滑液支原体WVU1853株热不稳定延伸因子的表达及黏附特性分析

doi:10.11843/j.issn.0366-6964.2022.02.018

摘要/Abstract

摘要： 旨在探究滑液支原体（Mycoplasma synoviae，MS）热不稳定延伸因子（elongation factor thermo unstable，EF-Tu）的黏附特性。参照GenBank中MS WVU1853株EF-Tu序列，设计引物对Tu-F/Tu-R，利用PCR扩增获得MS EF-Tu基因后，将其克隆入pET-28a（+）构建重组质粒pET-EF-Tu，继而转化大肠杆菌BL21（DE3）并经IPTG诱导表达。纯化表达产物免疫新西兰兔制备抗血清，进而利用Western blot、ELISA和免疫荧光试验分别分析重组蛋白的免疫原性及EF-Tu在MS中的分布，利用补体介导的杀菌试验分析重组蛋白抗血清的补体介导杀支原体活性，利用黏附及抑制试验分析EF-Tu的黏附特性。结果表明，重组蛋白在大肠杆菌中呈可溶性表达，其相对分子质量约43 ku，并具有良好的免疫原性，其抗血清具有补体介导的杀支原体活性；EF-Tu在MS的细胞膜和细胞质中均有分布，且是MS的一种黏附相关蛋白。EF-Tu是MS膜表面黏附相关的免疫原性蛋白，研究结果为深入研究MS EF-Tu生物学功能奠定了基础。

关键词: 滑液支原体, 热不稳定延伸因子, 免疫原性, 膜蛋白, 黏附

Abstract: The aim of this study was to investigate the adhesive function of elongation factor thermo unstable (EF-Tu) of Mycoplasma synoviae (MS). The primer pairs Tu-F/Tu-R were designed according to the sequence of EF-Tu gene of MS WVU1853 strain in GenBank. The MS EF-Tu gene was amplified by PCR and cloned into pET-28a (+). The recombinant plasmid pET-EF-Tu was constructed and transformed into Escherichia coli (E. coli) BL21(DE3). Then the recombinant proteins (rMSEF-Tu) were expressed after induction by IPTG, and the expression product was purified and used to immunize New Zealand rabbits to prepare anti-serum. Subsequently, the immunogenicity of the rMS EF-Tu and the distribution of EF-Tu in MS were respectively analyzed by Western blot, ELISA and immunofluorescence tests. The complement mediated mycoplasmacidal activity of recombinant protein antiserum was assessed by complement mediated bactericidal assay, and the adhesion function of MS EF-Tu was evaluated by adhesion and inhibition assay. The results showed that MS EF-Tu was expressed mainly in soluble form in E. coli. The relative molecular mass of rMS EF-Tu was about 43 ku, and had good immunogenicity. The anti-rMS EF-Tu serum has complement-mediated mycoplasmacidal activity. In addition, the MS EF-Tu was distributed both in MS membrane and cytoplasm, and was an adhesion-related protein of MS. This study showed that EF-Tu was an immunogenic protein related to MS membrane surface adhesion. The verification of its biological function laid a foundation for further study of MS EF-Tu.

Key words: Mycoplasma synoviae, elongation factor thermo unstable (EF-Tu), immunogenicity, membrane protein, adhesion

中图分类号:

S852.62

岳亚辉, 邢小勇, 武小椿, 温峰琴, 张宏燕, 龙翠琴, 张立, 马海云, 包世俊. 滑液支原体WVU1853株热不稳定延伸因子的表达及黏附特性分析[J]. 畜牧兽医学报, 2022, 53(2): 520-528.

YUE Yahui, XING Xiaoyong, WU Xiaochun, WEN Fengqin, ZHANG Hongyan, LONG Cuiqin, ZHANG Li, MA Haiyun, BAO Shijun. Expression of Elongation Factor Thermo Unstable of Mycoplasma synoviae WVU1853 Strain and Analysis of Its Adherence Characterization[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(2): 520-528.

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