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目的研制铜绿假单胞菌3型O-特异性多糖(O-specific polysaccharide,O-SP)-破伤风类毒素(Tetanus toxoid,TT)结合疫苗,并分析其免疫特性。方法采用热酚水法提取铜绿假单胞菌国际抗原分型系统(International Antigen Typing System,IATS)3型菌株的脂多糖(Lipopolysacchride,LPS),去除类脂A后纯化O-SP,用1-氰基-4-二甲胺基吡啶四氟硼酸盐(CDAP)活化O-SP,己二酸二肼(ADH)作为连接臂,在碳二亚胺(EDAC)作用下,与TT结合制备O-SP-TT结合疫苗,检测其理化性质,并分析结合疫苗的免疫原性和免疫保护性。结果 O-SP收获量相当于水解LPS量的30%,其蛋白质和核酸含量均低于1.0%,O-SP衍化度为3.83%,O-SP与TT的结合率约为40%,多糖蛋白质比(w/w)为1∶2.18。O-SP-TT结合疫苗可刺激小鼠产生高效价的IgG抗体,与O-SP组相比,差异有统计学意义(P<0.05);结合疫苗免疫小鼠能保护5 LD50和10 LD50活菌的攻击,小鼠免疫O-SP-TT后兔抗血清对3 LD50和5 LD50活菌攻击具有良好的保护作用。结论制备的铜绿假单胞菌3型O-SP-TT结合疫苗具有良好的免疫原性和免疫保护性,该疫苗有望成为防治IATS 3型铜绿假单胞菌感染的有效疫苗。
Objective To develop an O-specific polysaccharide (O-SP) -tetra tetanus toxoid (TT) conjugate vaccine against Pseudomonas aeruginosa and analyze its immunogenicity. Methods Lipopolysacchride (LPS) was isolated from hot spot of Pseudomonas aeruginosa type IATS by hot phenolic water method. O-SP was purified after removal of lipid A, Cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) activated O-SP, adipic acid dihydrazide (ADH) as a linker, prepared by combining with TT under carbodiimide (EDAC) The O-SP-TT conjugate vaccine was tested for its physico-chemical properties and analyzed for its immunogenicity and immunoprotective properties. Results The yield of O-SP was equivalent to 30% of the amount of hydrolyzed LPS, the content of protein and nucleic acid was less than 1.0%, the yield of O-SP was 3.83%, the binding rate of O-SP and TT was about 40% The ratio (w / w) is 1: 2.18. O-SP-TT conjugate vaccine stimulated mice to produce high titer of IgG antibody, compared with O-SP group, the difference was statistically significant (P <0.05); vaccinated mice can protect 5 LD50 and 10 LD50 live Bacteria challenge, rabbit anti-O-SP-TT rabbit antiserum after immunization against 3 LD50 and 5 LD50 live bacteria has a good protective effect. Conclusions The prepared Pseudomonas aeruginosa 3 type O-SP-TT conjugate vaccine has good immunogenicity and immunoprotection, and the vaccine is expected to become an effective vaccine against IATS type 3 Pseudomonas aeruginosa infection.