牛支原体0580基因C端截短体的原核表达及生物学功能初步分析

doi:10.11843/j.issn.0366-6964.2023.07.030

Abstract

Abstract: The purpose of this study was to analyze the biological function of 0580 gene of Mycoplasma bovis (Mb) PG45 strain. In the previous stage, we found that PG45、08M and 07801 strain of M. bovis can lyse mouse red blood cells. So, according to the amino acid sequence of the putative hemolysin related gene 0580 in PG45 strain, gene MBOVPG45_0580 presumed to be related to hemolysin was screened. Due to the protein encoded by this gene is predicted to have four transmembrane regions and is difficult to express, we constructed the prokaryotic expression vector Pcold III-0580-C terminal fragment without transmembrane region to induce expression and purification, The recombinant protein 0580-C was immunized in BALB/c to prepare polyclonal antibody. At last, we explored whether the 0580-C recombinant protein has the ability of lysing RBCs and influences the ability of the strain to adhere to cell; Besides, the obtained polyclonal antibody specificity and titer were detected. The putative hemolysin protein 0580 in strain PG45 was 100% homologous to M. bovis, and the C-terminal truncated form of protein 0580 which removed four transmembrane regions could be successfully expressed in E. coli. The relative molecular weight of the purified recombinant 0580-C protein was about 37 ku. The hemolytic test of mouse RBCs found that the recombinant 0580-C protein has no hemolytic activity, but the cell adhesion test show that the protein contributes to improve the adhesion ability of Mb to EBL cell, polyclonal antibody of 0580-C protein could effectively block the adhesion process. The titer of the polyclonal antibody against 0580-C protein was as high as 2¹¹. The polyclonal antibody could recognize the 0580 protein expressed by three strains of M. bovis (PG45, 08M, 07801), which indicated that the polyclonal antibody against 0580-C protein was specific. This study found that the C-terminus of protein 0580 may be a novel adhesive-related molecule, which provides new insights into the biological function of putative hemolysin associated protein and the pathogenic mechanism of Mycoplasma bovis.

Key words: Mycoplasma bovis, prokaryotic expression, adhesion, 0580-C recombinant protein

CLC Number:

S852.62

JING Tingting, YIN Hao, HUANG Rong, LAN Shimei, LI Zhangcheng, YOU Liuchao, HAO Huafang, FU Lei, CHU Yuefeng. Prokaryotic Expression and Biological Function Analysis of Mycoplasma bovis 0580 Gene C-terminal Truncated[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2991-3001.

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Prokaryotic Expression and Biological Function Analysis of Mycoplasma bovis 0580 Gene C-terminal Truncated

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