单核增生李斯特菌Internalin A的克隆表达与抗体制备

目的克隆表达单核增生李斯特菌Internalin A（InlA）,并以之为抗原制备检测单核增生李斯特菌的抗体。方法通过PCR技术从单核细胞增生李斯特菌4b中扩增出inlA基因,克隆筛选和测序鉴定后,最终构建该基因的原核表达质粒pGEX-4T-InlA,谷胱甘肽树脂亲和层析纯化表达产物后,免疫小鼠分别制备相应的多抗和单抗。结果在大肠埃希菌中成功表达了InlA,并对其进行了纯化,融合表达产物分子量约为110 kD;免疫小鼠获得的抗血清效价达到1∶1600;得到了3株抗InlA的单克隆抗体杂交瘤细胞株,腹水单抗效价为1∶1×10^5-1∶3×10^5。2种抗体与其他病原菌均无交叉反应。结论通过表达单核增生李斯特菌的特异性蛋白制备的抗体,能有效地消除交叉反应,提高检测的特异性。

Cloning, expression and antisera preparation of internalin a from Listeria monocytogenesis

Objective To clone and express protein InlA from Listeria monocytogenes and prepare the specific antibodies against Listeria monocytogenes. Method InlA gene of Listeria monocytogenesis serotype 4b was amplified by PCR technology, plasmid pGEX-4T-Li was constructed and expressed by molecular cloning method. Fusion protein InlA was purified by affinity chromatography and correspondingly, the antiserum was prepared by immunization.Result Fusion protein InlA with molecular weight of 110 kD was expressed, the titre of prepared antiserum was 1:1600. Three InlA Mab clones were obtained with a titer as high as 1:1×10~5 ～1:3×10~5 in ascities. The antibodies had no cross reaction with other pathogenic microorganisms. Conclusion Expression of the specific antigen from Listeria monocytogenes e.g. InlA and preparation of the polyclonal antibodies was a valid method to eliminate the cross-reaction from other species, and thus was beneficial to improve the specificity for the detection of Listeria monocytogenesis.