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

摘要/Abstract

摘要：

旨在研究牛支原体(Mycoplasma bovis, Mb)分子伴侣Dnak (chaperone protein Dnak)蛋白的免疫原性和黏附特性。根据GenBank中Mb HB0801株Dnak基因设计引物，利用Overlap PCR扩增获得Mb Dnak基因，构建重组质粒pET-Dnak, 经双酶切和测序鉴定正确后，将重组质粒转入大肠杆菌BL21(DE3)并通过异丙基硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside, IPTG)诱导表达。将纯化的重组蛋白免疫新西兰白兔获得抗rDnak血清，利用Western blot、ELISA和间接免疫荧光检测Dnak蛋白的免疫原性以及Dnak蛋白在Mb中的分布。通过补体介导的杀菌试验分析检测rDnak抗血清介导的激活补体杀支原体活性。利用黏附及黏附抑制试验分析Dnak蛋白对宿主细胞的黏附功能。结果表明，重组蛋白rDnak主要以上清可溶形式在大肠杆菌中成功表达，其相对分子质量约为70 ku，具有良好的免疫原性；其多抗血清可有效激活补体杀伤支原体；Dnak蛋白在Mb的细胞膜和细胞质中均存在，是Mb的一种黏附相关蛋白，参与对EBL细胞的黏附。综上，Dnak蛋白是Mb中具有良好免疫原性的黏附相关蛋白，为探究Mb的致病机制奠定基础。

关键词: 牛支原体, Dnak蛋白, 原核表达, 黏附, 免疫原性

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

中图分类号:

S852.62

赵云海, 张阳阳, 马海云, 王青, 何肖肖, 刘凯, 张钰婷, 刘玉东, 杨永宁, 武小椿, 邢小勇, 权国梅, 张志雄, 包世俊. 牛支原体分子伴侣Dnak的原核表达及黏附特性分析[J]. 畜牧兽医学报, 2025, 56(6): 2868-2878.

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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