溶血性曼氏杆菌截短型白细胞毒素的原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.06.032

中国畜牧兽医 ›› 2023, Vol. 50 ›› Issue (6): 2479-2486.doi: 10.16431/j.cnki.1671-7236.2023.06.032

溶血性曼氏杆菌截短型白细胞毒素的原核表达及多克隆抗体制备

宋越¹, 苏胜杰², 张帆¹, 白帆¹, 戴伶俐¹, 王娜¹, 王大伟³, 杨茜雯⁴, 赵世华¹, 张月梅¹

1. 内蒙古自治区农牧业科学院, 呼和浩特 010031;
2. 内蒙古自治区动物疫病预防控制中心, 呼和浩特 010010;
3. 通辽市动物疫病预防控制中心, 通辽 028000;
4. 辽宁省农业经济学校, 锦州 121007

收稿日期:2022-11-14 出版日期:2023-06-05 发布日期:2023-05-30
通讯作者: 张月梅 E-mail:zhangyuemei82@163.com
作者简介:宋越,E-mail:zsysongyue@163.com;苏胜杰,E-mail:shengjiesu.ok@126.com。
基金资助:
国家自然科学基金青年基金项目（31902305）；内蒙古农牧业科学院青年创新基金项目（31902305）；内蒙古自治区自然科学基金项目（2022MS03069）；内蒙古农牧业创新基金项目（2018CXJJM05）；内蒙古自治区科技计划项目（2022YFHH0141）

Prokaryotic Expression and Polyclonal Antibody Preparation of Mannheimia haemolytica Truncated Leukotoxin

SONG Yue¹, SU Shengjie², ZHANG Fan¹, BAI Fan¹, DAI Lingli¹, WANG Na¹, WANG Dawei³, YANG Qianwen⁴, ZHAO Shihua¹, ZHANG Yuemei¹

1. Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences, Hohhot 010031, China;
2. Inner Mongolia Animal Disease Control Center, Hohhot 010010, China;
3. Tongliao Animal Disease Control Center, Tongliao 028000, China;
4. Liaoning Agricultural Economics School, Jinzhou 121007, China

Received:2022-11-14 Online:2023-06-05 Published:2023-05-30

摘要/Abstract

摘要： 【目的】构建溶血性曼氏杆菌截短型白细胞毒素（LktA）原核表达系统，表达融合蛋白并进行纯化和鉴定，为溶血性曼氏杆菌检测方法的建立提供物质基础。【方法】设计并合成特异性引物，PCR扩增羊溶血性曼氏杆菌lkta截短基因；用限制性内切酶EcoR Ⅰ和Hind Ⅲ对PCR产物和pET-28b （+）载体进行双酶切，用T4 DNA连接酶将溶血性曼氏杆菌lkta 截短基因克隆到pET-28b （+）载体；使用IPTG诱导融合蛋白LktA的表达。通过Ni²⁺亲和层析纯化后采用SDS-PAGE和Western blotting对融合蛋白进行鉴定。用融合蛋白LktA免疫4月龄健康雌性新西兰大白兔制备多克隆抗体，4次免疫完成后进行抗体效价测定。【结果】测序结果表明，构建的表达载体中溶血性曼氏杆菌lkta基因序列正确；融合蛋白LktA的分子质量约为30 ku，在大肠杆菌中高效表达并以包涵体形式存在，在变性条件下纯化融合蛋白LktA电泳图显示条带单一，确定其纯度>90%；纯化的融合蛋白LktA可与His抗体进行特异性结合。将纯化的融合蛋白LktA免疫新西兰大白兔后成功制备出多克隆抗体，抗体效价达1：256 000以上。【结论】本研究成功构建了羊溶血性曼氏杆菌截短型LktA的原核表达载体并制备其多克隆抗体，为后期溶血性曼氏杆菌检测方法的建立提供试验材料。

关键词: 截短型蛋白; 原核表达载体; 毒力因子; 致病机理

Abstract: 【Objective】 The purpose of the test was to construct a prokaryotic expression system for the truncated leukotoxin (LktA) of Mannheimia haemolytica, express the fusion protein and purify and identify it, and provide a material basis for the establishment of detection methods for Mannheimia haemolytica.【Method】 The specific primers were designed and synthesized, and the lkta truncated gene of Mannheimia haemolytica was amplified by PCR.The PCR product and pET-28b(+) vector were digested with restriction endonucleases EcoR Ⅰ and Hind Ⅲ, and the lkta truncated gene of Mannheimia haemolytica was cloned into the pET-28b(+) vector using T4 DNA ligase.The expression of fusion protein LktA was induced using IPTG.After purification by Ni²⁺ affinity chromatography, the fusion protein was identified using SDS-PAGE and Western blotting.The polyclonal antibody was prepared by immunizing 4-month-old healthy female New Zealand White rabbits with fusion protein LktA.After four immunizations, the antibody titer was determined.【Result】 The sequencing results showed that the lkta gene sequence of Mannheimia haemolytica in the constructed expression vector was correct.The fusion protein LktA had a molecular weight of about 30 ku and was highly expressed in Escherichia coli.The fusion protein existed in the form of inclusion bodies, and the purified fusion protein LktA electrophoresis pattern under denaturation conditions showed a single band, with a purity of >90%.The purified fusion protein LktA could specifically bind to His antibody.After immunizing New Zealand White rabbits with purified fusion protein LktA, polyclonal antibodies were successfully prepared, with an antibody titer of over 1:256 000.【Conclusion】 In this study, we successfully constructed a prokaryotic expression vector containing truncated LktA from Mannheimia haemolytica and prepared polyclonal antibodies against it, providing a material basis for the establishment of a later detection method for Mannheimia haemolytica.

Key words: truncated protein; prokaryotic expression vector; virulence factor; pathogenic mechanism

中图分类号:

S852.61

宋越, 苏胜杰, 张帆, 白帆, 戴伶俐, 王娜, 王大伟, 杨茜雯, 赵世华, 张月梅. 溶血性曼氏杆菌截短型白细胞毒素的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(6): 2479-2486.

SONG Yue, SU Shengjie, ZHANG Fan, BAI Fan, DAI Lingli, WANG Na, WANG Dawei, YANG Qianwen, ZHAO Shihua, ZHANG Yuemei. Prokaryotic Expression and Polyclonal Antibody Preparation of Mannheimia haemolytica Truncated Leukotoxin[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2479-2486.

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溶血性曼氏杆菌截短型白细胞毒素的原核表达及多克隆抗体制备

Prokaryotic Expression and Polyclonal Antibody Preparation of Mannheimia haemolytica Truncated Leukotoxin

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