摘要应用差别pH值沉淀蛋白质的原理，建立了水稻条纹病毒病特异蛋白(SP)的两种提纯方法。这两种方法都可以从病叶中提纯到大量的SP，其粗提纯量分别为0.8和2.0mg/g病叶。通过SDS-PAGE分离后得到了精提纯的蛋白，其分子量为20.1kDa。将粗提纯和精提纯的SP分别免疫兔子，制备出效价为51200和6400的抗血清。将效价为6400的高度特异性的抗血清用于研究RSV-SP与RSV-CP及同属的水稻草状矮化病毒(RGSV)SP、CP之间的血清学关系，结果表明，RSV-SP的抗血清与RGSV-CP、RSV-CP之间无反应，但可与RGSV-SP微弱反应；而RGSV-SP、CP及RSV-CP的抗血清与RSV-SP之间都无血清学反应。结果证实了RSV和RGSV之间存在着进化上的亲缘关系。

Purification and Serological Characteristics of Disease-Specific

Protein of Rice Stripe Virus

Lin Hanxin Lin Qitian Wu Zujian

Lin Liming Lin Qiying Xie Lianhui

(Institute of Plant Virology of Fujian Agricultural University, Key Laboratory

of Plant Virology of Fujian Province, Fuzhou 350002)

Abstract According to the differential pH precipitation of proteins, two methods were established for purification of disease-specific protein (SP) in rice plants infected by rice stripe virus (RSV), a member of Tenuivirus. The yields obtained by the two methods were 0.8 and 2.0 mg SP per g of infected tissues, respectively. After separation by SDS-PAGE, further purified SP was obtained. The Mr of SP is 20.1 kDa. Antisera against the crude and further purified SP were prepared with the titer of 51 200 and 6 400, respectively. The antiserum against the further purified SP was proved to be highly specific, with which the serological relationships between RSV-SP and RSV-CP, CP and SP of rice grassy stunt virus (RGSV), another member of Tenuivirus were studied. Using PAS-ELISA, it was found that RSV-SP antiserum did not react with RSV-CP and RGSV-CP, but weakly reacted with RGSV-SP. No serological relationship existed between RSV-SP and the antisera raised against RSV-CP, RGSV-SP and RGSV-CP, respectively. These results confirmed that there is evolutionary relationship between RSV and RGSV.

Key words Rice stripe virus, Disease-specific protein, Purification, Serological characteristics

Rice stripe virus (RSV) has caused great decreases to rice yields in sixteen provinces in China^［1］. It also made severe damage to rice production in Japan, Korea and USSR^［2］. A large quantity of specific protein accumulated in infected rice tissues was found. The amount of this protein was proportional to the degree of severity of symptom and resistance of rice varieties, so the protein is designated as disease-specific protein (S-protein or SP)^［3］. The other members of Tenuivirus, rice grassy stunt virus (RGSV), rice hoja blanca virus (RHBV) and maize stripe virus (MStV), and the tentative members, Echinochloa hoja blanca virus (EHBV) and winter wheat striate mosaic virus (WWMV) also induce the accumulation of large amounts of SP in infected plants^［4-8］.

Analysis of the complete nucleotide sequence of RSV genome showed that SP is encoded by the viral-sense RNA4 segment^［9，10］. However, the role of SP remains unknown. Therefore, further studies on the protein are necessary. Here, we report the purification and serological characteristics of SP.

1 Materials and methods

1 Maintenance of vectors, plants and virus RSV isolated from Yunnan province was maintained and propagated in rice seedlings (Japonica variety, Hexi 28) by transmission with viruliferous small brown planthopper (Laodelphax striatellus Fallen). The plants with typical stripe symptoms were collected and used for purification.

2 Purification of SP We used two methods modified from those for MStV-SP^［4，5］ and RGSV-SP^［11］. Method I: fifteen grams RSV-infected leaves were ground in liquid nitrogen. The tissue powder was mixed with a phosphate-citrate buffer, pH5.0 (1 g tissue/3 mL buffer). Phosphate-citrate buffers of various pH's were prepared by combining 0.2 mol/L Na-2HPO₄ and 0.1 mol/L citric acid. The mixture was squeezed through three-layers cheesecloth and kept for 30 min at 4 ℃, then centrifuged at 15 000 g for 10 min (Beckeman JA-20 rotor). The pellet was resuspended with phosphate-citrate, pH7.0 (1mL/g tissue), then centrifuged at 27 000 g for 30 min. The pH of the supernatant was then lowered by adding an equal volume of phosphate-citrate, pH 3.0 and kept overnight at 4 ℃. After centrifugation at 12 000 g for 5 min, the pellet was resuspended with 20 mL phosphate-citrate, pH7.0, then centrifuged at 142 000 g for 3 h(Beckman Tyi 80 rotor). The supernatant was added with an equal volume of phosphate-citrate, pH 3.0 and held overnight at 4 ℃. During the night crystallization occurred. Crystals were pelleted at 12 000 g for 10 min and dissolved in 2 mL phosphate-citrate, pH 7.0. That was the crude purified SP preparation. Method II: Firstly, fifteen grams RSV-infected leaves were also ground in liquid nitrogen, then mixed with phosphate-citrate, pH 7.0 (1 g/2 mL buffer). The extract was squeezed and centrifuged at 12 000 g for 10 min. Twenty percent CCl₄ was added into the supernatant and stirred for 5 min, then centrifuged at 27 000 g for 30 min. The aqueous phase was separated. The next procedures were the same as method I.

SP obtained from differential pH precipitation was further purified by preparative gel electrophoresis. Samples were loaded without heating. After electrophoresis, the protein band was visualized by soaking the gel in 0.2 mol/L KCl solution for 5-15 min. The piece of gel containing the SP was excised and homogenized in phosphate-citrate buffer, pH7.0 with a mortar and pestle to extract the SP. Protein was precipitated by differential pH, saturated ammonium sulfate precipitation and centrifugation. Protein was also recovered from gel by using electro-elution, with procedures performed following the instruction manual (Bio-Rad model 422 electro-eluter). The recovered buffer was added with 2.5 volumes of methanol containing 0.1 mol/L ammonium acetate, stored in -20 ℃ for 2 h, then centrifuged at 13 000 g for 10 min. The pellet was suspended in 0.01 mol/L PBS, pH7.2, and dialyzed overnight against the same buffer.

3 Analysis of SP by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) One microliter for each of crude and further purified SP preparation was mixed with 4 μL loading buffer (1% SDS, 0.5% 2-mer-captoethanol, 2 mmol/L EDTA, 4% glycerol and 0.5% bromephenol blue). Samples were also made from the extracts prepared by griding infected leaves in 10 times loading buffer (w/v) and centrifugation at 15 000 g for 5 min. All the samples were heated at 100 ℃ for 3 min, and then electrophoresed. The standard molecular weights of markers were97.4, 66.2, 43.0, 31.0, 20.1 and 14.4 kDa.

4 Preparation of antiserum against SP Rabbits were immunized with the crude and further purified SP. Five intramuscular injections into the leg were given at weekly intervals with 0.7 mg crude SP and 0.2 mg further purified SP respectively, emulsified with an equal volume of incomplete Freund's adjuvant. Ten days after the last intramuscular injection 2.8 mg crude SP and 0.5 mg further purified SP were injected into the ear vein. Ten days after the intravenous injection, the antisera were collected, mixed with 20% glycerol and stored at 4 ℃.

5 Protein A sandwich-enzyme linked immunosorbent assay (PAS-ELISA) The procedures