分子佐剂 mC3d 增强了肠炎沙门氏菌血清型肠型菌毛 FimA 的免疫原性。
The molecular adjuvant mC3d enhances the immunogenicity of FimA from type I fimbriae of Salmonella enterica serovar Enteritidis.
机构信息
College of Veterinary Medicine, Yangzhou University, Yangzhou, China; Faculty of Veterinary Science, University of Nyala, Sudan.
College of Veterinary Medicine, Yangzhou University, Yangzhou, China.
出版信息
J Microbiol Immunol Infect. 2014 Feb;47(1):57-62. doi: 10.1016/j.jmii.2012.11.004. Epub 2013 Jan 24.
BACKGROUND
The fimbriae of Salmonella enterica serovar Enteritidis are used for colonization and invasion into host cells, and have drawn considerable interest because fimbriae can serve as potential immunogens against many pathogenic bacteria that colonize on epithelial surfaces. The purpose of the study is to use a molecular adjuvant, C3d, to enhance the immunogenicity of FimA proteins against Salmonella enterica serovar Enteritidis.
METHODS
FimA of type I fimbriae from Salmonella enteritidis and FimA with one copy of mC3d, two copies of mC3d2 and three copies of mC3d3 were cloned into the expression vector pCold-TF. Soluble fusion proteins of FimA with different copy of mC3d were induced by IPTG and expressed into Escherichia coli BL21 (DE3). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the recombinant proteins from pCold-TF-fimA, TF-fimA-mC3d, TF-fimA-mC3d2, TF-fimA-mC3d3 were 70 kDa, 100 kDa, 130 kDa and 160 kDa, respectively. The fusion protein was recognized by rabbit anti-fimbriae polyclonal antibodies, and then visualized by goat anti-rabbit polyclonal antibodies with a chrome appearance by enzyme-subtract interaction. The recombinant proteins were purified by Ni-TED (tris-carboxymethyl ethylene diamine), immobilized metal ion affinity chromatography (IMAC). Balb/c mice were subcutaneously immunized with the purified proteins and the immune response was monitored by an enzyme-linked immunosorbent assay (ELISA) for FimA-specific antibody. The immunized mice were challenged with a 10-fold LD50 dose (i.e., 100 CFU) of Salmonella enterica serovar Enteritidis standard strain (SD-2) 1 week after the second immunization.
RESULTS
The immunized mice with the fusion proteins FimA-mC3d2 and FimA-mC3d3 had increased levels of ELISA titer of antibody that were 2 and 4 logs, respectively, more immunogenic than the TF-FimA protein alone. The challenge results showed that immune protection rate in the mice immunized with 10 μg of FimA, FimA-mC3d2, and FimA-mC3d3 were 50%, 75% and 100%, respectively.
CONCLUSION
We conclude that mC3d can be expressed in a prokaryotic vector and enhance the immune response of the recombinant protein. FimA-mC3d3 is potentially a subunit vaccine against S. enterica serovar Enteritidis infection.
背景
肠炎沙门氏菌血清型 Enteritidis 的菌毛用于定植和入侵宿主细胞，由于菌毛可以作为潜在的免疫原，对抗定植在上皮表面的许多致病性细菌，因此引起了相当大的关注。本研究旨在使用分子佐剂 C3d 来增强 FimA 蛋白对肠炎沙门氏菌血清型 Enteritidis 的免疫原性。
方法
从肠炎沙门氏菌血清型 Enteritidis 的 I 型菌毛中克隆 FimA 以及带有一个拷贝 mC3d、两个拷贝 mC3d2 和三个拷贝 mC3d3 的 FimA，将其克隆到表达载体 pCold-TF 中。通过 IPTG 诱导可溶性融合蛋白 FimA 与不同拷贝的 mC3d 的表达，并在大肠杆菌 BL21（DE3）中表达。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）显示，来自 pCold-TF-fimA、TF-fimA-mC3d、TF-fimA-mC3d2 和 TF-fimA-mC3d3 的重组蛋白分别为 70 kDa、100 kDa、130 kDa 和 160 kDa。融合蛋白被兔抗菌毛多克隆抗体识别，然后通过酶消除相互作用，用山羊抗兔多克隆抗体显示出铬外观。通过 Ni-TED（三羧甲基乙二胺）、固定化金属离子亲和层析（IMAC）纯化重组蛋白。用纯化蛋白对 Balb/c 小鼠进行皮下免疫，通过酶联免疫吸附试验（ELISA）监测针对 FimA 特异性抗体的免疫反应。在第二次免疫后 1 周，用 10 倍 LD50 剂量（即 100 CFU）的肠炎沙门氏菌血清型 Enteritidis 标准株（SD-2）对免疫小鼠进行攻毒。
结果
与单独的 TF-FimA 蛋白相比，融合蛋白 FimA-mC3d2 和 FimA-mC3d3 免疫的小鼠的 ELISA 抗体滴度分别增加了 2 个和 4 个对数级，具有更高的免疫原性。攻毒结果表明，用 10 μg FimA、FimA-mC3d2 和 FimA-mC3d3 免疫的小鼠的免疫保护率分别为 50%、75%和 100%。
结论
我们得出结论，mC3d 可以在原核载体中表达，并增强重组蛋白的免疫反应。FimA-mC3d3 可能是肠炎沙门氏菌血清型 Enteritidis 感染的亚单位疫苗。
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