肺炎链球菌自溶素对感染性肺炎的诊断价值  

Value of autolysin of Streptococcus pneumoniae in diagnosis of infective pneumonia

作　　者：邹琴[1] 孙文平[2] 杨光[2] 周杰[3] 罗红[2] 

机构地区：[1]华中科技大学同济医学院附属普爱医院检验科,湖北武汉430033 [2]大连医科大学临床免疫与微生物教研室,辽宁大连116044 [3]大连市友谊医院检验科,辽宁大连116100

出　　处：《中华医院感染学杂志》2015年第21期4801-4804,共4页Chinese Journal of Nosocomiology

基　　金：辽宁省科技厅自然科技基金资助项目(201202053)

摘　　要：目的通过基因原核表达技术获取肺炎链球菌自溶素LytA重组蛋白(rLytA),探讨其对感染性肺炎患者的血清学诊断价值。方法根据Genbank中肺炎链球菌M66菌株lytA基因序列(FN549899.1)设计合成特异性引物;采用PCR技术以肺炎链球菌基因组DNA为模板扩增lytA基因序列;构建重组表达质粒pET32a(+)/lytA,经由IPTG诱导后通过等电点洗脱方法获取纯化的重组蛋白LytA;Western blot测定表达蛋白的抗体结合活性;以rLytA为抗原,建立ELISA反应模式,测定2013年3-6月36例临床诊断为CAP并经细菌学确定的患者血清标本中相应IgG和IgM抗体,评价对肺炎链球菌感染性肺炎的诊断价值。结果成功构建原核表达质粒pET32a(+)/lytA,表达的rLytA具有较好的抗体结合活性;CAP患者血清IgM和IgG类抗LytA的抗体滴度均高于健康对照组,差异有统计学意义(P<0.05),其诊断的敏感性均高于常规的痰培养和血培养(P<0.01)。结论以重组蛋白LytA为抗原建立ELISA反应模式可用于肺炎链球菌感染的血清学诊断,快速、客观,具有一定的应用价值。OBJECTIVE To construct the recombinant protein LytA（rLytA）of Streptococcus pneumoniae strain through prokaryotic expression system and investigate its value in serologic diagnosis of the patients with infective pneumonia.METHODS The specific primers were designed according to lytA gene sequence（FN549899.1）of Streptococcus pneumoniae M66 strain recorded in Genbank,and the PCR was employed to amplify the lytAgene by using the genome DNA of Streptococcus pneumoniae strain as template.The recombinant plasmid pET32a（＋）/lytA was constructed,after the induction by IPTG,the purified recombinant protein LytA was obtained with the use of isoelectric point elution method,the Western-blot was used to determine the antibody binding activity of the expressed protein.The ELISA reactive mode was established by setting the rLytA as antigen so as to determine the serum IgG and IgM antibodies in the 36 patients who were clinically diagnosed with CAP and conformed through bacteriological examination from Mar 2013 to Jun 2013,and the value in the diagnosis of Streptococcus pneumoniae pneumonia was evaluated.RESULTS The prokaryotic expression vector pET32a（＋）/lytA was successfully constructed,and the expressed rLytA had good antibody biding activity.The anti-LytA antibody titers of serum IgM and IgG of the patients with CAP were higher than those of the healthy controls,with significant difference（P〈0.05）,and their sensitivities in diagnosis were higher than those of the conventional sputum culture and blood culture（P〈0.01）.CONCLUSION The ELISA reactive mode established based on the recombinant protein LytA can be used for the serologic diagnosis of Streptococcus pneumoniae infection,it is rapid and objective and has certain application value.

关 键 词：肺炎链球菌 自溶素 原核表达 酶联免疫吸附试验 

分 类 号：R446[医药卫生—诊断学]