基于RdRp蛋白的兔出血症病毒感染与免疫鉴别诊断ELISA方法的建立

doi:10.11843/j.issn.0366-6964.2026.01.036

Abstract

Abstract:

To establish a diagnostic ELISA method for distinguishing RHDV infection and vaccination， the RdRp proteins of RHDV1 and RHDV2 were expressed by prokaryotic expression system. And these two soluble RdRp proteins were successfully purified. RdRp protein was used as coated antigen， an indirect ELISA method was established to detect serum antibody against RdRp. After purification and restriction enzyme digestion， the RdRp protein without tag was obtained with a size of 57 ku. Western blot analysis confirmed that both RdRp proteins reacted specifically with RHDV1 or RHDV2 infection sera. And there was no difference between the types of RdRp proteins. So RHDV2-RdRp was selected as a coating antigen at a coating concentration of 2.5 μg·mL^-1. The optimal serum dilution was 1∶100 and the incubation was 60 min at 37 ℃. The optimal dilution of enzyme-labeled antibody was 1∶10 000. The cut-off value was 0.225. It was proved that the ELISA method has good specificity and sensitivity. The coefficient of variation in both intra-batch and inter-batch repeatability test was less than 10%， which indicating a good stability. The samples after RHDV challenge and the serum samples after genetic engineering vaccine immunization were tested， and the results showed that RHDV infection can induce the production of serum antibodies against RdRp protein， but the serum tests after genetic engineering vaccine immunization were negative. In this study， the ELISA method based on RHDV-RdRp protein can distinguish between genetic engineering vaccine immunity and RHDV infection， which provides an effective detection method for clinical screening of RHDV infection.

Key words: RHDV, RNA-dependent RNA polymerase （RdRp）, indirect ELISA

CLC Number:

S852.61

ZHU Wei, QIU Rulong, CHEN Mengmeng, HU Bo, WEI Houjun, FAN Zhiyu, GE Lei, WU Mengting, SONG Yanhua, WANG Fang. Establishment and Application of Indirect ELISA Method based on RHDV RdRp Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2026, 57(1): 414-422.

Figures/Tables 7

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References 30

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序号 Number	株系 Strain	登录号 Accession No.	基因型 Gene type	国家 Country	分离年份 Isolation year
1	AUS/NSW/CAT_2-12/2007	KX357674.1	GI.4	澳大利亚	2007
2	AUS/NSW/LWR-1/2010	KX357676.1	GI.4	澳大利亚	2010
3	AUS/VIC/BM-60/2009	KX357705.1	GI.4	澳大利亚	2009
4	CD/China	AY523410.1	GI.1	中国	2004
5	GI.3/O cun/FR/2006/06-11	MN737115.1	GI.3	法国	2006
6	GI.3/Ocun/FR/2008/08-133	MN746289.1	GI.3	法国	2008
7	GI.3/O cun/FR/2009/09-48	MN737116.1	GI.3	法国	2009
8	HB	KY437668.1	GI.1	中国	2016
9	Italy-90	EU003579.1	GI.1	意大利	1990
10	LIB	MN853659.1	GI.2	波兰	2018
11	NJ-2009	HM623309.1	GI.1	中国	2009
12	RHDV2-NL2016	MN061492.1	GI.2	荷兰	2016
13	RHDV-V351	U54983.1	GI.1	捷克	1996
14	RHDV-N11	KM878681.1	GI.2	西班牙	2011
15	Rij06-12	KP129395.1	GI.2	西班牙	2014
16	RHDV2-SC2020/0401	MT586027.1	GI.2	中国	2020
17	SOS404	MG763950.1	GI.2	葡萄牙	2015
18	ZD0	KU882095.1	GI.1	波兰	2000

编号 Number	OD_{450 nm}值 OD_{450 nm}value	编号 Number	OD_{450 nm}值 OD_{450 nm}value	编号 Number	OD_{450 nm}值 OD_{450 nm}value	编号 Number	OD_{450 nm}值 OD_{450 nm}value
1	0.130 9	11	0.096 5	21	0.191 9	31	0.070 4
2	0.115 7	12	0.109 8	22	0.072 5	32	0.102 5
3	0.115 7	13	0.140 2	23	0.199 1	33	0.117 6
4	0.156 9	14	0.116 9	24	0.081 3	34	0.138 4
5	0.143 6	15	0.081 8	25	0.107 2	35	0.071 9
6	0.187 0	16	0.103 5	26	0.158 6	36	0.125 7
7	0.168 9	17	0.095 7	27	0.117 9	37	0.178
8	0.170 4	18	0.109 0	28	0.101 8	38	0.149 4
9	0.084 1	19	0.117 1	29	0.098 9	39	0.134 5
10	0.116 2	20	0.071 3	30	0.090 0	40	0.136 8

Establishment and Application of Indirect ELISA Method based on RHDV RdRp Protein

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