滑液囊支原体GA组件蛋白的原核表达及间接ELISA方法的建立

doi:10.16431/j.cnki.1671-7236.2024.06.036

摘要/Abstract

摘要： 【目的】体外表达滑液囊支原体(Mycoplasma synoviae,MS)的GA组件蛋白(GA module-containing protein),建立一种MS抗体检测方法,用于血清学检测及MS抗体水平监测。【方法】分析、筛选MS的GA组件蛋白长链保守结构域,合成重组质粒pET30a-ΔGA-L,转化大肠杆菌BL21(DE3)感受态细胞进行诱导表达,并优化表达条件;通过SDS-PAGE检测重组蛋白的表达,纯化重组蛋白ΔGA-L并进行Western blotting鉴定;以纯化后的重组蛋白ΔGA-L作为包被抗原,建立MS抗体的间接ELISA检测方法,优化反应条件,确定临界值,对其特异性、敏感性、重复性进行检验,并进行临床样品检测。【结果】重组蛋白ΔGA-L的分子质量大小为45.7 ku,最佳表达条件为25 ℃、0.2 mmol/L IPTG诱导表达5 h,以包涵体形式表达。Western blotting结果表明,重组蛋白ΔGA-L能与MS抗体发生特异性反应。以ΔGA-L抗原包被浓度为0.5 μg/mL,一抗稀释度为1∶400,二抗稀释度为1∶12 000为最佳条件,建立了MS抗体间接ELISA检测方法,其阴阳性临界值为0.283;所建立方法特异性强、灵敏度高,有较高稳定性;与商品化MS抗体检测试剂盒总符合率为96%。【结论】本研究成功表达了MS重组蛋白ΔGA-L,所建立的MS抗体间接ELISA检测方法具有良好的特异性、敏感性、重复性,为MS抗体检测提供了有效快捷的方法。

关键词: 滑液囊支原体; GA组件蛋白; 脂质相关膜蛋白; 原核表达; 间接ELISA

Abstract: 【Objective】 GA module-containing protein of Mycoplasma synoviae (MS) was expressed in vitro.A method for detecting MS antibody was established for serological detection and monitoring of MS antibody level.【Method】 The long-chain conserved domain of GA module-containing protein in MS was analyzed and screened,and the recombinant plasmid pET30a-ΔGA-L was synthesized and transformed into Escherichia coli BL21 (DE3) competent cells for induced expression,and the expression conditions were optimized.The expression of recombinant protein ΔGA-L was detected by SDS-PAGE.The recombinant protein ΔGA-L was purified,and identified by Western blotting.The purified recombinant protein ΔGA-L was used as the coating antigen to establish an indirect ELISA method for detection of MS antibody.The response conditions were optimized,the critical value was determined,the specificity,sensitivity and repeatability were tested,and clinical samples were tested.【Result】 The molecular weight of the recombinant protein ΔGA-L was 45.7 ku,and the optimal expression conditions were 25 ℃ and 0.2 mmol/L IPTG induced expression for 5 h,and the expression was in the form of inclusion bodies.Western blotting result showed that the recombinant protein ΔGA-L could react specifically with MS antibody.An indirect ELISA method for MS antibody detection was established with ΔGA-L antigen coating concentration of 0.5 μg/mL,primary antibody dilution ratio of 1∶400 and secondary antibody dilution ratio of 1∶12 000 as the optimal conditions,and the critical positive value was 0.283.The established method had strong specificity,high sensitivity and high stability.The total coincidence rate was 96% compared with commercial MS antibody detection kit.【Conclusion】 In this study,the MS recombinant protein ΔGA-L was successfully expressed,and the established indirect ELISA method for MS antibody detection had great specificity,sensitivity and repeatability,providing an effective and fast method for antibody detection of MS.

Key words: Mycoplasma synoviae; GA module-containing protein; lipid-associated membrane protein; prokaryotic expression; indirect ELISA

中图分类号:

S852.62

高乐, 司朵朵, 郭磊, 陈灿, 王玮, 王健霖, 王玲玲, 李继东. 滑液囊支原体GA组件蛋白的原核表达及间接ELISA方法的建立[J]. 中国畜牧兽医, 2024, 51(6): 2621-2632.

GAO Le, SI Duoduo, GUO Lei, CHEN Can, WANG Wei, WANG Jianlin, WANG Lingling, LI Jidong. Prokaryotic Expression of GA Module-containing Protein in Mycoplasma synoviae and Establishment of Indirect ELISA Method[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(6): 2621-2632.

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