Sommer P, Bruyère T, Ogier J A, Garnier J M, Jeltsch J M, Klein J P
Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Strasbourg, France.
J Bacteriol. 1987 Nov;169(11):5167-73. doi: 10.1128/jb.169.11.5167-5173.1987.
Genomic libraries from Streptococcus mutans OMZ175 were constructed in bacteriophage vectors. DNA fragments 1 to 2 kilobases in length were cloned in expression vector lambda gt11. S. mutans DNA fragments 15 to 20 kilobases in length were inserted in the BamHI site of phage EMBL3. Rabbit antiserum raised against an S. mutans saliva-interacting protein with a molecular weight of 74,000, designated 74K SR, was used to screen the lambda gt11 library. A recombinant phage carrying an S. mutans DNA sequence of 1.45 kilobases, lambda SmAD2, was detected and isolated. This fragment, named SmAD2, was used to construct the recombinant expression plasmid pSAD2-4 which encoded for the expression of a 60,000-molecular-weight protein controlled by the beta-galactosidase promoter from plasmid pUC8. The SmAD2 fragment and polyclonal anti-74K SR antibodies were used to screen the EMBL3 library. A total coincidence between the screening with antibodies and the DNA probe was observed, and two phages, lambda SmAD9 and lambda SmAD10, were isolated. They contained a common S. mutans DNA sequence of about 11.8 kilobases and coded for a protein with a molecular weight of about 195,000, which comigrated with a protein of an S. mutans cell wall extract. The expressed protein was purified, and a very strong relationship with the S. mutans 74K SR protein was found by competitive enzyme-linked immunosorbent assay. Thus, cloning of the 74K SR gene allowed us to demonstrate that the saliva receptor appears to be a part of an S. mutans precursor molecule with a molecular mass of 195,000 daltons.
利用噬菌体载体构建了变形链球菌OMZ175的基因组文库。将长度为1至2千碱基的DNA片段克隆到表达载体λgt11中。将长度为15至20千碱基的变形链球菌DNA片段插入噬菌体EMBL3的BamHI位点。用针对一种分子量为74,000的变形链球菌唾液相互作用蛋白（命名为74K SR）制备的兔抗血清筛选λgt11文库。检测并分离出携带1.45千碱基变形链球菌DNA序列的重组噬菌体λSmAD2。该片段命名为SmAD2，用于构建重组表达质粒pSAD2 - 4，该质粒编码由质粒pUC8的β - 半乳糖苷酶启动子控制的分子量为60,000的蛋白质的表达。用SmAD2片段和多克隆抗74K SR抗体筛选EMBL3文库。观察到抗体筛选与DNA探针筛选完全一致，并分离出两个噬菌体λSmAD9和λSmAD10。它们含有约11.8千碱基的共同变形链球菌DNA序列，编码一种分子量约为195,000的蛋白质，该蛋白质与变形链球菌细胞壁提取物中的一种蛋白质迁移率相同。对表达的蛋白质进行了纯化，并通过竞争性酶联免疫吸附测定发现其与变形链球菌74K SR蛋白有很强的相关性。因此，74K SR基因的克隆使我们能够证明唾液受体似乎是分子量为195,000道尔顿的变形链球菌前体分子的一部分。
