重组单增李斯特菌溶血素O在HEK293T细胞中的分泌表达及活性分析  

Secretory expression of recombinant listeriolysin O in HEK293 Tcells and its biological activity analysis

作　　者：常玉梅[1,2,3] 李文艳[2,3] 张永红[3] 张建楼[3] 温洁霞[3] 王利月[3] 仲飞[3] 

机构地区：[1]中国人民解放军保定第252医院检验科,河北保定071001 [2]河北大学基础医学院,河北保定071001 [3]河北农业大学动物医学院/河北省兽用生物制品工程技术研究中心,河北保定071001

出　　处：《河北农业大学学报》2015年第5期53-57,共5页Journal of Hebei Agricultural University

基　　金：河北省自然科学基金(C2013204130);河北大学医学学科专项资金建设项目(2015A2002)

摘　　要：为研究单增李斯特菌分泌的溶血素O(LLO)的免疫佐剂作用,需要构建其真核表达载体,使其在动物体内进行分泌表达,从而发挥其生物佐剂活性。为此,本试验通过PCR方法从LM基因组DNA中扩增了LLO基因,并将其插入到带有CD5信号肽的质粒pcDNA3.1ACD5sp的信号肽下游构建成真核表达载体,然后转染人胚肾细胞HEK293T细胞使其分泌表达,用Ni-NTA琼脂糖凝胶层析法从培养基中分离纯化重组LLO蛋白,通过Western-blot对其进行鉴定,并用溶血试验鉴定其生物活性。结果表明,扩增的LLO基因序列与GenBank中的基因序列完全一致,在HEK293T细胞中表达的重组LLO在CD5信号肽介导下能被分泌到细胞外,并具有较强的溶血活性,这为进一步研究LLO的功能提供了必要的条件。To test LLO adjuvant activity ,the construction of its eukaryotic expression vector is required for LLO expression in eukaryotic cells to perform its adjuvant activity .Thus ,the LLO gene was amplified by PCR from the genomic DNA of LM and inserted into pcDNA3 . 1ACD5sp plasmid at the downstream of human CD5 signal peptide ,fused with the peptide ,to construct the eukaryotic expression vector of LLO . The LLO vector was transfected into HEK293T cells for recombinant LLO secretory expression .The recombinant LLO was puri‐fied from cell culture supernatant by Ni‐NTA agarose affinity chromatography ,identified with Western blot .The biological activity was detected by hemolytic test .The results showed that the sequence of LLO gene amplified was completely consistent with that in GenBank .The re‐combinant LLO could be secreted into cell culture medium under the mediation of human CD5 signal peptide and possess the potent bioactivity ,which provided necessary conditions for fur‐ther investigation on LLO biological functions .

关 键 词：单增李斯特菌 溶血素O 真核表达 溶血活性 

分 类 号：S855.1[农业科学—临床兽医学]