鸡滑液囊支原体FtsZ蛋白的原核表达及间接ELISA抗体检测方法的建立

doi:10.11843/j.issn.0366-6964.2025.11.031

摘要/Abstract

摘要：

滑液囊支原体(Mycoplasma synoviae, MS) 是一种重要的禽类病原体，其丝状温度敏感蛋白FtsZ (filamentous temperature-sensitive protein Z) 具有良好的免疫原性。本研究旨在基于MS-FJ01株的FtsZ基因，建立一种特异性高、重复性好的间接ELISA抗体检测方法。根据GenBank中MS-FJ01菌株的FtsZ基因序列设计特异性引物，构建重组表达质粒pET-28a-FtsZ，利用大肠杆菌原核表达系统表达并纯化FtsZ蛋白。以20 μg·mL^-1的纯化蛋白作为包被抗原，通过优化反应条件和检测步骤，建立基于FtsZ蛋白的MS间接ELISA抗体检测方法，评价其特异性、重复性及临床适用性。结果显示，该方法表现出良好的特异性，仅与MS阳性血清发生特异性反应，对鸡毒支原体(Mycoplasma gallisepticum, MG)、新城疫病毒(Newcastle disease virus, NDV)、禽流感病毒(avian flu virus, AIV)H9亚型、大肠杆菌及沙门菌阳性血清均无交叉反应。阴阳性临界值设定为S/P≥0.498(${\bar x}$+3*s，${\bar x}$=0.132，s=0.122)。批内和批间变异系数为1.32%~4.78%和2.81%~4.72%，表明重复性良好；与IDEXX试剂盒相比，阳性符合率为82.1%，阴性符合率为95.8%，总符合率为82%。本研究成功建立了一种基于FtsZ蛋白的间接ELISA抗体检测方法，具有高特异性、稳定性及操作简便的特点，为MS感染的快速诊断和流行病学监测提供了高效、经济的技术手段。

关键词: 滑液囊支原体, 间接ELISA, 细胞分裂蛋白, 抗体检测

Abstract:

Mycoplama synoviae (MS) is a significant avian pathogen, and its FtsZ protein (filamentous temperature-sensitive protein Z) has good immunogenicity. This study aimed to establish an indirect enzyme-linked immunosorbent assay (ELISA) for detecting MS antibodies based on the FtsZ gene of the MS-FJ01 strain, a conserved cell division protein with high immunogenicity. The FtsZ gene from the MS-FJ01 strain was cloned into pET-28a vector and expressed in Escherichia coli. The purified recombinant protein was used as a coating antigen to optimize ELISA conditions. Specificity and reproducibility was evaluated using sera from MS-infected and other pathogen-infected chickens. The established MS indirect ELISA antibody detection method demonstrated high specificity, and only had specific reaction with the positive serum of MS. The method showed no cross-reactivity with serum of MG, NDV, AIV H9, E. coli and Salmonella. The cutoff value (S/P≥0.498) was determined based on 30 negative sera (${\bar x}$=0.132, s=0.122). Intra-and inter-assay coefficients of variation (CV) were 1.32%-4.78% and 2.81%-4.72%, repeatability. Compared with IDEXX, the positive, negative, and total coincidence rates were 82.1%, 95.8%, and 82%, repeatability. This study successfully established an indirect ELISA antibody detection method based on the FtsZ protein of MS. The method demonstrates high specificity and reproducibility, offering a cost-effective tool for MS diagnosis and epidemiological surveilance.

Key words: Mycoplama synoviae, indirect ELISA, FtsZ protein, antibody detection

中图分类号:

S852.62

宋鸿燕, 陈富友, 贾晨宇, 黄煜洲, 赖钰芳, 周琦, 黄立栋, 陈吉龙, 李训良. 鸡滑液囊支原体FtsZ蛋白的原核表达及间接ELISA抗体检测方法的建立[J]. 畜牧兽医学报, 2025, 56(11): 5732-5742.

SONG Hongyan, CHEN Fuyou, JIA Chenyu, HUANG Yuzhou, LAI Yufang, ZHOU Qi, HUANG Lidong, CHEN Jilong, LI Xunliang. Expression of FtsZ Protein of Mycoplasma synoviae and Establishment of an Indirect ELISA Method for Antibody Detection[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5732-5742.

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引物名称 Primer name	引物序列(5′→3′) Primer sequence	产物长度/bp Product length (bp)
MS-JD506	F：CTTCTATGCTTAAACTTTCC R：TAAAGATATTACAACGACAT	506
MSFtsZ-1	F: CGGGATCCAAAAACATATTACATAGC R：CATTAG$\underline{{\rm{CCA}}}$CATATCAAATTC	1 293
MSFtsZ-2	F: GAATTTGATATG$\underline{{\rm{TGG}}}$CTAATG R：TTCATCTAAGTC$\underline{{\rm{CCA}}}$TGTGAA	302
MSFtsZ-3	F: TTCACA$\underline{{\rm{TGG}}}$GACTTAGATGAA R：CCCTCGAGTTAGA$\underline{{\rm{ACC}}}$AACTATATTTATC	156

HLA	SB	WB
DRB1_0103	6	31
DRB1_0301	6	56
DRB1_0402	4	21
DRB1_0403	15	46
DRB1_0404	5	22
DRB1_0405	13	31

血清稀释倍数 Dilutions of serum	指标 Index	抗原包被浓度/(μg·mL^-1) Coating concentration of the antigen (μg·mL^-1)
血清稀释倍数 Dilutions of serum	指标 Index	20	10	5	2.5	1.25	0.625
1∶20	P	1.47	1.46	1.43	1.41	1.38	1.35
	N	0.28	0.27	0.28	0.22	0.21	0.29
	P/N	5.23	5.12	5.12	6.52	6.69	4.60
1∶100	P	1.225	1.172	1.146	1.086	1.013	0.980
	N	0.162	0.156	0.151	0.154	0.156	0.157
	P/N	7.580	7.518	7.594	7.065	6.496	6.235
1∶200	P	0.64	0.51	0.63	0.32	0.45	0.42
	N	0.19	0.13	0.12	0.12	0.11	0.11
	P/N	3.33	4.03	5.26	2.78	4.12	3.97
1∶500	P	0.493	0.398	0.303	0.368	0.316	0.238
	N	0.194	0.114	0.177	0.169	0.119	0.147
	P/N	2.538	3.498	1.718	2.171	2.664	1.614

二抗稀释倍数 Dilution of second antibody	指标 Index	反应时间Reaction time
二抗稀释倍数 Dilution of second antibody	指标 Index	30 min	45 min	60 min
1∶5 000	P	1.372	1.21	1.03
	N	0.187	0.13	0.12
	P/N	7.343	9.53	8.63
1∶10 000	P	1.511	1.64	1.17
	N	0.193	0.13	0.12
	P/N	7.811	10.82	9.57
1∶20 000	P	1.222	1.17	1.09
	N	0.173	0.14	0.12
	P/N	7.065	8.61	9.07

血清序号 Serum No.	S/P	血清序号 Serum No.	S/P	血清序号 Serum No.	S/P
1	0.288	11	0.045	21	0.109
2	0.320	12	0.036	22	0.195
3	-0.054	13	0.021	23	0.102
4	0.246	14	0.155	24	0.154
5	0.182	15	0.001	25	0.252
6	-0.093	16	0.275	26	0.116
7	0.129	17	0.161	27	0.025
8	0.146	18	0.075	28	0.315
9	0.015	19	0.247	29	-0.047
10	0.260	20	0.316	30	-0.021