Bleiweis A S, Lee S F, Brady L J, Progulske-Fox A, Crowley P J
Department of Oral Biology, University of Florida, Gainesville 32610.
Arch Oral Biol. 1990;35 Suppl:15S-23S. doi: 10.1016/0003-9969(90)90126-u.
To understand more fully the biological function(s) and investigate the reported cross-reactivity with heart tissue of antigen P1 (I/II) of Streptococcus mutans (serotype c), this molecular biological study of the responsible gene, spaP, was undertaken. A 5.2 kb Hin dIII fragment of strain NG5 was cloned into Escherichia coli JM109 by a shotgun procedure with pUC18 as the vector. Recombinant SM2949 expressed a P1 fusion protein under the control of the streptococcal promoter. Southern analysis revealed hybridization of pSM2949 with DNA from Strep. mutans (serotypes c, e, f), Strep. cricetus (a) and Strep. sobrinus (d), but not Strep. sobrinus (g), Strep. rattus (b) or Strep. downei (h). Recombinant (r) antigen was detected in E. coli periplasm, indicating the presence of a signal sequence. This product (of Mr 155K) showed partial identity to the native streptococcal P1 antigen by Ouchterlony double-diffusion analysis. The N-terminal 28 amino acid residues of rP1 were determined by Edman degradation analysis and an end-labelled oligonucleotide probe corresponding to residues 8-13 was used to determine the 5'-3' orientation of spaP by Southern hybridization with restriction enzyme digests of pSM2949. Rabbit antisera made against native and rP1 did not cross-react with human heart tissue. Isogenic mutants of strain NG8 were isolated after transformation with insertionally inactivated spaP. Each mutant was non-reactive with anti-P1 antisera. Selected mutants were shown to have a defective spaP gene incorporated into their chromosomal DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
为更全面地了解变形链球菌(血清型c)抗原P1(I/II)的生物学功能,并研究其与心脏组织的交叉反应性报道,我们对负责该抗原的基因spaP进行了分子生物学研究。将菌株NG5的一个5.2 kb HindIII片段通过鸟枪法克隆到以pUC18为载体的大肠杆菌JM109中。重组体SM2949在链球菌启动子的控制下表达P1融合蛋白。Southern分析显示pSM2949与变形链球菌(血清型c、e、f)、仓鼠链球菌(a)和远缘链球菌(d)的DNA杂交,但与远缘链球菌(g)、鼠链球菌(b)或唐氏链球菌(h)的DNA不杂交。在大肠杆菌周质中检测到重组(r)抗原,表明存在信号序列。通过免疫双扩散分析,该产物(Mr 155K)与天然链球菌P1抗原显示部分同一性。通过Edman降解分析确定了rP1的N端28个氨基酸残基,并使用与残基8 - 13对应的末端标记寡核苷酸探针通过与pSM2949的限制性酶切片段进行Southern杂交来确定spaP的5'-3'方向。针对天然和rP1制备的兔抗血清与人心脏组织无交叉反应。用插入失活的spaP转化后,分离出菌株NG8的同基因突变体。每个突变体与抗P1抗血清无反应。所选突变体显示其染色体DNA中整合了有缺陷的spaP基因。(摘要截短至250字)