Pilot-scale production and characterization of paramyosin, a vaccine candidate for schistosomiasis japonica
Pilot-scale production and characterization of paramyosin, a vaccine candidate for schistosomiasis japonica
复制标题
DOI：
10.1128/iai.00409-08
复制
发表时间：
2008-07-01
影响因子：
3.1
通讯作者：
Kurtis, Jonathan D.
中科院分区：
文献类型：
--
作者：
Jiz, Mario;Wu, Hai-Wei;Kurtis, Jonathan D.
尽管有有效的化疗，血吸虫病仍然是发展中国家的一个主要公共卫生问题，至少有 2 亿活动性感染导致严重发病。治疗后快速再感染，伴随着广泛的残余发病率，需要替代控制策略，包括疫苗开发。副肌球蛋白是一种仅在无脊椎动物中发现的肌原纤维蛋白，作为曼氏血吸虫和日本血吸虫的候选疫苗已被广泛研究。最近，我们证明对副肌球蛋白的 Th2 型免疫反应与人类对日本血吸虫再感染的抵抗力有关。然而，血吸虫副肌球蛋白中试规模生产的挑战阻碍了对这种有希望的候选疫苗的进一步研究。在这里，我们报告了重组日本血吸虫副肌球蛋白（rSj97）的中试规模表达和纯化方法。 rSj97 从大肠杆菌包涵体中提取，并通过顺序阴离子交换、羟基磷灰石和尺寸排阻色谱进行纯化。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析判断，纯化的rSj97纯度>95%，并且没有明显的内毒素污染。我们证明，与天然副肌球蛋白一样，rSj97 采用 α 螺旋卷曲螺旋三级结构并结合免疫球蛋白和胶原蛋白。感染日本血吸虫的幼鼠最早在感染后 4 周就产生抗 rSj97 免疫球蛋白 G (IgG) 抗体，而从日本血吸虫感染个体收集的血清含有抗 rSj97 IgE 抗体。我们的重组全长副肌球蛋白的中试规模生产方法将有助于副肌球蛋白作为血吸虫病疫苗的临床前评估。
Despite effective chemotherapy, schistosomiasis remains a major public health problem in the developing world, with at least 200 million active infections resulting in significant morbidity. Rapid reinfection after treatment, accompanied by extensive residual morbidity, mandates alternative control strategies, including vaccine development. Paramyosin, a myofibrillar protein found only in invertebrates, has been widely studied as a vaccine candidate for both Schistosoma mansoni and Schistosoma japonicum. Recently, we demonstrated that Th2-biased immune responses to paramyosin are associated with resistance to reinfection with S. japonicum in humans; however, challenges in the pilot-scale production of schistosome paramyosin have hampered further studies of this promising vaccine candidate. Here we report a method for the pilot-scale expression and purification of recombinant S. japonicum paramyosin (rSj97). rSj97 was extracted from Escherichia coli inclusion bodies and purified with sequential anion-exchange, hydroxyapatite, and size exclusion chromatography. The purified rSj97 was >95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis and was free of significant endotoxin contamination. We demonstrate that, like native paramyosin, rSj97 adopts an alpha-helical coiled-coil tertiary structure and binds immunoglobulin and collagen. Naive mice infected with S.japonicum produce anti-rSj97 immunoglobulin G (IgG) antibodies as early as 4 weeks postinfection, while sera collected from S. japonicum-infected individuals contain anti-rSj97 IgE antibodies. Our method for pilot-scale production of recombinant full-length paramyosin will facilitate preclinical evaluation of paramyosin as a vaccine for schistosomiasis.