肺炎链球菌小片段自溶素蛋白LytA′的体外抗菌活性探讨

目的 构建肺炎链球菌（ATCC49619）小片段自溶素基因lytA′的原核表达载体,通过大肠埃希菌表达系统进行原核表达获得一种新型肺炎链球菌小片段自溶素重组蛋白LytA′,初步探讨该蛋白的体外抗菌活性,并与青霉素G同时进行比较。方法 设计lytA基因的特异性引物,并通过PCR方法从肺炎链球菌（ATCC49619）中扩增lytA基因,并将其插入原核表达载体PGM-T中,构建PGM-T/lytA重组质粒,使用限制性核酸内切酶BamHⅠ和HindⅢ消化PGM-T / lytA获得小片段自溶素基因lytA′。再构建表达质粒pET-32a（+）/lytA′ 并转化到感受态细胞大肠埃希菌BL21（DE3）中,经IPTG诱导表达目的 蛋白,使用透析袋等电点洗脱技术纯化回收目的 蛋白LytA′;采用微量肉汤稀释法分别测定LytA′和青霉素G对肺炎链球菌（ATCC49619）的最小抑菌浓度（MIC）,并将其作用于青霉素G敏感菌株肺炎链球菌（ATCC49619）,初步探讨小片段自溶素蛋白LytA′的体外抗菌活性。结果 成功构建重组质粒PGM-T / lytA和pET-32a（+）/lytA′。小片段自溶素重组蛋白表达良好,并显示出预期的抗菌活性。与生长对照组相比,LytA′和青霉素G在3 h时显示出明显的抗菌活性（t=14.243 0,P<0.011 0;t=7.400 0,P<0.022 0）,抑菌效应持续至7 h （t=20.838 0,P<0.008 0;t=17.889 0,P<0.017 0）,抑菌效应逐渐增强。此外,在7 h时,LytA′ 的抑菌活性明显强于青霉素G（t=6.764 0,P<0.027 0）。结论 小片段自溶素重组蛋白LytA′对青霉素G敏感肺炎链球菌显示出一定的体外抑菌活性,且持续时间久,有成为抗肺炎链球菌感染治疗药物的可能性。

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

Objective To detect in vitro antibacterial activities of new short fragment protein LytA′ from Streptococcus pneumoniae ATCC49619 obtained by prokaryotic expression system, and compare with penicillin G at the same time. Methods Specific primers were designed in accordance with the lytA gene sequence, and the lytA gene from Streptococcus pneumoniae (ATCC49619) was amplified using PCR. The lytA gene was inserted into prokaryotic expression vector PGM-T to construct PGM-T/lytA recombinant plasmid. The new short fragment lytA′ gene was obtained through digesting PGM-T/lytA with BamHⅠ and HindⅢ. Then expressing plasmid pET-32a(+)/ytA′ which had been well constructed and sequentially confirmed respectively were transformed into E.coli BL21 and induced by IPTG to express the target protein LytA′. The target proteins LytA′ were purified by dialysis bag isoelectric point elution technology, detected by SDS-PAGE. The minimum inhibitory concentrations (MIC) of LytA’ and penicillin G against Streptococcus pneumoniae (ATCC49619) were determined by broth dilution method, and the antimicrobial activity of LytA’ was preliminarily studied by using LytA’ and penicillin G to penicillin G-sensitive Streptococcus pneumoniae. Results The recombinant plasmids PGM-T/lytA and pET-32a (+)/lytA′ were successfully constructed. The recombinant autolysin protein expressed well and showed expected antimicrobial activity. Compared with growth control group, LytA’ and penicillin G showed obvious antibacterial activity at 3 h (t=14.243 0, P<0.011 0; t=7.400 0, P<0.022 0), and the antibacterial effect lasted until 7 h (t=20.838 0, P<0.008 0; t=17.889 0, P<0.017 0). The antibacterial effect was gradually enhanced. In addition, the antibacterial activity of LytA’ at 7 h was significantly stronger than that of penicillin G (t=6.764 0, P<0.027 0). Conclusion s The short fragment autolysin recombinant protein LytA’ shows certain antibacterial activity against penicillin G-sensitive Streptococcus pneumoniae in vitro, and the duration is long. It has the possibility of becoming an anti-Streptococcus pneumoniae therapeutic agent.