肺炎链球菌菌体蛋白LytA和CbpA对感染小鼠的诊断价值  

Performance of pneumococcal LytA and CbpA for diagnosis in a rat model of pneumococcal infection

作　　者：霍琳红[1] 孙文平[1] 杨光[1] 陈晨[1] 邓燕燕[1] 罗红[1] 

机构地区：[1]大连医科大学临床免疫与微生物教研室,116044

出　　处：《中华微生物学和免疫学杂志》2011年第4期366-370,共5页Chinese Journal of Microbiology and Immunology

基　　金：辽宁省科技厅自然科学基金资助项目（20052171）

摘　　要：目的通过基因工程技术获取肺炎链球菌自溶素（autolysin，LytA）和胆碱结合蛋白A（choline binding proteinA，CbpA），探讨其对肺炎链球菌感染小鼠的血清学诊断价值。方法根据GenBank中lytA和cbpA基因保守序列设计合成特异性引物，采用PCR技术从肺炎链球菌基因组中扩增lytA和cbpA保守序列片段；经由IPTG诱导并通过等电点洗脱方法获取纯化的重组蛋白LytA和CbpA；Westernblot测定表达蛋白的抗体结合活性。以LytA和CbpA为抗原，建立ELISA反应模式测定感染小鼠血清中相应的IgG和IgM抗体，评价诊断价值。结果成功构建了原核表达载体pET-32a（＋）／lytA和pET-32a（＋）／cbpA，表达的重组蛋白LytA和CbpA具有较好的抗体结合活性。实验组小鼠（肺炎链球菌感染组）IgM和IgG类抗LytA抗体滴度高于对照组（正常对照组和乙型链球菌感染对照组），差异有统计学意义（P〈0．05），CbpA抗体与正常对照组差异有统计学意义，与乙型链球菌感染组差异无统计学意义（P〉0．05）。CbpA蛋白对感染小鼠诊断的敏感性较高（IgG：83．3％；IgM：75．0％），而LytA特异性较高（IgG：100％；IgM：100％）。结论协同利用重组蛋白CbpA诊断敏感性及LvtA的特异性，对肺炎链球菌感染小鼠的血清学诊断具有一定价值，为进一步用于临床检验奠定基础。Objective To obtain the pneumococcal autolysin （LytA） and choline binding protein A（CbpA） by prokaryotic expression system and investigate their diagnosis for infection caused by Streptococcus pneumoniae. Methods The specific primers were designed according to lytA and cbpA of Streptococcus pneumoniae gene sequence, lytA and cbpA were amplified by PCR form the pneumococcus genome. After IPTG inducing, the recombinant proteins were purified by electroeluting of bag filter, detected by SDS-PAGE and Western blot. Serum IgG and IgM antibodies accordingly of BALB/c mice infected with Streptococcus pneumoniae were detected by ELISA. Results The recombinant plasmid pET-32a（ ＋ ）/lytA and pET-32a （ ＋ ）/cbpA were constructed successfully. Fusion proteins LytA and CbpA were expressed and displayed expected antigenicity. IgM and IgG antibodies level anti LytA were significantly higher than the control group （ infections with B Streptococcus group and healthy mice）,, （ P 〈 0.05 ）, but antibodies level anti CbpA did not increase as compared with group infected with B Streptococcus（ P 〉 0.05 ） . Diagnostic sensitivity of Cb- pA was 83.3% （IgG） and 75.0% （IgM）. Diagnostic specificity of LytA was 100% （ IgG and IgM ）. Conclusion The synergistic use of specificity of LytA and sensitivity of CbpA may be worthy of serological diag- nosis for Streptococcus pneumoniae infection, and may be used for further clinical test.

关 键 词：肺炎链球菌 自溶素 胆碱结合蛋白A 酶联免疫吸附试验 

分 类 号：R515[医药卫生—内科学]