牛支原体NOX2的原核表达及黏附特性

doi:10.11843/j.issn.0366-6964.2020.11.028

Abstract

Abstract: To explore the biological function of NADH oxidase NOX2 from Mycoplasma bovis (Mb), according to the nox2 gene sequence of Mb strain Hubei (GenBank.CP002513.1), the primers were designed and the nox2 gene of Mb strain Lintao was amplified by PCR. Based on sequencing and gene optimization, the prokaryotic expression vector pET-nox2 was constructed, and was expressed in Escherichia coli Rosetta (DE3). Subsequently, the analysis of the enzymatic activity and the immunogenicity of recombinant proteins rMbNOX2 were completed. And then the subcellular localization of NOX2, complement-dependent bactericidal activity of anti-rMbNOX2 serum, and as well as inhibition effect of anti-rMbNOX2 serum to Mb adhering to host cells was determined. The results showed that the CDS sequence of the nox2 gene of Mb Lintao strain was 1 350 bp, and showed 99.93% homology with nox2 gene of all Mb except Mb JF4278 strain in GenBank. The result of SDS-PAGE displayed the optimized nox2 was successfully expressed in E. coli. The recombinant protein rMbNOX2 was about 67 ku. Enzyme activity analysis showed that the purified rMbNOX2 had good enzymatic activity. The results of ELISA and Western blot showed that the rMbNOX2 has excellent immunogenicity, and the Mb NOX2 distribute both in the cell membrane and cytoplasm, but it's more distributed in the cytoplasm. Complement-dependent mycoplasmacidal assay and adherence inhibition assay confirmed anti-rMbNOX2 serum has distinct complement-dependent mycoplasmacidal activity and can also effectively inhibit the adherence of Mb to host cells. The results of this study lay a foundation for further study on the biological function of Mb NOX2.

Key words: Mycoplasma bovis, nox2 gene, prokaryotic expression, function analysis

CLC Number:

S852.62

BAO Shijun, ZHU Caihong, XING Xiaoyong, DING Xiaoqin, WEN Fengqin, WU Xiaochun, XUE Huiwen. Prokaryotic Expression of NOX2 of Mycoplasma bovis and Its Adherence Characterization[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(11): 2895-2902.

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Prokaryotic Expression of NOX2 of Mycoplasma bovis and Its Adherence Characterization

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