绵羊痒螨兔亚种MMP2基因的转录水平及其重组蛋白间接ELISA的建立

doi:10.11843/j.issn.0366-6964.2022.02.023

摘要/Abstract

摘要： 本研究旨在分析绵羊痒螨兔亚种MMP2基因（PocMMP2）的虫体转录水平，及建立针对重组PocMMP2（rPocMMP2）蛋白抗体的间接ELISA检测方法（iELISA）。通过实时荧光定量PCR（qRT-PCR）测定PocMMP2在不同发育阶段虫体、饱食与饥饿虫体的转录水平，采用原核表达系统获得rPocMMP2，经方阵滴定法优化反应条件，建立rPocMMP2-iELISA，并用该方法检测成都地区有痒螨病流行病史兔场的86份兔血清。结果显示：PocMMP2在雌虫、雄虫、若虫和幼虫阶段均有表达，且在饥饿虫体的转录水平显著高于饱食虫体（P<0.05）；rPocMMP2约72 ku，主要存在于包涵体中；rPocMMP2-iELISA临界值为0.223，敏感性和特异性分别为86.0%和83.3%，受试者工作曲线下面积（AUC）为0.876，批内与批间重复性变异系数均小于10.00%；该方法的临床检测呈现74.42%的血清阳性检出率（64/86），高于病原学检出率（10.47%，9/86）。PocMMP2可能参与了绵羊痒螨兔亚种发育和入侵宿主过程，且所建立的rPocMMP2-iELISA有良好的敏感性、特异性和较高的重复性，可用于兔痒螨病的血清抗体检测。

关键词: 绵羊痒螨兔亚种, MMP2, 转录水平, 原核表达, 间接ELISA

Abstract: The object of this study was to analyze the transcription profiles of the MMP2 in Psoroptes ovis var. cuniculi (PocMMP2), and to establish an indirect ELISA method (iELISA) for detecting IgG antibody against recombinant PocMMP2 (rPocMMP2). The transcription profiles of PocMMP2 across four life-cycle stages, "fed" and "starved" mites were investigated by quantitative real-time PCR. The rPocMMP2 was obtained in Escherichia coli expression system, and this recombinant protein was used to develop rPocMMP2-iELISA by the checkerboard titration tests. Finally, the field investigation using this iELISA was performed for analysis of 86 sera samples collected from a rabbit farm with a previous history of psoroptic mange in Chengdu. Results were as follows:PocMMP2 was expressed across four life-cycle stages, and expression level of MMP2 was significantly higher in "starved" mites than in "fed" mites (P<0.05). The rPocMMP2 was mainly presented in inclusion bodies with the size of~72 ku. The cut-off value for rPocMMP2-iELISA was 0.223, the sensitivity and specificity for the test were 86.0% and 83.3%,respectively. Additionally, the area under the receiver operating curve (AUC) for the test was 0.876,and both the intra-and inter-coefficients of variation were less than 10.00%. Further field study showed 74.42% (64/86) seropositive rate, which was higher than etiological detection rate (10.47%, 9/86). In summary, PocMMP2 might play an important role in the developmental physiology and the invasion process. The rPocMMP2-iELISA appeared good sensitivity, specificity and high repeatability, and could be used to detect serum antibody of psoroptic mange in rabbits.

Key words: Psoroptes ovis var. cuniculi, MMP2, transcription level, prokaryotic expression, indirect ELISA

中图分类号:

S852.746

葛优, 郑晶, 蒲善秋, 周宇, 黄翠锐, 杨光友, 谢跃, 何冉, 徐璟, 古小彬. 绵羊痒螨兔亚种MMP2基因的转录水平及其重组蛋白间接ELISA的建立[J]. 畜牧兽医学报, 2022, 53(2): 567-575.

GE You, ZHENG Jing, PU Shanqiu, ZHOU Yu, HUANG Cuirui, YANG Guangyou, XIE Yue, HE Ran, XU Jing, GU Xiaobin. The Transcription Level of MMP2 in Psoroptes ovis var. cuniculi and Establishment of the Indirect ELISA Based on Its Recombinant Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(2): 567-575.

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