马铃薯Y病毒衣壳蛋白抗原表位分析及其快速ELISA检测方法的建立

doi:10.3864/j.issn.0578-1752.2021.06.007

Abstract

Abstract:

【Objective】Potato virus Y (PVY) is one of the most serious viruses that affect the yield and quality of potato. At present, no effective virus treatment agent has been found, and application of virus-free seed potato is the main control measure to prevent PVY damage. The objective of this study is to establish a rapid detection method with high sensitivity and specificity, and to provide technical support for quality control of virus-free seed potato. 【Method】PVY coat protein (CP) was expressed as small polypeptides with overlapping parts, and the epitopes of PVY-CP were analyzed by Western blot. Matching experiments were performed on monoclonal antibodies that recognized different epitopes by conventional double antibody sandwich ELISA (DAS-ELISA), and a pair of monoclonal antibodies with the best detection effect was screened. At the same time, the captured and detected antibodies were confirmed. According to the standard of maximum P/N value, the working concentration of antibody was determined by square titration. The optimal co-incubation time of antibody and antigen was determined by control variable method. The sensitivity of rapid DAS-ELISA was tested with different concentrations of PVY-CP protein as antigen. Potato samples infected with different viruses were used as antigens to detect the specificity of rapid DAS-ELISA. Through rapid DAS-ELISA and RT-PCR detection of 50 samples of potato samples with suspected infection PVY collected in the field, the coincidence rate was tested. Finally, the established rapid DAS-ELISA was used to detect potato samples infected by different PVY strains. 【Result】A panel of monoclonal antibodies (9G6 and 3D3) that can be used for DAS-ELISA were screened using the expressed peptides of PVY CP, and a rapid DAS-ELISA was established based on this panel of MAbs. The ELISA plate coated with the capture antibody 9G6, and then co-incubated with detector antibody HRP labeled 3D3 (2 μg·mL^-1) and samples for 5 min at 37℃ after blocking the plate. The detection limit was 0.5 ng·mL^-1. The results of specific analysis showed that the established method was only positive for potato samples infected with PVY, and negative for potato virus S (PVS), potato virus M (PVM) and potato leaf-roll virus (PLRV). In comparison with RT-PCR, DAS-ELSIA had the coincidence rate of 96% through testing 50 potato simples, and the results of PVY^N and PVY^O potato samples were positive. 【Conclusion】 The established PVY detection method has high sensitivity and specificity, and can be completed within 30 min, which is convenient and fast, and provides key technical support for high-throughput detection of PVY and the production of virus-free seed potato.

Key words: potato virus Y (PVY), monoclonal antibody, antigen epitope, double antibody sandwich ELISA (DAS-ELISA)

YuXin LIANG,JianXiang WU,XiaoYu LI,ChunYu ZHANG,JiChao HOU,XuePing ZHOU,YongZhi WANG. Mapping of Epitopes and Establishment of Rapid DAS-ELISA for Potato Virus Y Coat Protein[J].Scientia Agricultura Sinica, 2021, 54(6): 1154-1162.

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References 35

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片段 Segment	引物 Primer	引物序列Primer sequence (5′-3′)
PVY-CP_1-180	PVY-AF	GGGATCCCCGGAATTCATGGGAAATGACACAATCGATG
PVY-CP_1-180	PVY-AR	GATGCGGCCGCTCGAGTTAGTGGTGGTGGTGGTGGTGCGGTTCCTTTTTGTTGCGC
PVY-CP_90-281	PVY-BF	GGGATCCCCGGAATTCCAGTTTGATACGTGGTATGAAGC
PVY-CP_90-281	PVY-BR	GATGCGGCCGCTCGAGTTAGTGGTGGTGGTGGTGGTGTGCGGCCGCAAGCTTGTCGA
PVY-CP_1-59	PVY-CF	GGGATCCCCGGAATTCATGGGAAATGACACAATCGATG
PVY-CP_1-59	PVY-CR	GATGCGGCCGCTCGAGTTAGTGGTGGTGGTGGTGGTGCTTAGGCATCCTCATTTTGGAC
PVY-CP_31-89	PVY-DF	GGGATCCCCGGAATTCAAGGAAAAGGACGTGAATGTTGG
PVY-CP_31-89	PVY-DR	GATGCGGCCGCTCGAGTTAGTGGTGGTGGTGGTGGTGTGATTGAGTTGCTCGAGTATTTG
PVY-CP_90-148	PVY-EF	GGGATCCCCGGAATTCCAGTTTGATACGTGGTATGAAGC
PVY-CP_90-148	PVY-ER	GATGCGGCCGCTCGAGTTAGTGGTGGTGGTGGTGGTGTGGTTTCAGTGGGTATTCGAC
PVY-CP_120-180	PVY-GF	GGGATCCCCGGAATTCTGCATTGAAAATGGAACCTCG
PVY-CP_120-180	PVY-GR	GATGCGGCCGCTCGAGTTAGTGGTGGTGGTGGTGGTGCGGTTCCTTTTTGTTGCGC

分段表达蛋白 Segmented expression protein	单克隆抗体Monoclonal antibody
分段表达蛋白 Segmented expression protein	3D3	9G6	3E4
PVY-CP_1-180	+	+	+
PVY-CP_90-281	+	+	-
PVY-CP_1-59	-	-	+
PVY-CP_31-89	-	-	+
PVY-CP_90-148	+	+	-
PVY-CP_120-180	+	-	-

检测抗体浓度 Concentration of detection antibody (μg·mL^-1)	捕获抗体浓度Concentration of capture antibody (μg·mL^-1)
检测抗体浓度 Concentration of detection antibody (μg·mL^-1)	4	2	1	0.5
2	12.72±0.95*	7.56±0.93	5.10±0.53	4.05±0.13
1	7.44±0.77	4.42±0.07	2.82±0.12	2.46±0.01
0.5	5.17±0.36	3.31±0.04	2.34±0.19	2.03±0.07
0.25	2.87±0.25	2.14±0.01	1.81±0.18	1.59±0.15

Mapping of Epitopes and Establishment of Rapid DAS-ELISA for Potato Virus Y Coat Protein

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