【摘要】 目的克隆肺炎链球菌自溶酶(LytA)的基因序列,并进行重组表达。方法分别提取不同临床分离株的基因组DNA,根据已知肺炎链球菌野生R6株LytA基因序列设计引物,进行PCR扩增,将获得的目的基因片段克隆入原核表达质粒,然后测序;利用生物信息学方法,对不同临床分离株LytA基因序列进行比较分析,同时进行LytA基因的重组表达。结果从不同临床分离株的基因组中均扩增出完整的LytA基因片段,成功构建了重组质粒pGEX-4T-1-L-ytA。比较不同临床分离株LytA基因的DNA序列及推测的氨基酸序列,发现各株LytA基因序列及氨基酸序列间存在差异。通过异丙基巯基半乳糖(IPTG)诱导,LytA基因在大肠埃希菌JM109中得到高效表达,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDSPAGE)结果显示pGEX-4T-1-L-ytA融合蛋白相对分子质量约62000,与理论预测一致。结论序列分析发现各分离株的LytA基因序列及氨基酸序列间存在差异,但同源性极高,推测LytA基因序列保守,可用于疫苗研发。更多还原

【Abstract】 Objective To clone the gene of autolysin(LytA) which are from different clinical strains of Streptococcus pneumoniae and express them in Escherichia coli. Methods The LytA gene was amplified by PCR from the total DNA of S.pneumoniae. Primers were designed according to the LytA gene sequence of R6. Recombinant plasmids were constructed and the sequences of different clinical strains were analyzed through method of bioinformatics. The cloned genes were expressed in E.coli and detected by SDS-PAGE. Results Complete LytA gene were amplified from all of the different clinical strains of S.pneumoniae and recombinant plasmids pGEX-4T-1-LytA were constructed successfully. After comparing the sequence of DNA and supposed protein, we find some differences. Induced by IPTG, LytA gene was expressed effectively in E.coli Jm109. Result of SDS-PAGE showed that the molecular weight of expressed protein was 62 kD, the same as calculated. Conclusions The sequences encoding LytA from different clinical strains of S.pneumoniae were cloned, the recombinant plasmids pGEX-4T-1-LytA was constructed successfully. Sequence analysis showed that there have difference among the gene and amino acid sequences of LytA from different clinical strains. Further studies should be focused on whether the difference contributes to activity of autolysin and the drug-resistance of S.pneumoniae.更多还原