肺炎链球菌溶血素对感染性肺炎的诊断价值

Role of Streptococcus pneumoniae pneumolysin in the diagnosis of infective pneumonia

目的利用原核表达技术获得肺炎链球菌溶血素(PLY)重组蛋白,探讨其对肺炎链球菌感染性肺炎的血清学诊断价值。方法根据Gen Bank中肺炎链球菌ply基因序列,切除羧基端33个核苷酸,设计合成特异性引物,从标准菌株的肺炎链球菌中提取DNA为模板,扩增目的基因ply,克隆后构建原核表达载体PET32a-ply,然后将其转化到大肠埃希菌DH5a BL21(DE3)中,以异丙基硫代半乳糖苷(IPTG)诱导其大量表达,获得融合6个组氨酸标签、无溶血活性的重组蛋白,经Ni-NTA树脂纯化,获得重组蛋白,经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定后,采用免疫印迹法检测其免疫原性;以重组蛋白PLY为抗原,采用酶联免疫吸附试验(ELISA)检测临床诊断为社区获得性肺炎(CAP)并经细菌学确诊的患者血清中相应的Ig M抗体,评价其对肺炎链球菌感染性肺炎的快速诊断价值。结果成功构建出原核表达载体PET32a-ply,并实现了无溶血活性的PLY蛋白的可溶性表达,其具有较好的免疫原性。CAP患者血清抗PLY-Ig M抗体阳性率高于体检健康者(P<0.05),其诊断的敏感性均高于常规的痰培养和血培养(P<0.05)。结论以重组蛋白PLY为抗原的ELISA可用于肺炎链球菌感染的血清学快速诊断,在临床上具有重要的应用价值。

Objective To obtain Streptococcus pneumoniae pneumolysin（PLY） through prokaryotic expression system,and to investigate its serological diagnosis role for infective pneumonia with Streptococcus pneumoniae. Methods The specific primers with cutting 33 nucleotides of carboxyl end were designed according to ply gene sequence of Streptococcus pneumoniae in GenBank. The ply gene was amplified by using the genome DNA of Streptococcus pneumoniae as template. The recombinant plasmid PET32a-ply was constructed,and it was expressed after transformation in Escherichia coli DH5aBL21（DE3）. Overexpression of recombinant protein without hemolysis activity in gene labeled with 6 infused amino acids was induced by isopropyl phosphorothioate （IPTG）. The immunocompetence of recombinant protein PLY was determined by western blotting after it was purified with Ni-NTA resin and identified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）. Enzyme-linked immunosorbent assay（ELISA） reactive mode was established by setting PLY as IgM antigen so as to determine community acquired pneumonia（CAP）,and the role of PLY in the diagnosis of infective pneumonia with Streptococcus pneumoniae was evaluated. Results The prokaryotic expression vector PET32a-ply was successfully constructed. The recombinant protein PLY without hemolysis activity had soluble expression, with good immunogenicity. The positive rate of anti-PLY IgM antibody in CAP patients was higher than that in healthy subjects（P〈0.05）,and the sensitivities were higher than those of conventional sputum culturing and blood culturing（P〈0.05）.Conclusions ELISA reactive mode based on the recombinant protein PLY could be used for the serological diagnosis of infective pneumonia with Streptococcus pneumoniae.