Prokaryotic Expression Polyclonal Antibodies Preparation And Characteristic Analysis Of Rice Stripe Virus Nsvc2Gene Products

Rice stripe disease, which is caused by rice stirpe virus(RSV), is an important virus disease of rice production. RSV is transmitted by the small brown planthopper(Laodelphax striatellus Fallen, SBPH). It distributed in a large scale of China and brought severe crop losses in rice fields. At present, the transmission mechanisms of RSV by SBPH remained unclear, and it is crucial for cutting off virus transmission and disease control to research the mechanisms how RSV is transmitted specifically by SBPH. RSV has genomic structure similarity to Tomato spotted wilt virus(TSWV), and NSvc2protein of RSV is corresponded to glycoprotein(determinants for thrips transmission) of TSWV. So author hypothesized that NSvc2was determinant for SBPH transmission of RSV.To elucidate the function of NSvc2, the topology structure of NSvc2protein was analyzed firstly, and then R-GN, R-GC, R-GNS and R-GCS genes were cloned from RSV-infected rice via RT-PCR. Fusion protein expression recombinants were constructed by using prokaryotic expression vectors PGEX-4T-3and pET-32a, and transformed into E.coli BL21(DE3) to express proteins via IPTG inducing. The expression products were evaluated by SDS-PAGE, and results showed that they had expected sizes. The antisera of R-GNS and R-GCS were prepared using purified fusion proteins, and Western-blot analysis showed that the antisera could react with expression products specifically. Then antisera were used to detect the expression of NSvc2gene in RSV-infected rice tissue and viruliferous SBPH. The results demonstrated that RSV NSvc2protein existed in two different forms in rice and in SBPH. NSvc2was cleaved into two proteins R-GN (40kDa) and R-GC(53or56kDa) in rice, but in SBPH, NSvc2produced an intact protein (94kDa) without cleavage. The interaction relations between RSV CP and R-GN, R-GC were analyzed by using yeast two-hybrid assay. The results showed that both R-GN and R-GC could interact with RSV-CP, while interaction relations between R-GN and R-GC did not exist, which laid a foundation for identifying NSvc2as determinant for SBPH transmission of RSV.Additionally, yeast two-hybrid cDNA library of high-viruliferous (RSV-infected) SBPH populations was constructed with author’s participation. Viruliferous SBPH were maintained in our laboratory. Detection of the library indicated that it contained3.68×107independent clones, and the titer of the amplified library was2.62×1010cfu/mL. The recombination rate was above95%, and the average size of inserts was above1kb in the cDNA library. The results demonstrated that the library database meets the requirements of the standard cDNA library. The yeast two-hybrid cDNA library of high-viruliferous SBPH will be useful for the future research on the interaction between insect vector and R.S V.