原核表达MurA蛋白并制备其多克隆抗体用于免疫磁珠快速检测单增李斯特菌  

Prokaryotic expression of MurA protein and development of polyclonal antibody for rapid detecting Listeria monocytogenes using immunomagnetic beads

作　　者：童吉宇[1] 李志清[1] 闻一鸣[1] 李雨辰[1] 向军俭[1] 

机构地区：[1]广东省分子免疫与抗体工程重点实验室暨南大学抗体工程研究中心,广东广州510632

出　　处：《微生物学通报》2014年第6期1234-1242,共9页Microbiology China

摘　　要：【目的】制备MurA多抗,结合免疫磁珠与选择平板进行单增李斯特菌的快速检测,建立单增李斯特菌的免疫磁珠快速检测方法。【方法】构建MurA的原核表达载体,转化大肠杆菌进行优化表达。镍柱纯化表达产物,质谱鉴定重组蛋白,再免疫小鼠,制备其多克隆抗体。用所获多抗制备免疫磁珠,建立单增李斯特菌免疫磁珠-选择性培养基检测方法,并对人工污染牛奶样品进行检测。【结果】在大肠杆菌中高效表达了分子量约为72 kD的可溶性融合蛋白,质谱鉴定其为MurA蛋白;免疫小鼠获得的抗血清效价达1:10 000,与伤寒沙门氏菌、副溶血弧菌、大肠杆菌及属内其它病原菌均无交叉;所建立的免疫磁珠-选择性培养基检测法可检出浓度为103 CFU/mL及以上的单增李斯特菌,仅与英诺克李斯特菌存在一定交叉反应;牛奶样品单次仅需9 h增菌就能被检出,较常规增菌时间缩短39 h;检测限为0.4 CFU/mL。【结论】表达并纯化得到高纯度的单增李斯特菌MurA蛋白,制备的鼠源多克隆抗体亲和力高,特异性好;建立了快速检测单增李斯特菌的免疫磁珠联合选择性培养基法,在灵敏度不变的情况下,实现24 h内成功对牛奶样品的检测,较国标法减少42 h以上。[Objective] To develop polyclonal antibody against MurA protein and combine immunomagnetic with selective plate to rapidly detect Listeria monocytogenes （LM）. [Methods] Prokaryotic expression vector of MurA was constructed and transformed into E. coli to optimally express. Product after expression was purified by nickel aff＇mity chromatography, mass spectrometry analysis was used to identify the recombinant protein; after correct identification, the protein was applied to immune mice in order to prepare polyclonal antibodies. The antibodies we acquired were used to develop immunomagnetic beads, which combined with selective medium todetect artificial contaminated milk samples. [Results] A soluble fusion protein with a molecular weight of 72 kD was expressed in E. coli, this protein was identified as MurA protein; antiserum possessed a titer as high as 10 000, with no cross-reaction to Salmonella typhi, Vibrio prholyticus, E. coli and other pathogenic bacteria. Despite a little cross reaction with Listeria innocua, themethod combined specific immunomagnetic beads with selective medium managed to detect LM of a concentration of 10^3 CFU/mL or above. After nine hours enrichment, milk samples within an original concentration of more than 0.4 CFU/mL were successfully detected, which was 39 hoursless than the regular enrichment method. [Conclusion] Recombinant MurA protein was highly expressed in E. coli and showed high purity after purification, the polyclonal antibodies showed high affinity and specificity against LM. The immunomagnetic beads-selective medium method fordetecting LM was able to detect milk samples within 24 hours, which was 42 hours less than national standard method with the same sensitivity.

关 键 词：单增李斯特菌 MurA 多克隆抗体 免疫磁珠 

分 类 号：TS207.4[轻工技术与工程—食品科学]