B群Nm 3407菌株外膜蛋白复合物(OMPC)的提取及免疫原性

Extraction and immunogenicity of outer membrane protein complex from the strain 3407 of Nm serogroup B

目的Ｂ群脑膜炎奈瑟氏菌Ｎｍ荚膜多糖作为菌苗候选成分效果不理想，有些非荚膜的抗原成分存在血清型和亚型的限制，为了能提取一种免疫原性强、并具有广泛的抗原交叉反应性的Ｂ群Ｎｍ外膜蛋白复合物（ｏｕｔｅｒｍｅｍｂｒａｎｅｐｒｏｔｅｉｎｃｏｍｐｌｅｘ，ＯＭＰＣ）以研制更有效的Ｂ群Ｎｍ菌苗提供依据。方法比较了从病人分离的Ｂ群Ｎｍ３４０７菌株提取ＯＭＰＣ的３种方法，用ＥＬＩＳＡ、杀菌力试验、小鼠免疫试验、Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ测定ＯＭＰＣ的抗原滴度和免疫原性。结果在提取ＯＭＰＣ的３种方法中，选用７０％饱和硫酸铵初步沉淀细菌培养物的上清液，再经ＳｅｐｈａｃｒｙｌＳ┐３００ＨＲ纯化的方法，获得了３个洗脱峰。其中第一个洗脱峰中ＯＭＰＣ产量最高，溶解性最好。经ＳＤＳ┐ＰＡＧＥ及Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ测定，其主要含有分子量为（４２，３９，３０，２６和１９）×１０３的蛋白和少量约（９２和７２）×１０３的蛋白。用ＥＬＩＳＡ测定此ＯＭＰＣ与兔抗菌株３４０７和５４２８５２血清反应的滴度分别为１∶３２０００和１∶６４０００；与小鼠抗此ＯＭＰＣ血清反应的滴度为１∶２５６００。另外，上述抗ＯＭＰＣ血清与菌株３４０７和５４２８５２反应的?

Objectives The capsular polysaccharide of Neisseria meningitidis serogroup B is not an effective components for a vaccine candidate because there are some limits of serotype and subserotype in some noncapsular antigens, it will be significant to prepare an effective group B vaccine if a high immunogenic and broadly cross reactive outer membrane protein complex (OMPC) can be extracted from Nm serogroup B. Methods Three methods for extracting OMPC from the strains 3407 of Nm serogroup B isolated from a clinical case were compared. ELISA, Bactericidal test, immunologic experiments with mice and Western blotting were used for testing the antigenic titers and immunogenicity of OMPC. Results Among the three methods for extracting OMPC, the method that the cultural supernatant was primarily precipitated with 70% saturated ammonium sulfate and further purified on Sephacryl S 300 HR column was chosen, and three elution peaks were obtained. The OMPC in the peak 1 was of the largest yield and of the best solubility. The 42, 39, 30, 26, 19kD and minor 92,72kD proteins in the OMPC were determined by SDS PAGE and Western blotting. The reactive titers of the OMPC with the sera of rabbit anti the strains 3407 and 542852, and with the sera of the mice anti OMPC were 1∶32000, 1∶64000, and 1∶25600,respectively. In addition, the reactive titers of the strains 3407 and 542852 as well as some differant serotype and subserotype strains of Nm serogroup B with the sera of the mice anti OMPC were 1∶25600, 1∶25600 and 1∶640 to 1∶3200, respectively , when ELISA was used for testing the above reactions. The bactericidal titers of anti OMPC sera to the strains 3407 and 542852 were both 1∶256. The OMPC extracted with other two methods showed lower antigencity , solubility and immunogenicity. Conclusions The above OMPC of Nm serogroup B possessed high immunogenicity, good solubility and broadly cross reactivity. It seems to be hopeful that the OMPC could be chosen as a vaccine candidate of Nm serogroup B, and could be also useful as a carrier of protein coupling with capsular polysaccharide.