摘要
目前国际上的着丝粒蛋白研究工作几乎全是以酵母和高等生物为材料进行的。为了从起源与进化的角度考察着丝粒蛋白，我们以人喉癌培养细胞ＨｅｐⅡ作为对照材料，以两种ＡＣＡ血清和ＣＥＮＰ－Ｂ单抗、多抗以及ＣＨＯ动粒蛋白单抗为探针，用间接免疫荧光和免疫印迹技术对嗜热四膜虫（Ｔｅｔｒａｈｙｍｅｎａｔｈｅｒｍｏｐｈｉｌａ）作检查。免疫荧光结果表明，ＨｅｐⅡ细胞的着丝粒抗原在间期核中呈点状分布：与ＨｅｐⅡ细胞的不同，嗜热四膜虫的着丝粒抗原在问期核中的分布呈不规则的斑块状。免疫印迹结果表明，作为单细胞生物的纤毛虫虽然在进化上距离哺乳动物和人甚远，但却已具备了与哺乳动物和人的相似的着丝粒／动粒点抗原蛋白。这说明着丝粒和动粒的蛋白组分可能是发生得很早的，而且是相当保守的。
UP to now,the studies on the centromere proteins are almost carried out in yeast andhigher organisms in the world. In this paper we report our studies on centromere proteins inone kind of ciliates, which is much lower than yeast in the evolutionary position mainly withindirect immunofluorescent microscopy and western immunoblotting techniques using twoACA sera, ra-ACA-2, mACA-2, and mAb37A5 as probes. The control material in thestudy is human HepⅡ cell.With immunofluorescent stain with Chinese ACA antiserum, in the interphase nucleus ofHepⅡ cells only very minute pots-precentromere could be observed.This means that theACA antiserum can actually and specifically recognize the protein components ofcentromer/kinetochore. When Tetrahymena,was stained with the same technique,therewere only patched within nuclei composed of minute fluorescent spots,no precentromerecould be distinguished, although all the nuclei gave positive reaction.After immunobloted with Chinese ACA serum and SH serum the positive band-pattern(from 14 kD to 140 kD) of Tetrahymena was highly similar to the band-pattern of humanHepⅡ cells. The blotting results with mACA-2 antibody showed that HepⅡ cells gave twobands (80 kD, stained deely and 120 kD, stained slightly), Tetrahymena, gave three positivebands (80 kD, 120 kD, and 150 kD). The blotting results with ra-ACA-2 antiserum showedthat Hep Ⅱ and Tetrahymena all gave 50 kD, 60 kD and 80 kD bands.In the immunoblotting with monoclonal antibody mAb37A5, HepⅡ cells gave two bands very similarto those of CHO cells. Tetrahymena gave only one band with molecular weight somewhatlower than 140 kD.The results described above indicat the high similarity in the centromere/ kinetochoreprotein components between HepⅡ cells and the Tetrahymena. must have emerged earlier inlife evolutionary history.
关键词
四膜虫
着丝粒蛋白
Tetrahymena, Centromere protein, ACA serum
