肺炎链球菌重组蛋白LytA体外抗菌效应探讨

In vitro antibacterial activity of the recombinant protein LytA of Streptococcus pneumoniae

目的构建肺炎链球菌(ATCC49619)自溶素lytA基因的原核表达载体,并在大肠埃希菌表达系统中表达,获取LytA重组蛋白(rLytA),初步探讨其体外抗菌效应。方法根据GenBank中的肺炎链球菌M66菌株lytA基因序列(FN549899.1)设计合成特异性引物,采用PCR技术从肺炎链球菌ATCC49619标准株基因组中扩增lytA基因序列,分别构建克隆载体PGM-T/lytA和表达载体pET32a(+)/lytA,将pET32a(+)/lytA转化至感受态细胞E.coli BL21(DE3),经IPTG诱导后用透析袋等电点洗脱技术纯化目的蛋白rLytA;采用微量肉汤稀释法测定rLytA和青霉素G对肺炎链球菌及其青霉素G耐药株的最低抑菌浓度(MIC),动态测定肺炎链球菌及其耐药株对rLytA和青霉素G的药物敏感性,评价rLytA的体外抗菌活性。结果成功构建了原核表达载体pET32a(+)/lytA,获得的rLytA对肺炎链球菌标准株和青霉素G耐药株的MIC分别为8μg/ml和16μg/ml;以rLytA对标准株及耐药株MIC的3倍浓度,分别作用于标准株及耐药株,与未加药物的对照组相比,rLytA作用于标准株3h后细菌数显著减少(P<0.05),且效果持续至7h(P<0.05);对青霉素G耐药株作用3h时显示具有抑菌效果(P<0.01),且抑菌作用持续至7h(P<0.01)。结论一定浓度的rLytA对肺炎链球菌标准株和耐药株均显示出抑菌作用,且持续时间长,具有作为抗菌药物的可能性。

Objectives To construct a prokaryotic expression system to obtain the recombinant protein LytA（rLytA）of Streptococcus pneumoniae and to investigate its antibacterial activity in vitro in order to provide a basis for the examination of the in vitro antibacterial activity of autolysin fragments. Methods Specific primers were designed in accordance with the lytA gene sequence of S.pneumoniae M66 in GenBank（FN549899.1）,and the lytA gene from S.pneumoniae（ATCC49619）was amplified using PCR.The recombinant plasmids PGM-T/lytA and pET-32а（＋）/lytA were constructed.The expression plasmid pET-32а（＋）/lytA was transformed into E.coli BL21（DE3）to express LytA.After induction with IPTG,the expressed protein LytA was further analyzed using SDS-PAGE and purified by electroelution into a dialysis bag.The MIC of rLytA was determined for ATCC49619 and penicillin G-resistant S.pneumoniae using broth microdilution.S.pneumoniae ATCC49619 and penicillin G-resistant S.pneumoniae（final concentration of 5×10^5 CFU/ml）were exposed to 3times the original MIC of rLytA in broth（including 10% sterile calf serum）and colonies were counted at different times to gauge antibacterial activity in vitro. Results The recombinant protein LytA was successfully expressed and purified.The recombinant protein had a relative molecular weight of 53 ku and a concentration of 0.58mg/ml.The MIC of rLytA was 8μg/ml for the ATCC49619 strain and 16μg/ml for the penicillin G-resistant strain.An in vitro test revealed that 3times the original MIC of rLytA inhibited the growth of ATCC49619 and the penicillin G-resistant strain after 3h（P=0.020 and P=0.001,respectively）.This inhibition lasted for up to 7h（P=0.020 and P=0.001,respectively）. Conclusion This study revealed that rLytA has certain bactericidal action on the ATCC49619 strain of S.pneumoniae and a penicillin G-resistant S.pneumoniae strain,suggesting that rLytA may serve as an antimicrobial agent.