Okwumabua Ogi, Chinnapapakkagari Sharmila
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53705-4494, USA.
Clin Diagn Lab Immunol. 2005 Apr;12(4):484-90. doi: 10.1128/CDLI.12.4.484-490.2005.
In our continued effort to search for a Streptococcus suis protein(s) that can serve as a vaccine candidate or a diagnostic reagent, we constructed and screened a gene library with a polyclonal antibody raised against the whole-cell protein of S. suis type 2. A clone that reacted with the antibody was identified and characterized. Analysis revealed that the gene encoding the protein is localized within a 2.0-kbp EcoRI DNA fragment. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 445 amino acid residues with a calculated molecular mass of 46.4 kDa. By in vitro protein synthesis and Western blot experiments, the protein exhibited an electrophoretic mobility of approximately 38 kDa. At the amino acid level the deduced primary sequence shared homology with sequences of unknown function from Streptococcus pneumoniae (89%), Streptococcus mutans (86%), Lactococcus lactis (80%), Listeria monocytogenes (74%), and Clostridium perfringens (64%). Except for strains of serotypes 20, 26, 32, and 33, Southern hybridization analysis revealed the presence of the gene in strains of other S. suis serotypes and demonstrated restriction fragment length differences caused by a point mutation in the EcoRI recognition sequence. We confirmed expression of the 38-kDa protein in the hybridization-positive isolates using specific antiserum against the purified protein. The recombinant protein was reactive with serum from pigs experimentally infected with virulent strains of S. suis type 2, suggesting that the protein is immunogenic and may serve as an antigen of diagnostic importance for the detection of most S. suis infections. Pigs immunized with the recombinant 38-kDa protein mounted antibody responses to the protein and were completely protected against challenge with a strain of a homologous serotype, the wild-type virulent strain of S. suis type 2, suggesting that it may be a good candidate for the development of a vaccine that can be used as protection against S. suis infection. Analysis of the cellular fractions of the bacterium by Western blotting revealed that the protein was present in the surface and cell wall extracts. The functional role of the protein with respect to pathogenesis and whether antibodies against the antigen confer protective immunity against diseases caused by strains of other pathogenic S. suis capsular types remains to be determined.
为持续寻找可作为疫苗候选物或诊断试剂的猪链球菌蛋白，我们构建并筛选了一个基因文库，该文库使用针对2型猪链球菌全细胞蛋白产生的多克隆抗体。鉴定并表征了一个与该抗体发生反应的克隆。分析表明，编码该蛋白的基因位于一个2.0-kbp的EcoRI DNA片段内。核苷酸序列包含一个开放阅读框，其编码一个由445个氨基酸残基组成的多肽，计算分子量为46.4 kDa。通过体外蛋白质合成和蛋白质印迹实验，该蛋白的电泳迁移率约为38 kDa。在氨基酸水平上，推导的一级序列与肺炎链球菌（89%）、变形链球菌（86%）、乳酸乳球菌（80%）、单核细胞增生李斯特菌（74%）和产气荚膜梭菌（64%）中功能未知的序列具有同源性。除20、26、32和33血清型菌株外，Southern杂交分析显示该基因存在于其他猪链球菌血清型菌株中，并证明了由EcoRI识别序列中的点突变引起的限制性片段长度差异。我们使用针对纯化蛋白的特异性抗血清，证实了杂交阳性分离株中38-kDa蛋白的表达。该重组蛋白与经2型猪链球菌强毒株实验感染的猪血清发生反应，表明该蛋白具有免疫原性，可能作为检测大多数猪链球菌感染的重要诊断抗原。用重组38-kDa蛋白免疫的猪对该蛋白产生抗体反应，并对同源血清型菌株（2型猪链球菌野生型强毒株）的攻击完全具有抵抗力，表明它可能是开发可用于预防猪链球菌感染疫苗的良好候选物。通过蛋白质印迹分析细菌的细胞组分，发现该蛋白存在于表面提取物和细胞壁提取物中。该蛋白在致病机制方面的功能作用以及针对该抗原的抗体是否能对其他致病性猪链球菌荚膜型菌株引起的疾病提供保护性免疫，仍有待确定。
