Characterization of regular array protein in the cell wall of Lactobacillus

1. 嗜酸乳杆菌是由许多具有不同生物学和生化特性的菌株组成的。我们发现除了DNA-DNA同源性分类外，有机体的规则阵列蛋白的特征也可能用于分类嗜酸乳杆菌菌株。只有DNA同源类群pa的菌株产生RA蛋白，其他类群不产生RA蛋白。所有A-1亚群菌株产生的RA蛋白具有相同的分子量和肽图。此外，它们在免疫学特性上具有高度的同源性。相比之下，RA亚群A-2、A-3和A-4蛋白在分子量和肽定位上存在差异。虽然A-2亚型RA蛋白的免疫特性表现出相当的同质性，但A-3和A-4亚型RA蛋白的免疫特性表现出异质性。根据RA蛋白的性质，我们认为有必要将A-3和A-4亚群进行分类。我们试图克隆短乳杆菌ATCC8287的规则阵列蛋白基因。将EcoRI部分酶切产生的染色体DNA片段（3- 9kb）与g11ecori酶切DNA结扎，体外包装。用抗ra蛋白抗体免疫筛选，从2638个斑块中筛选出16个阳性克隆。通过Western blotting检测10个阳性克隆的裂解液，7个克隆产生分子量大于130 kDa的免疫阳性蛋白，3个克隆产生分子量小于130 kDa的阳性蛋白。前者含有2.4 kb的EcoRI片段，后者含有3.2 kb的EcoRI片段。将2.4 kb的EcoRI片段克隆到pU19质粒中构建pLB1，在大肠杆菌JM109中表达，得到与纯化后的RS蛋白分子量相同的RA蛋白。pLB1的延迟突变体已经产生，目前正在对克隆DNA的2.4 kb片段进行测序。

1. Lactobacillus acidophilus was composed of many bacterial strains with very heterogeneous biological and biochemical characteristics. We showed possibility that in addition to classification by DNA-DNA homology,character of regular array protein of the organism could be used to classify L. acidophilus strains. Only the strains of DNA homology groupA produced RA proteins and no other groups produced. All A-1 subgroup strains produced RA proteins with same molecular weight and peptidemapping. Furthermore, they showed high homology between their immunological properties. In contrast, RA proteins of subgroup A-2, A-3 and A-4 were differed from each other in their molecular weight and peptide mapping. Although, immunological properties of subgroup A-2 RA proteins showed considerably homogeneous, those of subgroup A-3 and A-4 strains appeared heterogeneous. We conclude that there are necessity to classify subgroup A-3 and A-4 by means of properties of RA proteins.2. We tried to clone the regular array protein gene of L. brevis ATCC8287. The chromosomal DNA fragments(3-9 kb) produced bypartial digestion with EcoRI was ligated with gt 11 EcoRI digested DNA and then packaged in vitro. Sixteen positive clones were screened from 2638 plaques by immunoscreening with anti-RA protein antibody. When we examined the lysate of ten positive clones by Western blotting, seven clones produced immunopositive protein with molecular weight more than 130 kDa, and three clones made positive protein with a molecular weight lower than 130 kDa. The former phage DNA contained 2.4 kb EcoRI fragment and the latter contained 3.2 kb EcoRI fragment. The 2.4 kb EcoRI fragment was cloned in the plasmid pU19 to construct pLB1, which was expressed in E.coli JM109 and produced the RA protein identical in molecular weight to the purified RS protein. The delated mutant of pLB1 was produced and work is in progress to sequence the 2.4 kb fragment of the cloned DNA.