链球菌溶血素O抗原的原核表达及条件优化  

Prokaryotic expression of streptolysin O antigen and its condition optimization

作　　者：胡锡林[1] 花欢[1] 邓群英[1] 

机构地区：[1]南京军区鼓浪屿疗养院检验科,福建厦门361002

出　　处：《国际检验医学杂志》2014年第19期2581-2583,共3页International Journal of Laboratory Medicine

摘　　要：目的构建链球菌溶血素O抗原(SLO)的重组表达质粒,并优化其在大肠杆菌中的最佳表达条件。方法以A群链球菌DNA为模板,采用PCR法扩增SLO基因,将扩增的基因连接pET-32a(+)表达载体构建pET-32a(+)-SLO重组质粒,重组质粒转化到大肠杆菌BL21(DE3)中,转化后的表达菌经异丙基-β-D-硫代半乳糖苷(IPTG)诱导和自诱导的方式分别诱导及纯化,最终确定最佳的表达条件。结果 PCR扩增的基因大小与SLO基因大小一致,测序完全正确。不同浓度的IPTG诱导均为包涵体表达,且表达量均较低;自诱导的方式,不仅表达量明显提高,且实现了部分可溶性表达。结论成功构建了SLO的原核表达质粒,优化筛选出能高效可溶性表达的诱导条件,纯化获得了SLO重组融合蛋白质,为进一步的基础及临床研究应用奠定基础。Objective To construct the recombinant streptolysin O antigen（SLO） prokaryotic expression plasmid and establish its best expression condition in Escherichia coli .Methods The DNA fragment encoding SLO was amplified from streptococcal ge-nomic DNA template by PCR ,and then incorporated into pET-32a（＋ ） vector to construct pET-32a（＋ ）-SLO recombinant plas-mid .pET-32a（＋ ）-SLO was transformed into Escherichia coli BL21（DE3） and SLO protein was expressed and purified by isopro-pyl-β-D-thiogalactoside（IPTG）-induction and auto-induction ,respectively .Results The results of DNA electrophoresis and DNA sequencing showed that pET-32a（＋ ）-SLO recombinant plasmid was constructed successfully .When IPTG at different concentra-tion was used ,SLO expressed as inclusion body and its expression efficiency was low .Under auto-induction condition ,SLO ex-pressed as partly soluble manner ,and its expression efficiency increased .Conclusion The prokaryotic expression plasmid pET-32a （＋ ）-SLO is constructed successfully and the best condition for SLO expression and purification from Escherichia coli culture is es-tablished ,which lay the foundation for further basic and clinical application research with SLO .

关 键 词：链球菌溶血素O抗原 包涵体 自诱导 蛋白纯化 

分 类 号：R392.1[医药卫生—免疫学]