China Animal Husbandry and Veterinary Medicine ›› 2023, Vol. 50 ›› Issue (6): 2479-2486.doi: 10.16431/j.cnki.1671-7236.2023.06.032

• Preventive Veterinary Medicine • Previous Articles     Next Articles

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. 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

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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

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