肺炎链球菌自溶素蛋白LytA对社区获得性肺炎的血清学诊断意义

Diagnostic value of Streptococcus pneumoniae autolysin rLytA for community acquired pneumonia

目的通过原核表达技术获取肺炎链球菌（ATCC49619）自溶素LytA重组蛋白（rLytA），以此为抗原检测其抗体在健康人及社区获得性肺炎（CAP）患者外周血中的水平，为CAP的血清学诊断奠定基础。方法根据Genbank中公布的肺炎链球菌M66菌株LytA基因序列设计合成特异性引物。以ATCC49619株基因组DNA为模板，采用原核表达技术获取相对分子质量为56 000的重组蛋白rLytA。以rLytA为抗原，建立ELISA反应模式，测定健康人及CAP患者相应IgM抗体，并与痰培养结果比较。应用χ2检验评价结果差异。结果通过原核表达技术成功获得肺炎链球菌重组蛋白rLytA。以此为抗原，测得CAP患者血清中IgM类抗LytA的抗体滴度高于健康对照组，差异有统计学意义（P＝0.000），诊断的敏感度、特异度分别为27.8%和100.0%，痰培养的敏感度和特异度分别为19.4%和72.2%。血清学诊断的敏感度与痰培养相比差异无统计学意义（χ2＝0.693，P＝0.405），但特异度显著高于常规痰培养结果（χ2＝14.316，P＝0.000）。阳性预测值为100%，IgM类抗LytA抗体的阳性结果对肺炎链球菌的感染具有确诊价值。结论以重组蛋白LytA为抗原建立ELISA反应模式，检测IgM类LytA抗体，可能为链球菌感染性肺炎提供一条早期快速、客观诊断的途径。（中华检验医学杂志，2017, 40：212－215）

Objective To obtain the recombinant protein LytA （rLytA） of Streptococcus pneumoniae strain （ATCC49619） through prokaryotic expression system and to investigate their diagnostic value for patients with community acquired pneumonia （CAP）. Methods The specific primers were designed according to LytA gene sequence of Streptococcus pneumoniae M66 strain recorded in Genbank. The recombinant plasinid pET32a（ ＋ ）/LytA was constructed and transfirmed into BI21 （DE3） to express LytA. The expressed protein LytA was purified by electroeluting of bag filter. Serum IgM of anti-LytA accordingly of patients with CAP were detected by ELISA. The results were evaluated by Chi-square test. Results The recombinant protein LytA was expressed and purified successfully with a relative molecular weight of 56 000. The IgM antibodies level of anti-LytA was significantly higher than the healthy control group （ P = 0. 000 ）. Diagnostic sensibility and specificity of LytA-IgM were 27.8% and 100. 0% ,while sensibility and specificity of sputum culture were 19. 4% and 72.2% , respectively. The sensibility of LytA-IgM was equal to sputum culture（χ2 = 0.693, P= 0.405）, but the specificity was higher than it （χ2 = 14. 316 P= 0.000）. Conclusions A rLytA-ELISA assay maybe has clinical value for diagnosis of pneumococcal infections. It is more rapid and objective than the culture method. （ Chin J Lab Med, 2017, 40： 212-215）