Naka A, Honda T, Miwatani T
Research Institute for Microbial Diseases, Osaka University, Japan.
Microbiol Immunol. 1992;36(4):419-23. doi: 10.1111/j.1348-0421.1992.tb02040.x.
A new simple purification method (I) for Vibrio cholerae non-O1 hemagglutinin/protease (NAG-HA/P) was developed. The method (I) requires only an immunoaffinity column chromatography using a monoclonal antibody against NAG-HA/P. The method (I) is much simpler than previously reported purification method (II) (Honda, T. et al, Infection and Immunity 57: 2799-2803, 1989) which required four or more complicated chromatographic procedures. Method (I) also gave an improved recovery rate (about 27%) compared with (II). The molecular weight of NAG-HA/P purified by method (I) was mainly 34 kilodaltons (kDa) with a little of 32 kDa, whereas that of NAG-HA/P purified by (II) was usually 32 kDa. Immunological analysis by the Ouchterlony double gel diffusion test and Western blotting test using polyclonal antibody against 32 kDa protein revealed that the 34 and 32 kDa proteins are immunologically indistinguishable and thus it is supposed that 34 K protein is an isoform or a preform of the 32 K protein.
开发了一种用于霍乱弧菌非O1型血凝素/蛋白酶（NAG-HA/P）的新型简单纯化方法（方法I）。该方法（方法I）仅需使用抗NAG-HA/P的单克隆抗体进行免疫亲和柱层析。该方法（方法I）比先前报道的纯化方法（方法II）（本田，T.等人，《感染与免疫》57: 2799 - 2803，1989）简单得多，后者需要四个或更多复杂的层析步骤。与方法（II）相比，方法（I）的回收率也有所提高（约27%）。用方法（I）纯化的NAG-HA/P的分子量主要为34千道尔顿（kDa），有少量32 kDa，而用方法（II）纯化的NAG-HA/P的分子量通常为32 kDa。使用针对32 kDa蛋白的多克隆抗体通过双向琼脂扩散试验和蛋白质免疫印迹试验进行的免疫学分析表明，34 kDa和32 kDa的蛋白在免疫学上无法区分，因此推测34 kDa蛋白是32 kDa蛋白的异构体或前体形式。
