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

Abstract

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

CLC Number:

S852.62

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.

Figures/Tables 8

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References 43

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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

Expression of FtsZ Protein of Mycoplasma synoviae and Establishment of an Indirect ELISA Method for Antibody Detection

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