SRBSDV结构蛋白P10和非结构蛋白P9-1抗体的制备及应用

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Abstract Southern rice black-streaked dwarf virus (SRBSDV), the genus Fijivirus in the family Reoviridae, is transmitted mainly by white backed planthopper (Sogatella furcifera, WBPH) in a persistent- propagative manner. SRBSDV encodes at least 6 putative structural proteins (P1, P2, P3, P4, P8 and P10) and 7 putative nonstructural proteins (P5-1, P5-2, P6, P7-1, P7-2, P9-1 and P9-2). P10 are putative major outer capsid proteins, while P9-1 can form viroplasms which is the putative sites of viral replication. In this study, to enrich the prediction system of SRBSDV, polyclonal antibodies of P10 and P9-1 were prepared. By using a Gateway prokaryotic system, firstly, part fragment of P10 (591 bp) and P9-1 (714 bp) were recombined to the pDONR221 vector, respectively. After verified by sequencing, they were recombined to the expression vector pDEST17, respectively. Then they were transformed into Escherichia coli strain Rosetta. By isopropyl-β-d-thiogalactoside (IPTG) inducing, recombinant proteins of 27 kD (for P10) and 33 kD (for P9-1) were obtained. The recombinant protein was used to immunize New Zealand white rabbits (Oryctolagus cuniculus) for preparation of polyclonal antibodies. By an indirect enzyme-linked immunosorbent assay (in-ELISA) method, the antiserum titer of P10 and P9-1 were determined to be 1∶6 400 and 1∶3 200, respectively. Western blot analysis showed that P10 and P9-1 antibodies could capture proteins of 63 and 39.9 kD in infected rice (Oryza sativa) plants, respectively, which were consistent of the actual length of the P10 and P9-1. There were no any protein captured by the 2 antibodies in healthy rice plants. Our results revealed that both antibodies could detect the infected rice plants specifically. To compare the application effect of the 2 antibodies in the detection of infected rice plants and viruliferous percent of WBPHs, immunocapture-RT-PCR (IC-RT-PCR) and dot-ELISA assays were done. In the IC-RT-PCR assays, using infected rice plants and viruliferous WBPHs as template and specific primers for P9-1 and P10, target fragments of P9-1 and P10 could obtained. In the dot-ELISA assays, a 78.7% viruliferous percent of WBPHs was obtained by using P9-1 or P10 antibodies, which showed a consistent result of the RT-PCR. Meanwhile, both the antibodies could capture the virus protein in infected rice plants by using the dot-ELISA. And there were no any blot in healthy rice plants. Our results revealed that both structural protein and nonstructural protein of SRBSDV could be used for the viral field detection, which provide support for forecast of the virus disease.

Key words： Southern rice black-streaked dwarf virus (SRBSDV) Antibody Immunocapture-RT-PCR (IC-RT-PCR) Dot enzyme-linked immunosorbent assay (dot-ELISA)

Received: 19 January 2016 Published: 01 July 2016

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	Articles by authors
	WU Jin-Hong
	CHEN Xiao-Min
	LIN Wen-Wu
	YUAN Bai-Jie
	WU Jie-Jian
	ZHANG Ji

Cite this article:

WU Jin-Hong,CHEN Xiao-Min,LIN Wen-Wu, et al. Preparation and Application of Polyclonal Antibodies Against Structral Protein P10 and Non-structral Protein P9-1 Encoded by SRBSDV[J]. , 2016, 24(8): 1190-1198.

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http://journal05.magtech.org.cn/Jwk_ny/EN/ OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2016/V24/I8/1190

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