副溶血性弧菌鞭毛蛋白FlaA的重组表达与多克隆抗体制备.doc

文档介绍：
副溶血性弧菌鞭毛蛋白 FlaA 的重组表达与
多克隆抗体制备#
王燕,夏雨,王周平**
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(江南大学食品学院,江苏无锡 214122)
摘要:副溶血性弧菌是广泛存在于水产品中的革兰阴性致病菌。鞭毛蛋白 FlaA 是副溶血性
弧菌所特有的蛋白并在其侵染宿主、引起宿主免疫反应过程中起到重要作用,由鞭毛蛋白免
疫动物得到的抗体可以作为检测副溶血性弧菌污染的探针。本研究通过分子生物学方法构建
了重组 FlaA 的表达载体 pET28a-FlaA,经 IPTG 诱导获得 FLaA 的大量表达。该重组蛋白以
可溶性蛋白形式存在,经 Ni-NTA 亲和柱纯化后对家兔进行免疫,得到浓度为 mg/mL、
效价为 1:125,000 的 FlaA 抗体。通过间接 ELISA 实验,测得该抗体与副溶血性弧菌反应的
灵敏度为 1E4 cfu/mL,且该抗体与五种其它常见致病菌无明显交叉反应。
关键词:副溶血性弧菌;鞭毛蛋白;克隆表达;抗体制备
中图分类号:;
binant expression and polyclonal antibody
preparation of Vibrio parahaemolyticus flagellin A
WANG Yan, XIA Yu, WANG Zhouping
(School of Food Science and Technology, Jiangnan University, JiangSu WuXi 214122)
Abstract: The Gram-negative bacterium Vibrio parahaemolyticus is mon pathogenic
bacteria in aquatic products. Flagellin A, which is a unique protein in this strain, plays an
important role during infection and induction of the host immune response. The production of
polyclonal antibody against FlaA protein can be used as a probe in V. parahaemolyticus fast
detection. The binant plasmid pET28a-FlaA was constructed and the FlaA was expressed
with induction of IPTG. The soluble FlaA protein was purified by Ni-NTA affinity
chromatography and the polyclonal antibody against FlaA was prepared by immunning rabbit. The
final concentration of FlaA antibody was mg/mL with the titer of