Isolation And Identification Of Host Factors Interacted With The Disease-specific Protein (SP) Of Rice Stripe Virus And Production And Application Of A Monoclonal Antibody Against Rice Ragged Stunt Virus

In recent years, Rice stripe virus (RSV) has caused severe damage to rice production in china. This virus is a typical member of genus tenuivirus.The shape of the virus particles is filamentous. This virus is transmitted by Laodelphax Striatellus Fallen in a persistent, circulative-propagative manner and spread to their offsprings. To analyze the pathogenic mechanisms of rice stripe virus, a host factor interacted with disease-specific protein (SP) of RSV was isolated and identified.A yeast two-hybrid screen was performed to identify host proteins from a rice cDNA library, using the RSV SP as a bait. The plasmid pGBKT7-SP and rice cDNA library pGADT7-Rec-cDNA were co-transformed into yeast strain Y2H Gold. Based on the growth situations of yeasts in the nutrition detective media, the gene segments of candidate host factors were preliminarily selected. Through sequencing and gene blastx analysis, a R3H domain protein was selected as a candidate rice host factor that interacted with RSV SP. The full length gene of R3H was cloned from healthy rice. The full length R3H gene was inserted into the yeast vector pGADT7and constructed recombinant vector pGADT7-R3H. The yeast strain Y2H Gold with pGADT7-R3H and pGBKT7-SP could grow in the nutrition defective media and this result indicated that SP protein could interact with R3H domain protein. YFPN-SP and YFPC-R3H recombinant plasmids were constructed for a bimolecular fluorescence complementation experiment (BiFC) and transformed into agrobacterium strain C58C1. Under the confocol microscopy, Nicotiana benthamiana plant leaves agrobacterium-infiltrated produced obviously yellow fluorescence. So the interaction between R3H and SP proteins was further confirmed in Nicotiana benthamiana plant cells. Isolation and identification of R3H domain protein laid a foundation for finding out the mechanisms of pathogenicity of RSV.Rice ragged stunt virus (RRSV) was a member of genus Oryzavirus in family Reoviride and transmited through Nilaparvata lugens in a propagative and persistant manner. RRSV causes rice ragged stunt disease. In recent years, rice ragged stunt disease has caused severe damage to rice production in China. The coat protein gene (CP) of RRSV was amplified by RT-PCR from the total RNA of RRSV-infected rice leaves and inserted into prokaryotic expression vector pMAL-C2X to construct recombinant expression vector pMAL-C2X-CP. The recombinant expression vector pMAL-C2X-CP was transformed into E. coli BL21(DE3) strain and expressed by0.5mM IPTG induction for3hours. After purified with Amylose Resin affinity column, an about80KD recombinant fusion protein was obtained. The purified recombinant fusion CP was used to immunize BALB/c mice for producing monoclonal antibodies (MAbs). One hybridoma cell line secreting MAb against RRSV was obtained after cell fusion, cell selection and cell cloning. The ascitic fluid of the hybridoma was developed. A dot-ELISA method based on MAb4H5was set up for RRSV detection, and could successfully detect minimum viruses in rice and brown planthopper samples at1:320(w/v, g/mL) and1:400(a brown planthopper/uL) dilution, respectively. The MAb against RRSV and dot-ELISA serological method for RRSV detection had important significance for forecast and early warning of the virus disease and establishment of a scientific prevent and control system of RRSV.