Faezi Sobhan, Bahrmand Ahmad Reza, Mahdavi Mehdi, Siadat Seyed Davar, Sardari Soroush, Nikokar Iraj, Khanaki Korosh, Mirzajani Ebrahim, Goudarzi Gholamreza
Departments of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran; Mycobacteriology Research Center (MRS), Pasteur Institute of Iran, Tehran, Iran.
Departments of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran; Mycobacteriology Research Center (MRS), Pasteur Institute of Iran, Tehran, Iran.
Biologicals. 2017 May;47:11-17. doi: 10.1016/j.biologicals.2017.04.001. Epub 2017 Apr 10.
Mucoid strains of Pseudomonas aeruginosa are closely associated with chronic pulmonary infections. In this report we describe a straightforward approach to conjugate high molecular weight alginate to type b-flagellin (FLB) and investigation of its bioactivity. The conjugation process was performed by using ADH and EDAC. The endotoxin was eliminated from the candidate vaccine by LPS removal resin followed by LAL test. The bioconjugate molecules were verified by simultaneously determination of polysaccharide/protein content followed by gel filtration chromatography and FTIR spectroscopy. Groups of eight BALB/c mice were injected intranasally with 5 μg (per each nostril) of purified alginate, FLB and conjugated alginate-FLB with two week intervals. The functional activity of the vaccine was evaluated by ELISA and opsonophagocytosis tests. Vaccination with the alginate-FLB conjugate induced a significant (P = 0.0033) rise in alginate specific IgG in mice. At all dilution ranges, the opsonic activity of the conjugate vaccine antisera was significantly higher than alginate alone (61.9% vs. 17.3% at 1:4 dilution; P = 0.0067). The alginate-FLB conjugate could elicit high specific antibodies titer against alginate by improving its immunogenicity. In addition, the antisera raised against conjugate vaccine act as a suitable opsonin for phagocytosis of the mucoid strains of P. aeruginosa.
铜绿假单胞菌的黏液样菌株与慢性肺部感染密切相关。在本报告中，我们描述了一种将高分子量藻酸盐与b型鞭毛蛋白（FLB）偶联并研究其生物活性的直接方法。偶联过程通过使用ADH和EDAC进行。通过LPS去除树脂去除候选疫苗中的内毒素，随后进行LAL检测。通过同时测定多糖/蛋白质含量，然后进行凝胶过滤色谱和FTIR光谱法来验证生物偶联分子。将八组BALB/c小鼠每隔两周经鼻内注射5μg（每个鼻孔）纯化的藻酸盐、FLB和偶联的藻酸盐-FLB。通过ELISA和调理吞噬作用试验评估疫苗的功能活性。用藻酸盐-FLB偶联物接种疫苗可使小鼠体内藻酸盐特异性IgG显著升高（P = 0.0033）。在所有稀释范围内，偶联疫苗抗血清的调理活性均显著高于单独的藻酸盐（在1:4稀释时为61.9%对17.3%；P = 0.0067）。藻酸盐-FLB偶联物可通过提高其免疫原性引发针对藻酸盐的高特异性抗体滴度。此外，针对偶联疫苗产生的抗血清可作为吞噬铜绿假单胞菌黏液样菌株的合适调理素。
