铜绿假单胞菌3型O-特异性多糖-破伤风类毒素结合疫苗的制备及其免疫特性分析

Preparation and immune characteristics of Pseudomonas aeruginosa-IATS 3 O-specific polysaccharide-tetanus toxoid conjugate

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 prepare Pseudomonas aeruginosa-IATS（International Antigen Typing System）3 O-specific polysaccharide-tetanus toxoid conjugate vaccine and analyze its immune characteristics.Methods Lipopolysaccharide（LPS）was isolated from P.aeruginosa IATS 3 by hot phenol method,in which the lipid A was removed.The obtained O-specific polysaccharide（O-SP）was purified and ac-tivated with CDAP,then conjugated to tetanus toxoid（TT）in mediation of EDAC using adipic acid dihydrazide（ADH）as a linker.The prepared conjugate vaccine was analyzed for physic-chemical property,immunogenicity and immune protection.Results The yield of O-SP was equivalent to 30% of hydrolyzed LPS.Both the protein and nucleic acid contents in the obtained O-SP was less than 1.0%.The derivation of O-SP was 3.83%,while the binding rate to TT was about 40%,and the ratio（w / w）of polysaccharide to protein was 1 ∶ 2.18.O-SP-TT vaccine induced high titer IgG in mice,which was significantly higher than that induced with O-SP（P 0.05）.The conjugate vaccine protected mice against the challenge with live bacteria at dosages of 5 and 10 LD50.Conclusion The prepared O-SP-TT conjugate showed high immunogenicity and immune protection,which might be used as an effective vaccine for prevention and treatment of infection with P.aeruginosa IATS 3.