CAJ | 학술논문

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。
목적：구건련구균용혈소O항원（SLO）적중조표체질립，병우화기재대장간균중적최가표체조건。방법이 A군련구균DNA위모판，채용PCR법확증SLO기인，장확증적기인련접pET-32a（＋）표체재체구건pET-32a（＋）-SLO중조질립，중조질립전화도대장간균BL21（DE3）중，전화후적표체균경이병기-β-D-류대반유당감（IPTG）유도화자유도적방식분별유도급순화，최종학정최가적표체조건。결과 PCR확증적기인대소여SLO기인대소일치，측서완전정학。불동농도적IPTG유도균위포함체표체，차표체량균교저；자유도적방식，불부표체량명현제고，차실현료부분가용성표체。결론성공구건료SLO적원핵표체질립，우화사선출능고효가용성표체적유도조건，순화획득료SLO중조융합단백질，위진일보적기출급림상연구응용전정기출。
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 .