牛支原体分子伴侣Dnak的原核表达及黏附特性分析

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中图分类号：S852.62 文献标志码：A 文章编号：0366-6964（2025）06-2868-11

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

ZHAO Yunhai1，ZHANG Yangyang1，MA Haiyun1，WANG Qing1，HE Xiaoxiao 1 ，LIU Kai¹ ， ZHANG Yuting1，LIU Yudong1，YANG Yongning²，WU Xiaochun1，XING Xiaoyong1 ， QUAN Guomei¹，ZHANG Zhixiong1，BAO Shijun1 * （1. College of Animal Medicine， Gansu Agricultural University，Lanzhou 73oo7o， China ; 2.Qinghai Agri-animal Husbandry Vocational College， Xining 8121oo，China）

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 HBo8Ol strain in GenBank. The Dnak gene of Mycoplasma bouis 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 bouis. 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 （20 70ku ， 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 bois 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.

Keywords： Mycoplasma bovis ; Dnak protein; prokaryotic expression; adhesion; immunogenicity ∗ Corresponding author： BAO Shijun，E-mail： bsjdy@126. com

牛支原体是一种重要的牛致病性病原体，可造成牛的呼吸系统疾病综合征、关节炎综合征、中耳炎、结膜炎、脑膜炎以及奶牛的乳腺炎等疾病[1-2]，还可引起生殖系统疾病，导致难产、流产等[3]。（剩余17711字）