Pirestani Majid, Dalimi Abdolhossein, Sarvi Shahabodin, Khoramabadi Nima
Dept. of Parasitology and Medical Entomology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Dept. of Parasitology and Mycology, Sari Medical School, Mazandaran University of Medical Sciences, Sari, Iran.
Iran J Parasitol. 2014 Oct-Dec;9(4):491-502.
Echinococcosis is a zoonotic parasitic disease of humans and various herbivorous domestic animals transmitted by the contact with domestic and wild carnivores, mainly dogs and foxes. The aim of this study is the production, purification and evaluation immunogenicity of new construction of EG95 protein.
The recombinant plasmid pET32-a+ used for Eg95 expression was constructed with the EG95 gene of Echinococcus granulosus fused with the thioredoxin tag. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The purification was performed under denaturing conditions in the presence of 8M urea by Ni-NTA column and dialysis. The purified recombinant proteins were confirmed with western blot analysis using polyclonal antiserum. To find out the immunogenicity of the purified protein, the BALB/c mice (10 mice/group) were immunized by injecting 20 μg rEG95 protein formulated in Freund's and alum adjuvant.
Immunization of mice with rEG95 using CFA/IFA and alum adjuvant generated high level of total antibody. In proliferation assay, the lymphocytes were able to mount a strong proliferative response with related production of IFN-γ, IL-12 and TNF-α but with low secretion of either IL-4 or IL-10. The humoral and cellular immune responses against rEG95 suggested a mixed Th1/Th2 response with high intensity toward Th1.
Our findings suggest that new construct of rEG95 formulated with CFA/IFA and alum adjuvant elicited strong cellular and humoral responses supporting further development of this vaccine candidate.
棘球蚴病是一种人畜共患的寄生虫病，通过人与家养和野生食肉动物（主要是狗和狐狸）接触传播给人类和各种草食性家畜。本研究的目的是制备、纯化并评估新构建的EG95蛋白的免疫原性。
构建用于表达Eg95的重组质粒pET32-a+，将细粒棘球绦虫的EG95基因与硫氧还蛋白标签融合。该重组克隆在大肠杆菌BL-21（DE-3）中过量表达。在细胞裂解物中发现表达的融合蛋白几乎完全以不溶性形式（包涵体）存在。在8M尿素存在下于变性条件下通过Ni-NTA柱和透析进行纯化。使用多克隆抗血清通过蛋白质印迹分析确认纯化的重组蛋白。为了确定纯化蛋白的免疫原性，用20μg以弗氏佐剂和明矾佐剂配制的rEG95蛋白免疫BALB/c小鼠（每组10只小鼠）。
使用CFA/IFA和明矾佐剂用rEG95免疫小鼠产生了高水平的总抗体。在增殖试验中，淋巴细胞能够产生强烈的增殖反应，并产生相关的IFN-γ、IL-12和TNF-α，但IL-4或IL-10的分泌量较低。针对rEG95的体液和细胞免疫反应表明是一种混合的Th1/Th2反应，对Th1的强度较高。
我们的研究结果表明，用CFA/IFA和明矾佐剂配制的新构建的rEG95引发了强烈的细胞和体液反应，支持该候选疫苗的进一步开发。
