单增李斯特菌内化素A单克隆抗体制备及胶体金免疫层析试纸条的研制

Monoclonal Antibody Production Against Listeria Monocytogenes Internalin A and Colloid Gold Immunochromatographic Strip Development

将单增李斯特菌的内化素A（InlA）基因片段进行密码子优化后，合成基因并插入到质粒载体DET28a中，构建原核表达重组质粒。经过乳糖自诱导表达后，获得了大小为48kD的重组蛋白。用Ni亲和层析方法纯化重组表达后的InlA蛋白片段。用该蛋白免疫BALB／C小鼠，筛选高效的InlA单克隆抗体细胞株，并制备单克隆抗体，共筛选出3株亲和力较高的单克隆抗体，筛选得到的InlA单克隆抗体经过ProteinG亲和层析纯化。研制双抗夹心法胶体金免疫层析试纸条，并对其敏感性、特异性等指标进行评价。该试纸条灵敏度达到1x10^6CFU／mL，用实际样本加标后，对该胶体金试纸条进行验证，并用国标方法做了对比。结果表明，该试纸条能满足样本中单增李斯特菌的检测要求。

Internalin A gene from listeria monocytogenes was synthesized after codon optimization and inserted into pET28a vector. Recombinant internalin A protein of 48kD was expressed by lactose self-induction. The protein was purified by Ni-a- garose affinity chromatography. BALB/C mouse was immunized by the recombinant InlA protein and 3 monocle cell lines were got. Monoclonal antibodies from the 3 cell lines were purified by protein G affinity chromatography. Colloid Gold Immunochro- matographic strip of double antibody sandwich was developed.The sensitivity of the strip was lxl0sCFU/mL and the specificity was also detected. By contrast test with Chinese standard method for listeria monocytogenes. The result showed that the strip can be used in listeria monocytogenes detection.