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
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摘要
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。
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 BL21star(DE3) E.coli.The antiserum against Pns10 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 Pns10 and its antiserum provide important materials for the function of Pns10 and the antiserum was also used for the detection of infected rice and the vector insect in the field and the disease diagnosis.
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 BL21star(DE3) E.coli.The antiserum against Pns10 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 Pns10 and its antiserum provide important materials for the function of Pns10 and the antiserum was also used for the detection of infected rice and the vector insect in the field and the disease diagnosis.
引文
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[13]周雪平,李德葆.双链RNA技术在植物病毒研究中的应用[J].生物技术,1995,5(1):1-4.ZHOU Xueping,LI Debao.Plant virus research based on dsR-NA methods[J].Biotechnology,1995,5(1):1-4.
[2]谢联辉.近年来我国新发现的水稻病毒病[J].植物保护,1984,10(3):2-3.XIE Lianhui.Rice virus diseases recently found in China[J].Plant Protection,1984,10(3):2-3.
[3]吴美爱.水稻齿叶矮缩病毒三个分离物S6-S10片段的基因结构特征分析[D].硕士学位论文,福建农林大学,2008.WU Meiai.Genome structure analysis on segment 6 to segment10 of Rice ragged stunt virus Chinese isolates[D].Dissertationfor Master’s Degree,Fujian Agriculture and Forestry University,2008.
[4]郑璐平,谢荔岩,连玲丽,等.水稻齿叶矮缩病毒的研究进展[J].中国农业科技导报,2008,10(5):8-12.ZHENG Lupin,XIE Liyan,LIAN Lingli,et al.Research pro-gress on Rice Ragged Stunt Virus(RRSV)[J].Journal of Agri-cultural Science and Technology,2008,10(5):8-12.
[5]叶永发.水稻锯齿叶矮缩病的症状表现[J].福建农业科技,2010,(2):47.YE Yongfa.Symptoms of rice ragged stunt disease[J].FujianAgricultural Science and Technology,2010,2:47.
[6]KAWANO S,SHIKATA E,SENBOKU T.Purification and mor-phology of rice ragged stunt virus[J].Journal of the Faculty ofAgriculture,Hokkaido University,1983,61:209-218.
[7]KAWANO S,UYEDA L,SHIKATA E.Particle structure anddouble-stranded RNA of rice ragged stunt virus[J].Journal ofthe Faculty of Agriculture,Hokkaido University,1984,61:408-418.
[8]陆惠华,吴建华,龚祖埙,等.水稻齿叶矮缩病毒双链RNA的体外翻译[J].生物化学与生物物理学报,1987,19(5):354-359.LU Huihua,WU Jianhua,GONG Zuxun,et al.In vitro transla-tion of 10 segments of the ds-rna of rice ragged stunt virus(rrsv)[J].Acta Biochimica et Biophysica Sinica,1987,19(5):354-359.
[9]UPADHYAYA N M,RAMM K,GELLATLY J A,et al.Riceragged stunt oryzavirus genome segments S7 and S10 encodenon-structural proteins of M(r)68,025(Pns7)and M(r)32,364(Pns10)[J].Arch Virol,1997,142(8):1719-1726.
[10]刘瑛.两种植物呼肠孤病毒的小管蛋白鉴定及其功能结构域研究[D].硕士学位论文,福建农林大学,2011.LIU Ying.Identification and studies on the tubule--forming pro-teins of two kinds of retroviruses[D].Thesis for Master’S De-gree,Fujian Agriculture and Forestry University,2011.
[11]邵朝纲.水稻齿叶矮缩病毒的分子生物学研究及新城疫病毒检测新技术[D].博士学位论文,中国科学院研究生院(上海生命科学研究院),2004.SHAO Zhaogang.Molecular biological studies on rrsv&ndv-a-nalysis-software development[D].Dissertation for PHD,Chi-nese Academy of Science,2004.
[12]韩昭.水稻齿叶矮缩病毒Pns10的定位及其自身互作研究[D].硕士学位论文,福建农林大学,2011.HAN zhao.Studies on the cellular location and self-interactionof rice ragged stunt virus pns10[D].Thesis for Master’s De-gree,Fujian Agriculture and Forestry University,2011.
[13]周雪平,李德葆.双链RNA技术在植物病毒研究中的应用[J].生物技术,1995,5(1):1-4.ZHOU Xueping,LI Debao.Plant virus research based on dsR-NA methods[J].Biotechnology,1995,5(1):1-4.