金黄色葡萄球菌肠毒素SEK的纯化及DAS-ELISA检测方法的建立与应用

Prokaryotic Expression, Purification, Development and Application of a Double-antibody Sandwich Enzyme Linked Immunosorbent Assay for Staphylococcal Enterotoxin K

摘要:

本研究目的为建立一种快速检测金黄色葡萄球菌新型肠毒素SEK的双抗夹心酶联免疫吸附方法。将构建的原核表达载体pET-28a(+)-ΔNspSEK转化入BL 21(DE3) pLysS细胞中诱导表达，经Ni2+-NTA亲和层析柱纯化获得重组SEK蛋白作为抗原。利用双抗夹心酶联免疫方法检测程序，确定抗SEK单克隆抗体及抗SEK多克隆血清的最佳稀释度，并使用该方法应用于检测SEK人工污染样品的加标回收率和茶多酚及乳酸链球菌素（Nisin）对7株sek阳性菌株的SEK蛋白分泌影响。结果表明，原核表达载体得到可溶性表达，重组SEK蛋白分子量约为27.7 ku；建立的双抗夹心酶联免疫检测方法中，单克隆抗体最佳包被浓度为2.89 μg/mL、抗血清的最佳稀释度为1:500，回归方程为y=0.2165x+0.1627，相关系数R2=0.9993，最低检测限为0.1 μg/mL；检测SEK人工污染脱脂奶、LB肉汤和牛肉糜中的加标回收率高达97%以上；并且，采用建立的方法测得茶多酚和Nisin的添加浓度在其MIC及以下浓度时，茶多酚对7株sek阳性菌株蛋白分泌抑制效果更明显。

Abstract:

To measure a newly recognized staphylococcal enterotoxin K (SEK), a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was established in this study. The recombinant protein SEK was expressed in BL21 (DE3) pLysS cells and purified by Ni2+-NTA affinity chromatography. Using the protein as an antigen, the monoclonal antibody and polyclonal antibodies were prepared. They were employed for detection and capture of SEK in the DAS-ELISA system that was developed to be capable of detecting SEK in piked skimmed milk, LB medium and minced beef. Subsequently, the ELISA system was applied to determine SEK secretion for 7 sek positive S. aureus isolates with tea polyphenols and Nisin treatment. The results showed that the recombinant protein SEK was successfully expressed and purified with the expected molecular weight of 27.7 ku. The DAS-ELISA for SEK was developed with the optimal anti-SEK monoclonal antibody concentration 2.89 μg/mL, the serum dilution ratio 1:500, the regression equation y=0.2165x+0.1627(R2=0.9993), and the sensitivity 0.1 μg/mL. Using the developed ELISA assay, the recovery rates of SEK in spiked skimmed milk, LB medium and minced beef were more than 97%. Furthermore, both tea polyphenols and Nisin, especially tea polyphenols, inhibited the secretion of SEK at the concentration of MIC.