Sequence Characterization, Prokaryotic Expression and Immunogenicity Analysis of Gallibacterium anatis tolC Gene
SHI Zi-Cong1,*, LIU Pan-Pan1,2,*, WANG Xin-Wei1, ZHAI Xue-Ying1, CHEN Hua-Yuan1, LIU Hong-Ying1, MA Li-Jing1, YANG Xia1,**, CHEN Lu1
1 College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China; 2 College of Agricultural, Ningxia University, Yinchuan 750021, China
Abstract：The tolC gene is widespread in gram-negative bacteria, and TolC family protein (TolC) expressed by tolC is an outer membrane channel protein, which mediates the extracellular transport of various compounds and plays an important role in the biological functions of bacteria. The identification of the characteristic of tolC gene of Gallibacterium anatis and its protein would play crucial roles in efforts to prevent and control this disease. In order to analyze the structural characteristics of gene and amino acid and potential as an antigen of tolC of G. anatis, in this study, the sequences and phylogenetic analysis of tolC gene of 28 G. anatis isolates were carried out. Taking G. anatis PDS-RZ-1-SLG as the research object, the physicochemical properties and structures of TolC were predicted by bioinformatics methods. In addition, the tolC gene sequences were cloned and expressed, and its antigenic properties were analyzed. The results showed that the tolC gene, with 1 371 bp in length, was ubiquitous in Chinese isolates of G. anatis and its nucleotide sequences were highly conserved (similarity: 92.1%~100%), which had a close relationship with G. genomosp 1 and 2. Moreover, the tolC gene of G. anatis encoded a protein of 456 amino acids, with a calculated molecular mass of 51.1 kD, and the TolC protein was a stable, hydrophilic, α-type and secretory membrane protein without transmembrane domains, but with a signal peptide sequence and 10 main B cell epitopes. SDS-PAGE and Western blot analysis showed the recombinant protein was about 45 kD, and the recombinant protein could react specifically with positive serum of G. anatis and the polyclonal anti-TolC antiserum raised in rabbits had a specific binding with G. anatis, which verified the reactogenicity and immunogenicity of the TolC protein. This study provides basic information for further research on the biological functions of the TolC, and establishes a theoretical basis for development of new vaccines against G. anatis.
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