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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。
Objective To construct the prokaryotic expression vector of lytA gene of Streptococcus pneumoniae (ATCC49619) and express it in E.coli expression system. LytA recombinant protein (rLytA) was obtained and its in vitro antibacterial activity was discussed. Methods According to the lytA gene sequence of Streptococcus pneumoniae M66 in GenBank (FN549899.1), specific primers were designed and synthesized. The lytA gene was amplified from the genome of Streptococcus pneumoniae ATCC49619 by PCR. The cloned vector PGM-T / pET32a (+) / lytA was transformed into competent cells E.coli BL21 (DE3) by lytA and lytic vector pET32a (+) / lytA. After induced by IPTG, the target protein rLytA was purified by isoelectric point eluting technique The minimum inhibitory concentration (MIC) of rLytA and penicillin G against Streptococcus pneumoniae and penicillin G resistant strains was determined by micro broth dilution method. The drug susceptibility of S. pneumoniae and its resistant strains to rLytA and penicillin G was determined dynamically. Evaluation of rLytA in vitro antibacterial activity. Results The prokaryotic expression vector pET32a (+) / lytA was successfully constructed. The MICs of rLytA against Streptococcus pneumoniae and Penicillin G resistant strains were 8μg / ml and 16μg / ml, respectively. The rLytA against standard strains and resistant strains MIC of three times the concentration, respectively, on the standard strains and drug-resistant strains, compared with the drug-free control group, rLytA 3h after the standard strain significantly reduced the number of bacteria (P <0.05), and the effect continued until 7h <0.05). The antibacterial activity of penicillin G resistant strains was inhibited at 3h (P <0.01), and the antibacterial activity continued to 7h (P <0.01). Conclusion A certain concentration of rLytA has antibacterial activity against standard strains and resistant strains of Streptococcus pneumoniae, and has a long duration and possesses the possibility of being an antibacterial agent.