi: 10.1007/s00253-017-8579-8. Epub 2017 Nov 2.
Japanese cedar pollinosis is a seasonal allergic disease caused by two major pollen allergens: Cry j 1 and Cry j 2 antigens. To develop an oral vaccine to treat pollinosis, we constructed recombinant Lactococcus lactis harboring the gene encoding fused T cell epitopes from the Cry j 1 and Cry j 2 antigens. The recombinant T cell epitope peptide was designed to contain the fused cholera toxin B subunit as an adjuvant and a FLAG tag at the C-terminus. An expression plasmid was constructed by inserting the T cell epitope peptide gene into the multiple cloning sites of plasmid pNZ8148, an Escherichia coli-L. lactis shuttle vector. The constructed plasmid was transformed into L. lactis NZ9000 for expression induced by nisin, an antibacterial peptide from L. lactis. The expression of the epitope peptide was induced with 10-40 ng/mL nisin, and the expressed T cell epitope peptide was detected by western blot analysis using an anti-FLAG antibody and an antibody against the T cell epitopes. The concentration of the epitope peptide was estimated to be ~ 22 mg/L of culture in the presence of 40 ng/mL nisin, although it varied depending on the nisin concentration, the culture time, and the bacterial concentration when nisin was added. The expression of the recombinant epitope peptide in L. lactis, an organism generally recognized as safe, as demonstrated in this study, may contribute to the development of an oral vaccine for the treatment of pollinosis.
Cry j 1 和 Cry j 2 抗原引起的季节性过敏性疾病。为了开发治疗花粉症的口服疫苗，我们构建了携带编码来自 Cry j 1 和 Cry j 2 抗原的融合 T 细胞表位的基因的重组乳球菌 Lactococcus lactis。重组 T 细胞表位肽设计含有融合霍乱毒素 B 亚基作为佐剂和 C 末端的 FLAG 标签。通过将 T 细胞表位肽基因插入大肠杆菌-乳球菌穿梭载体 pNZ8148 的多克隆位点，构建了表达质粒。构建的质粒被转化为 L. lactis NZ9000，以诱导乳链菌肽（一种来自 L. lactis 的抗菌肽）表达。用 10-40ng/mL 的乳链菌肽诱导表位肽的表达，并通过使用抗 FLAG 抗体和针对 T 细胞表位的抗体进行 Western blot 分析来检测表达的 T 细胞表位肽。在存在 40ng/mL 乳链菌肽的情况下，表位肽的浓度估计约为 22mg/L 培养物，尽管它因乳链菌肽浓度、培养时间和添加乳链菌肽时的细菌浓度而异。本研究表明，在通常被认为是安全的生物体乳球菌中表达重组表位肽可能有助于开发治疗花粉症的口服疫苗。
