金黄色葡萄球菌LukF-PV基因的原核表达及血清特异性抗体的检测

Prokaryotic Expression and Detection of Serum Specific Antibody of Staphylococcal LukF-PV Gene

目的:在大肠杆菌中表达金黄色葡萄球菌(Staphylococcus aureus,SA)LukF-PV基因,纯化重组蛋白并以其为抗原检测儿童SA感染者血清特异性IgG抗体,分析不同部位SA感染患者血清LukF-PV抗体水平。方法:将Luk F-PV克隆至pET-28a(+)载体,IPTG诱导重组蛋白表达、His柱纯化重组蛋白后,用间接ELISA检测儿童SA感染者与健康对照者血清特异性IgG抗体水平。结果:成功表达纯化LukF-PV蛋白,儿童SA感染组与健康对照组血清特异性抗体均值分别为(0.309±0.063)、(0.505±0.261),具有统计学意义(P<0.05)。不同部位来源标本的感染者中血液组和扁桃体组抗体OD均值分别为(0.634±0.225)、(0.481±0.264)与健康对照组有显著差异(P<0.05),其余各组与健康对照组无显著差异(P>0.05)。抗体阳性率统计分析中血液组分别与脑脊液组、扁桃体组、咽拭子组、痰液组有统计学意义(P<0.05),其余各组间均无显著差异(P>0.05)。结论:LukF-PV成功表达纯化,,儿童SA感染组LukF-PV特异性IgG抗体显著高于健康对照组,其中来源于血液和扁桃体部位的SA感染者抗体水平与健康者有显著差异,尤以血液感染者最显著。

Objective： To express Staphylococcus aureus Luk F-PV gene in E.coli BL21, purify the recombinant protein, measure and analyze serum specific Ig G antibody in children with different site SA infection by using recombinant Luk F-PV as antigen. Methods：Luk F-PV gene was cloned into p ET-28a（＋） vector, recombinant protein expression was induced by IPTG and recombinant protein was purified by His colum, the SA infection patients＇ serum specific antibody level was measured by ELISA assay. Results： Recombinant Luk F-PV was successfully expressed and purified, the Luk F-PV fusion protein was used as antigen for ELISA. The mean values of the antibody levels of the subjects with SA-infected children group and the healthy control group were（0.309 ±0.063）,（0.505 ±0.261）respectively（P〈0.05）.The mean OD values of the antibody of the subjects with blood and tonsi were（0.634±0.225）,（0.481±0.264）,respectively. There were significant differences compared with healthy control（P〈0.05）. while the other subjects showed no significant difference with healthy control（P〉0.05）. Antibody positive rate of the subject blood showed significant difference with the subjects of CSF, tonsil, throat swab, and sputum（P〈0.05）, while antibody positive rate of other subjects showed no significant difference（P〉0.05）.Conclusions： The recombiant Luk F-PV protein was successfully expressed and purified, the Luk F-PV-specific Ig G antibody in SA infection children was significantly higher than healthy control, especially in blood and tonsil patients, with the blood the highest.