水稻齿叶矮缩病毒Pns10蛋白的原核表达、抗血清制备及初步应用

摘要

The genome of RRSV comprises 10 segments of double-stranded RNA ( dsRNA ) and the segment 10 encodes a non-structural protein Pns10. The dsRNA was extracted from infected RRSV Shaxian isolates( RRSV-F ). A set of PCR primers were designed according to the ORF in S10 of RRSV Thailand isolate. RRSV-F S10 was cloned and analyzed, the 35 kD product was expressed using Directional TOPO Vector in BL21 star ( DE3 ) E. coli. The antiserum a-gainst PnslO was prepared in rabbits at a titer of 1:7 680 by indirect ELISA, and its specificity was demonstrated by Western blotting. The fusion proteins PnslO and its antiserum provide important materials for the function of PnslO and the antiserum was also used for the detection of infected rice and the vector insect in the field and the disease diagnosis.%水稻齿叶矮缩病毒Pns10蛋白由基因组片段S10编码.从RRSV福建沙县分离物(RRSV-F)中获取该病毒的全基因组,根据RRSV泰国分离物核苷酸序列设计特异性引物获得S10编码区,利用Directional TOPO克隆技术,将S10连接至克隆表达载体pET100/D-TOPO构建重组质粒,并进行了序列测定和分析.重组质粒经IPTG诱导在BL21star(DE3)E.coli中高效表达约35 kD Pns10蛋白.将Pns10蛋白免疫新西兰大白兔制备了特异性的抗血清,间接ELISA法测定抗血清效价为1:7 680,Western blot 分析表明抗血清特异性强.表达产物及抗血清在Pns10蛋白的结构和功能研究中具有重要的应用价值,制备的抗血清可用于水稻病株和传播介体的检测以及病毒病的诊断.