摘要
目的构建金黄色葡萄球菌PGRP基因片段的原核表达,对表达产物进行生物信息学分析。方法根据GenBank中收录的金黄色葡萄自溶素(autolysin,AtlA)基因序列(AC:D17366),设计其中pgrp基因片段的特异性引物,并通过NCBI Blast,在收录的所有金黄色葡萄球菌菌株中对相应的基因序列进行保守性分析。提取金黄色葡萄球菌(ATCC25923)的全基因组DNA为模板,采用PCR技术扩增pgrp基因序列,构建该基因的克隆载体pEASY-T1/pgrp及表达载体pET-32а(+)/pgrp,通过IPTG诱导表达重组蛋白PGRP。利用生物信息学相关软件(ProtParam、ProtScale、SignalP 4.1server、Signal-3L、TMpred、DAS、NetPhos 2.0Server)分析rPGRP蛋白的理化性质、信号肽、跨膜域及磷酸化位点等生物学信息。结果成功构建重组质粒pEASY-T1/pgrp及表达载体pET-32а(+)/pgrp,构建的克隆质粒测序结果与GenBank中金黄色葡萄自溶素AtlA基因序列(AC:D17366)的相似性为95.7%。获得的rPGRP分子质量单位约为20×103。生物信息学方法分析rPGRP片段由155个氨基酸组成,分子质量单位(Mr)为17.440 95×103,理论pI为5.45,是一种稳定的亲水性蛋白,该蛋白无信号肽且为非跨膜蛋白,属于胞外蛋白。结论成功构建金黄色葡萄球菌自溶素酰胺酶PGRP的原核表达系统。获得的约20×103肽聚糖识别蛋白片段为稳定的亲水性胞外蛋白。
Objective To create a system for prokaryotic expression of a peptidoglycan recognition protein（PGRP）from Staphylococcus aureus and to bioinformatically analyze the expressed product. Methods Based on the sequence of the autolysin gene of S.aureus in GenBank,primers specific for a fragment of the pgrp gene were designed.NCBI Blast was used to analyze the conservation of the gene fragment relative to S.aureus strains in the database.The genomic DNA of S.aureus（ATCC25923）served as a template,and PCR was used to amplify the pgrp gene sequence.The recombinant plasmid pET-32а（＋）/pgrp was constructed and transformed into BL21（DE3）to express PGRP.After expression was induced with IPTG,the expressed protein PGRP was further analyzed with SDS-PAGE and purified via electroelution into a dialysis bag.Characteristics of the PGRP protein,such as its physical and chemical properties,signal peptides,transmembrane domains,and phosphorylation sites,were bioinformatically analyzed with bioinformatics software. ResultsNCBI Blast results indicated that pgrp was conserved in S.aureus.The pgrp gene produced a band of about 470 bp in length.Concordance between the results of recombinant plasmid sequencing and the gene sequence in GenBank（AC：D17366）was 95.7%.The recombinant protein PGRP was expressed and successfully purified with a relative molecular weight of 20×103 and a concentration of 456μg/ml.PGRP consisted of 155 amino acids and is a stable hydrophilic protein with an instability index of 30.83 and a grand average of hydropathicity of-0.644.Its relative molecular mass is17440.95 and its theoretical pI is 5.45.It has no signal peptides and PGRP is not a transmembrane protein. Conclusion The pgrp gene is highly conserved in S.aureus strains,and a recombinant PGRP protein can be obtained by genetic engineering.rPGRP is a stable hydrophilic protein with no signal peptides and it is a type of small extracellular protein.
作者
孟圆
刘思雨
马秋虹
王运铎
杨光
孙文平
罗红
MENG Yuan;LIU Si-yu;MA qiu-hong;WANG Yun-duo;YANG Guang;SUN Wen-ping;LUO Hong(Clinical Laboratory, Central Hospital of ZiBo, China;Department of Clinical Immunology and Microbiology, Dalian Medical University;Dalian Municipal Central Hospital)
出处
《中国病原生物学杂志》
CSCD
北大核心
2017年第11期1062-1065,共4页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.51372029)
关键词
金黄色葡萄球菌
自溶素
肽聚糖识别蛋白
Staphylococcal aureus
autolysin
peptidoglycan recognition proteins （PGRPs）
