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

Abstract

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

CLC Number:

S852.61

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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Prokaryotic Expression and Polyclonal Antibody Preparation of Mannheimia haemolytica Truncated Leukotoxin

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