摘要
根据Sanger研究所公布的猪链球菌2型(SS2)P1/7株的自溶素序列,设计检测引物,取SS2我国2次流行株、其它临床分离株和参考株,及猪链球菌1型、1/2型、7型和9型,共33株,分别以其DNA为模板,PCR扩增。结果表明,SS2除无毒株T15阴性外,其他临床分离株27株(含人源2株)均阳性;其它猪链球菌为,SS7阳性,SS1、SS1/2和SS9均阴性。同时设计引物向两侧扩增,以四川流行株ZY05719和江苏流行株HA9801的DNA为模板,扩增自溶素ORF完整的编码基因,软件分析结果显示,该基因含有6个重复的"GBS_Bsp-like"域和1个"N-乙酰胞壁酰-L-丙氨酸酰胺酶"域,与SS2欧洲株有较高同源性(99.8%),但与SS2加拿大株差异较大。在DNASTAR分析所编码蛋白的抗原性的基础上,另设计引物,以ZY05719株DNA为模板,PCR扩增具有良好免疫原性的片段基因,并定向克隆至表达载体pET30a(+)中,进行重组表达,SDS-PAGE和Western blot表明,所获得重组自溶素具有良好反应原性。
Streptococcus suis serotype 2 （SS2） is an economically important, zoonotic agent causing death and disease in both human and swine. According to published SS2 European strain PI/7 complete genomic sequence, primers for detection and amplification autolysin were designed. Autolysin gene was detected by PCR with genomic DNA of HA9801 （Jiangsu isolate）, ZY05719 （Sichuan isolate）, ATCC43765 （reference strain）, other SS2 isolates from different regions, and strains of other different serotypes （such as serotype 1, 1/2, 7 and 9）. PCR results showed that all 27 SS2 virulent isolates harbored gene autolysin, but not in non-virulent SS2 strain. Among other serotypes, only serotype 7 strain had this gene. The complete autolysin genes of HA9801 and ZY05719 were respectively amplified by PCR and sequenced. Their putative protein sequences were analyzed through online software, results showed both had six repeated domain ＂GBS_Bsp-like＂ and one domain ＂N-acetylmuramoyl-L-alanine amidase＂. In addition, sequence similarity analysis demonstrated that autolysins of the two strains （HA9801 and ZY05719） showed high homologue to that of European strain Pl/7（99.8%）, but obviously differed from Canada strain 89/1589, the latter lacked one domain ＂GBS Bsp-like＂. Software DNASTAR was used to analyze autolysin protein sequence and predicted its putative antigenicity. Then partial antigentic segment of autolysin was amplified, cloned and inserted into expression vector pET30a（＋）, and induced by IPTG to express recombinant autolysin. Its reactivity was analyzed by SDS-PAGE and western blot with swine convalescent sera, blot result showed that the recombinant protein had good reactivity and could be considered as a vaccine candidate.
出处
《微生物学报》
CAS
CSCD
北大核心
2008年第1期68-72,共5页
Acta Microbiologica Sinica
基金
国家"973项目"(2006CB504403)~~
关键词
猪链球菌2型
感染相关因子
自溶素
Streptococcus suis serotype 2
infection-related factor
cell wall hydrolase/autolysin
