牛支原体分子伴侣Dnak的原核表达及黏附特性分析

doi:10.11843/j.issn.0366-6964.2025.06.030

Abstract

Abstract:

This study aimed to study the immunogenicity and adhesive properties of the Dnak protein of Mycoplasma bovis (Mb). Primers were designed according to the Dnak gene of Mycoplasma bovis HB0801 strain in GenBank. The Dnak gene of Mycoplasma bovis was amplified by overlap PCR and the recombinant plasmid pET-Dnak was constructed. After double enzyme digestion and sequencing, the recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by Isopropyl-β-D-thiogalactopyranoside (IPTG) for expression. Western blot, ELISA, and indirect immunofluorescence assay were used to detect the immunogenicity of Dnak protein and the distribution of Dnak protein in Mycoplasma bovis. The complement-mediated bactericidal assay detected the activity of rDnak antiserum mediatedantiserum-mediated activation of mycoplasma. Adhesion and adhesion inhibition tests were used to analyze the adhesion function of Dnak protein to host cells. The results showed that the recombinant protein rDnak was successfully expressed in E. coli, the main soluble form was pure, the relative molecular size was about 70 ku, and the recombinant protein had good immunogenicity. Its multi-antiserum can effectively activate complement to kill mycoplasma; Dnak protein exists in both the membrane and cytoplasm of Mycoplasma bovis and is an adherence-related protein of Mycoplasma bovis, involved in adhesion to EBL cells. Dnak protein is an adhesion-related protein with good immunogenicity in Mycoplasma bovis, which lays a foundation for exploring the pathogenic mechanism of Mycoplasma bovis.

Key words: Mycoplasma bovis, Dnak protein, prokaryotic expression, adhesion, immunogenicity

CLC Number:

S852.62

ZHAO Yunhai, ZHANG Yangyang, MA Haiyun, WANG Qing, HE Xiaoxiao, LIU Kai, ZHANG Yuting, LIU Yudong, YANG Yongning, WU Xiaochun, XING Xiaoyong, QUAN Guomei, ZHANG Zhixiong, BAO Shijun. Prokaryotic Expression and Adhesion Characteristics of Molecular Chaperone Dnak of Mycoplasma bovis[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2868-2878.

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引物 Primer	序列(5′→3′) Sequence	产物大小/bp Product size
Dnak-F	CGC$\underline{{\rm{GGATCC}}}$ATGGCAAAAGAAGTTATTATTG	650
Dnak-R1	TTTTGATTAAATCAATTAACCATTTTACAATTTCATTG
Dnak-F1	CAATGAAATTGTAAAATGGTTAATTGATTTAATCAAAA	1 182
Dnak-R	CCG$\underline{{\rm{CTCGAG}}}$TTATTCGAATGTTTCTTCATC

稀释倍数 Dilution factor	OD₄₅₀ _nm Optical density
1∶3 000	1.202 5
1∶6 000	1.097 7
1∶12 000	0.983 2
1∶24 000	0.880 6
1∶48 000	0.721 9
1∶96 000	0.667 4
阴性对照 Negative control	0.302 5
空白对照Blank control	0.299 4

试验分组 Experiment groups	Mb CFU平均(CFU×10⁴) Mean CFU (×10⁴)	杀支原体率/% Mycoplasmacidal rates
rDnak多抗血清 rDnak polyclonal antibody serum	60	75.8
Mb菌体多抗血清 Mb bacterial polyclonal antibody serum	44	82.3
补体对照组 Complement control group	236	—
未免血清 Uninimmune serum	230	—
PBS	248	—

试验分组 Experiment groups	Mb CFU平均值(CFU×10³) Mean CFU (×10³)	黏附抑制率/% Adhesion inhibition rate
rDnak多抗血清 rDnak polyclonal antibody serum	151	85.16
Mb菌体多抗血清 Mb bacterial polyclonal antibody serum	98	90.37
免疫前血清 Preimmune serum	1018	—
未感染组 Uninfected group	0	0

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Prokaryotic Expression and Adhesion Characteristics of Molecular Chaperone Dnak of Mycoplasma bovis

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