China Animal Husbandry and Veterinary Medicine ›› 2024, Vol. 51 ›› Issue (2): 689-699.doi: 10.16431/j.cnki.1671-7236.2024.02.025

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Prokaryotic Expression of EF-Tu Protein of Mycoplasma ovipneumoniae and Preparation of Polyclonal Antibody

WANG Yongfei1,2, DENG Bowen1,2, LIU Xiaoyan1,2, HAERLEHA·Amantai1,2, GUO Jiadong1,2, ZHOU Zhengguo3, CAI Jiang1,2, LI Youwen1,2   

  1. 1. Key Laboratory of Tarim Animal Husbandry Science and Technology Corps of Xinjiang Production and Construction Corps, College of Animal Science and Technology, Tarim University, Alaer 843300, China;
    2. Engineering Laboratory for Diagnosis and Prevention and Control of Animal Diseases in South Xinjiang of Xinjiang Production and Construction Corps, Alaer 843300, China;
    3. Aksu Vocational and Technical College, Aksu 843000, China
  • Received:2023-07-26 Online:2024-02-05 Published:2024-01-29
  • Contact: 新疆生产建设兵团区域创新基金项目(2021BB014);塔里木大学校长基金项目(TDZKCX202305)
  • Supported by:
    The project was supported by the National Key Research and Development Program of China (2019YFC1905301);National Natural Science Foundation of China (22078115,21776108,21690083,22008078).

Abstract: 【Objective】 The purpose of this study was to clone EF-Tu gene in Mycoplasma ovipneumoniae (Mo), obtain EF-Tu protein by prokaryotic expression, prepare rabbit-derived polyclonal antibody against EF-Tu protein, laying the foundation for the study of the structure and function of Mo EF-Tu protein.【Method】 TGA codon in the middle of EF-Tu gene in pET-28a-EF-Tu plasmid was mutated to TGG by overlapping extension PCR, and the similarity alignment and genetic evolution analysis were conducted between the sequencing results and other Mycoplasma reference strains, and the putative protein sequences were analyzed for bioinformatics using online software.The mutated recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells for prokaryotic expression, identified by SDS-PAGE and Western blotting, and purified using Ni-chelating affinity chromatography.The polyclonal antibodies were prepared by immunizing rabbits with the purified EF-Tu fusion protein.The polyclonal antibody potency and immunological reactivity were detected by indirect ELISA and Western blotting.【Result】 The TGA site in EF-Tu gene was successfully mutated, and a prokaryotic expression vector expressing the His tag pET-28a-EF-Tu' was constructed.Bioinformatics analysis results showed that the cloned EF-Tu gene had the highest similarity and the closest genetic relationship to Mycoplasma ovipneumoniae strain MoGH3-3, and encoded 387 amino acids, without N-glycosylation site and transmembrane region.There were 10 serine, 20 threonine and 4 tyrosine phosphorylation sites of EF-Tu protein, and the secondary structure consists of random coil (35.14%), alpha helix (26.87%), extended chain (26.87%) and beta turn (11.11%).SDS-PAGE and Western blotting results showed that the size of the target protein was about 43 ku, and the protein purification concentration was 0.615 g/L.ELISA and Western blotting results showed that the prepared polyclonal antibody had a potency of up to 1∶128 000, and could specifically recognize the EF-Tu fusion protein, which had good immunological reactivity.【Conclusion】 The TGA codon of EF-Tu gene was successfully mutated in this study, the EF-Tu fusion protein was obtained by prokaryotic expression and purification, and its polyclonal antibody was prepared with a potency of 1∶128 000.This result provided an experimental basis for the subsequent study of the structure and biological function of Mo EF-Tu protein and its vaccine development.

Key words: Mycoplasma ovipneumoniae; overlapping extension PCR; EF-Tu gene; polyclonal antibody

CLC Number: