弧菌外膜蛋白Ompk基因序列分析及其多克隆抗体的制备

doi:10.7506/spkx1002-6630-201623024

摘要/Abstract

摘要： 弧菌外膜蛋白（outer membrane protein，OMP）OmpK不仅可激发机体的体液免疫和细胞免疫，而且可在不同血清型菌株之间产生免疫交叉反应。本研究利用克隆测序和查找GenBank获得20 株弧菌的Ompk基因序列并构建Ompk系统进化树，对比分析20 株弧菌Ompk基因序列；利用原核表达系统表达野生株Vibrio parahaemolyticus B的OmpK，纯化OmpK后制备兔多克隆抗体；采用酶联免疫吸附分析法和蛋白质印迹法（Western-blotting）分别检测抗体效价和特异性。系统进化树和基因序列比对结果显示Ompk基因在弧菌中高度保守，核苷酸序列相似性在77%以上；原核表达系统成功表达了弧菌OmpK，蛋白经纯化后成功制备的兔多抗效价达1∶16 000；Western-blotting实验证实抗体具有很好的特异性；OmpK多克隆抗体在弧菌中普遍存在免疫交叉性。因此，弧菌Ompk基因序列分析及其抗体研制，不仅为弧菌快速免疫学检测提供可能性，也为以OmpK为靶点的水产疫苗研制奠定了基础。

关键词: 弧菌, Ompk, 原核表达, 纯化, 多克隆抗体, 免疫学检测

Abstract: Outer membrane protein (OmpK) in Vibrio not only can stimulate humoral immunity and cellular immunity in the
body, but also can produce immune cross-reaction among strains of different serotypes. In this paper, cloning of sequences
and comparison with Genebank sequences were carried out to build Ompk phylogenetic trees of 20 Vibrio strains. A
prokaryotic expression system was used to express the OmpK of Vibrio parahaemolyticus B, and rabbit polyclonal antibody
was prepared after OmpK was purified and used to immunize rabbits. Antibody titer and specificity were detected by enzyme
linked immunosorbent assay (ELISA) and Western-blotting. Phylogenetic tree and gene sequence alignment results showed
that the Ompk gene was highly conservative in Vibrio and the similarity of nucleotide sequences was approximately 77%.
OmpK was successfully expressed in the prokaryotic expression system and rabbit polyclonal antibody was successfully
induced by immunization with the purified protein with a titer of 1:16 000 as detected by ELISA. Western-blotting results
confirmed that the antibody had good specificity. Vibrio Ompk gene sequence analysis and anti-Vibrio antibody preparation
will provide a theoretical basis for rapid and high-throughput detection of Vibrio in aquatic products..

Key words: Vibrio, Ompk, prokaryotic expression, purification, polyclonal antibody, immunological detection

中图分类号:

TS201.3

丁承超，谢曼曼，曾海娟，刘箐. 弧菌外膜蛋白Ompk基因序列分析及其多克隆抗体的制备[J]. 食品科学, doi: 10.7506/spkx1002-6630-201623024.

DING Chengchao, XIE Manman, ZENG Haijuan, LIU Qing. Gene Sequence Analysis and Polyclonal Antibody Preparation of Outer Membrane Protein, Ompk, in Vibrio[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201623024.

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弧菌外膜蛋白Ompk基因序列分析及其多克隆抗体的制备

Gene Sequence Analysis and Polyclonal Antibody Preparation of Outer Membrane Protein, Ompk, in Vibrio

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