Sefidi Mozhgan Derakhshan, Rasooli Iraj, Owlia Parviz, Talei Daryush, Astaneh Shakiba Darvish Alipour, Nazarian Shahram
Mozhgan Derakhshan Sefidi, Iraj Rasooli, Department of Biology, Shahed University, Tehran 3319118651, Iran.
World J Methodol. 2016 Sep 26;6(3):190-9. doi: 10.5662/wjm.v6.i3.190.
To study immunogenicity of Pseudomonas N terminal flagellin as an adjuvant for Acinetobacter baumannii (A. baumannii) biofilm associated protein (Bap).
The N terminal flagellin gene was amplified. The pET28a (+) and polymerase chain reaction products were digested with HindIII and EcoR I. The ligation of N terminal flagellin into pET28a (+) was performed using T4 DNA ligase and was then transformed into Escherichia coli BL21 (DE3) as a suitable expression host. pET28a (+) vector harboring a conserved region of Bap from our previous work was used. The recombinant proteins were expressed, analyzed by SDS-PAGE method and was purified by affinity chromatography with His-Tag residues followed by confirmation with western blotting. Mice were immunized with recombinant N terminal flagellin and Bap subunits. The immunized animals were intranasally (i.n) challenged with A. baumannii and Pseudomonas aeruginosa (P. aeruginosa).
The flagellin enhanced the immunogenicity of Bap causing an increase in specific IgG titers in serum (P < 0.001). Internal organs, i.e., liver, lung and spleen of the Bap-Flagellin immunized group challenged with A. baumannii showed significantly lower bacterial load compared to the control group. The bacterial loads were studied in internal organs. A. baumannii infected immunized animals with Bap-Flagellin exhibited internal organs with minor bacterial load while P. aeruginosa PAO1 infected group showed heavy bacterial load of (4.3 ± 0.12) × 10(6), (1.1 ± 0.01) × 10(6) and (2.2 ± 0.22) × 10(6) per gram of lungs, liver and spleen respectively. Bacterial loads were detected per gram of lungs, liver and spleen of the mice group immunized with Bap were (1.2 ± 0.06) × 10(7), (11.1 ± 0.041) × 10(5) and (3.6 ± 0.42) × 10(6) respectively. In vivo neutralization assay indicated that all experimental mice groups, except for Flagellin administered group was significantly (P < 0.05) protected against A. baumannii.
These results demonstrate that P. aeruginosa Flagellin as an adjuvant for Bap A. baumannii could be a useful model to evaluate new vaccine against A. baumannii.
研究铜绿假单胞菌N端鞭毛蛋白作为鲍曼不动杆菌生物膜相关蛋白（Bap）佐剂的免疫原性。
扩增N端鞭毛蛋白基因。用HindIII和EcoR I对pET28a（+）和聚合酶链反应产物进行酶切。使用T4 DNA连接酶将N端鞭毛蛋白连接到pET28a（+）中，然后转化到大肠杆菌BL21（DE3）作为合适的表达宿主。使用我们之前工作中含有Bap保守区域的pET28a（+）载体。表达重组蛋白，通过SDS-PAGE方法进行分析，并用His-Tag残基通过亲和层析进行纯化，随后通过蛋白质印迹法进行确认。用重组N端鞭毛蛋白和Bap亚基免疫小鼠。对免疫动物进行鼻内（i.n）鲍曼不动杆菌和铜绿假单胞菌攻击。
鞭毛蛋白增强了Bap的免疫原性，导致血清中特异性IgG滴度升高（P < 0.001）。与对照组相比，用鲍曼不动杆菌攻击的Bap-鞭毛蛋白免疫组的内脏器官，即肝脏、肺和脾脏显示出明显较低的细菌载量。在内脏器官中研究细菌载量。用Bap-鞭毛蛋白感染免疫动物的鲍曼不动杆菌在内脏器官中的细菌载量较小，而铜绿假单胞菌PAO1感染组每克肺、肝和脾的细菌载量分别为（4.3±0.12）×10⁶、（1.1±0.01）×10⁶和（2.2±0.22）×10⁶。用Bap免疫的小鼠组每克肺、肝和脾中检测到的细菌载量分别为（1.2±0.06）×10⁷、（11.1±0.041）×10⁵和（3.6±0.42）×10⁶。体内中和试验表明，除鞭毛蛋白给药组外，所有实验小鼠组均对鲍曼不动杆菌有显著（P < 0.05）的保护作用。
这些结果表明，铜绿假单胞菌鞭毛蛋白作为鲍曼不动杆菌Bap的佐剂可能是评估新型抗鲍曼不动杆菌疫苗的有用模型。
