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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。
Objective To clone the gene sequence of Streptococcus pneumoniae lytic enzyme (LytA) and to express it recombinantly. Methods The genomic DNAs of different clinical isolates were extracted respectively. According to the LytA gene sequence of Streptococcus pneumoniae R6 wild strain, primers were designed and amplified by PCR. The target gene fragment was cloned into prokaryotic expression plasmid and sequenced. Using bioinformatics Methods The LytA gene sequences of different clinical isolates were compared and analyzed, and the recombinant LytA gene was also expressed. Results The complete LytA gene was amplified from the genomes of different clinical isolates. The recombinant plasmid pGEX-4T-1-L-ytA was successfully constructed. Comparing the DNA sequence and deduced amino acid sequence of LytA gene of different clinical isolates, we found that LytA gene sequence and amino acid sequence of each strain were different. LytA gene was highly expressed in Escherichia coli JM109 induced by isopropylmercapto galactose (IPTG). SDSPAGE results showed that pGEX-4T-1-L- ytA fusion protein relative molecular mass of about 62000, consistent with the theoretical prediction. Conclusion The sequence analysis showed that the LytA gene sequences and amino acid sequences of the isolates were different, but the homology was very high. It is presumed that the LytA gene sequence is conserved and can be used in vaccine development.