牛结节性皮肤病病毒ORF002蛋白原核表达及间接ELISA抗体检测方法的建立

doi:10.11843/j.issn.0366-6964.2025.11.038

Abstract

Abstract:

The aim of this study was to establish an indirect ELISA antibody detection method based on the ORF002 protein of the Lumpy Skin Disease Virus (LSDV). By constructing the recombinant plasmid pET-30a(+) -ORF002 and inducing its expression, a protein with a molecular mass of approximately 13 ku was obtained. The ORF002 protein, verified by SDS-PAGE and Western blot, was used as the encapsulation antigen to establish an indirect ELISA antibody detection method for LSDV and to optimize the conditions. The optimized conditions for the indirect ELISA were as follows: antigen encapsulation concentration of 2.5 μg·mL^-1, serum dilution of 1∶100, secondary antibody dilution of 1∶120 000, and the OD_{450 nm} value of the sample≥standard positive value×20.34% was determined as positive, conversely, OD_{450 nm} value below this threshold were considered negative. The coefficient of variation from the repeatability test was < 10%, and there was no cross-reactivity observed with positive sera from various viruses. This method demonstrates good sensitivity, specificity, and reproducibility, making it suitable for the detection of LSDV in clinical serum samples and for evaluating the effectiveness of vaccine immunization.

Key words: lumpy skin disease, ORF002, prokaryotic expression, indirect ELISA

CLC Number:

S852.659.1

ZHANG Yaling, JING Wei, ZHAO Yan, HE Xiaobing, FANG Yongxiang, SU Yang, LI Xiaoming, ZHANG Hui, JING Zhizhong, CHEN Guohua. Establishment of Prokaryotic Expression of the ORF002 Protein of Lumpy Skin Disease Virus and an Indirect ELISA Antibody Detection Method[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5817-5825.

Figures/Tables 7

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References 16

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引物名称 Primers’ name	序列(5′→3′) Sequence
ORF002-Nde Ⅰ-F	$\underline{{\rm{GGAATTC}}}$CATATGAAGCTGATTAGCTTTATGTTCGG
ORF002-Xho Ⅰ-R	CTACTCGAGTTATTCCACCATATTCATCATACGTTC

血清稀释度 Serum dilution	样品 Sample	抗原包被浓度 Antigen concentration
血清稀释度 Serum dilution	样品 Sample	20.0	10.0	5.0	2.5	1.25	0.625
1∶100	P	1.361	1.308	1.258	1.290	1.263	1.266
	N	0.293	0.290	0.271	0.278	0.270	0.264
	P/N	4.578	4.641	4.736	5.088	4.663	3.981
1∶200	P	1.000	0.949	0.926	0.937	0.94	0.925
	N	0.231	0.219	0.203	0.195	0.202	0.193
	P/N	4.331	4.681	4.722	5.109	4.470	3.875
1∶400	P	0.628	0.601	0.564	0.564	0.562	0.579
	N	0.181	0.162	0.162	0.149	0.151	0.141
	P/N	3.583	3.627	3.908	3.930	2.694	2.586
1∶800	P	0.396	0.384	0.370	0.365	0.357	0.303
	N	0.145	0.127	0.122	0.114	0.122	0.115
	P/N	2.868	3.114	2.685	2.689	2.452	2.060

方法 Method	血清 Serum	竞争 ELISA Competitive ELISA		合计 Total	符合率/% Concordance level
方法 Method	血清 Serum	阳性血清 Positive serum	阴性血清 Negative serum	合计 Total	符合率/% Concordance level
间接ELISA Indirect ELISA	阳性血清 Positive serum	38	4	42	90.47
	阴性血清 Negative serum	0	12	12	100.00
	合计 Total	38	16	54	92.59

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Establishment of Prokaryotic Expression of the ORF002 Protein of Lumpy Skin Disease Virus and an Indirect ELISA Antibody Detection Method

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