南方水稻黑条矮缩病原核表达及多克隆抗体制备
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摘要
南方水稻黑条矮缩病毒（SRBSDV）Rice black streaked dwarf virus是近年来在我国南部较多省份大面积爆发的一种水稻病毒，属呼肠孤病毒科(Reoviridae)斐济病毒属(Fijivirus)成员。其发病症状与水稻黑条矮缩病（Rice black streakeddwarf virus）类似，其田间主要的传毒介体是白背飞虱(Sogatella furcifera)。
SRBSDV基因组由10个dsRNA组成，目前已完成了该病毒基因组的全部测序。在基因组序列水平上，SRBSDV与RBSDV最为相似，其S5-ORF2、S7编码的两个产物及S9编码的两个产物均为非结构蛋白，S10编码产物为病毒外壳蛋白，影响着病毒的致病性和介体传播特性，S1所编码的蛋白可能为Rdrp，其他几个基因编码的蛋白功能正处于研究当中。本文报道了福建农林大学植物病毒研究所分离纯化的SRBSDV福建南平分离株的基因组S2、S8、S10片段多克隆抗体的制备，其抗血清可用于田间SRBSDV的快速诊断，也为进一步对蛋白功能以及病毒传病机理的研究提供素材和依据。
用生物信息学软件分析SRBSDV-P2、P8、P10基因所编码的蛋白并对其进行预测，选取抗原区域分布较多的一段用于多克隆抗体的制备。
RT-PCR扩增SRBSDV-P2、P8、P10三个基因片段，用gateway系统进行载体构建，先将目的片段重组到中间载体pDONR221，经二次重组构到原核表达载体pdest17，筛选鉴定获得目的载体重组子pDEST17-SRBSDV-P2、pDEST17-SRBSDV-P8、pDEST17-SRBSDV-P10，转入表达菌Rosseta菌中，分别在一定的IPTG诱导条件下表达目的蛋白。
弗氏佐剂乳化目的蛋白，采取肌肉多点注射的方法免疫实验兔；4～5次免疫后，分别获得SRBSDV-P2、SRBSDV-P8和SRBSDV-P10的三个抗血清。综合Elisa和Western blot的检测结果表明SRBSDV-P8和SRBSDV-P10抗血清特异性强，病毒检测的最优稀释倍数分别为1:400和1:100~1:200；SRBSDV-P2的抗体效价和特异性有待进一步研究。
SRBSDV（Rice black streaked dwarf virus）, a rice virus outbreak in a large areaof many provinces in southern China in recent years, is one of the Fijivirus memberswhich belong to Reoviridaes family. The onset of symptoms similar to the rice blackstreaked dwarf disease, and its main insect mediator in the field is Sogatella furcifera.
The genome of SRBSDV consists of10double-stranded RNAs (dsRNAs), andall of the viral genome sequencing has completed. In the genome, SRBSDV andRBSDV has a great similarity, according to the study of RBSDV, we can speculatethat the product of S5-ORF2and S7and S9are non-structural protein, S10encodingproducts of the virus coat protein affect virus pathogenicity and propagationcharacteristics of mediator, the protein encoded by the S1may be Rdrp, function ofother genes is in the research.
This paper reports polyclonal antibody preparation of fragment SRBSDV-S2&SRBSDV-S8&SRBSDV-S10of Fujian Nanping isolates, they can be used in thefield as a rapid method of detection of SRBSDV, but also to provide the material andthe basis for further study of protein function and mechanism of transfering disease.
Bioinformatics software for analysis SRBSDV-S2, S8, S10gene, then we selecta period gene of more antibody antigen regional distribution for preparation.
Sequences were amplified by RT-PCR, then inserted into the Prokaryoticexpression vector pDEST17, resulting pDEST17-SRBSDV-P2&pDEST17-RRSV-P8and pDEST17-RRSV-P10respectively. Later we transferred the Recombinants toRosseta, express the target protein under various IPTG induction conditions.
With Freund's adjuvant to emulsify target protein, multi-point injected themuscles of immunized rabbits, after4to5immunization, Obtained resistantSRBSDV-P2, RRSV-P8and RRSV-P10three antiserum. Elisa and Western blot testresults show that the SRBSDV-P8and SRBSDV-P10have a strong antiserumspecificity, and the optimal dilution factor of the virus detection were1:400and1:100~1:200. Antiserum specificity were detected by Western blot and using Elisa to testtheir potency. Antibody titer and specificity of SRBSDV-P2should be further study.
SRBSDV基因组由10个dsRNA组成，目前已完成了该病毒基因组的全部测序。在基因组序列水平上，SRBSDV与RBSDV最为相似，其S5-ORF2、S7编码的两个产物及S9编码的两个产物均为非结构蛋白，S10编码产物为病毒外壳蛋白，影响着病毒的致病性和介体传播特性，S1所编码的蛋白可能为Rdrp，其他几个基因编码的蛋白功能正处于研究当中。本文报道了福建农林大学植物病毒研究所分离纯化的SRBSDV福建南平分离株的基因组S2、S8、S10片段多克隆抗体的制备，其抗血清可用于田间SRBSDV的快速诊断，也为进一步对蛋白功能以及病毒传病机理的研究提供素材和依据。
用生物信息学软件分析SRBSDV-P2、P8、P10基因所编码的蛋白并对其进行预测，选取抗原区域分布较多的一段用于多克隆抗体的制备。
RT-PCR扩增SRBSDV-P2、P8、P10三个基因片段，用gateway系统进行载体构建，先将目的片段重组到中间载体pDONR221，经二次重组构到原核表达载体pdest17，筛选鉴定获得目的载体重组子pDEST17-SRBSDV-P2、pDEST17-SRBSDV-P8、pDEST17-SRBSDV-P10，转入表达菌Rosseta菌中，分别在一定的IPTG诱导条件下表达目的蛋白。
弗氏佐剂乳化目的蛋白，采取肌肉多点注射的方法免疫实验兔；4～5次免疫后，分别获得SRBSDV-P2、SRBSDV-P8和SRBSDV-P10的三个抗血清。综合Elisa和Western blot的检测结果表明SRBSDV-P8和SRBSDV-P10抗血清特异性强，病毒检测的最优稀释倍数分别为1:400和1:100~1:200；SRBSDV-P2的抗体效价和特异性有待进一步研究。
SRBSDV（Rice black streaked dwarf virus）, a rice virus outbreak in a large areaof many provinces in southern China in recent years, is one of the Fijivirus memberswhich belong to Reoviridaes family. The onset of symptoms similar to the rice blackstreaked dwarf disease, and its main insect mediator in the field is Sogatella furcifera.
The genome of SRBSDV consists of10double-stranded RNAs (dsRNAs), andall of the viral genome sequencing has completed. In the genome, SRBSDV andRBSDV has a great similarity, according to the study of RBSDV, we can speculatethat the product of S5-ORF2and S7and S9are non-structural protein, S10encodingproducts of the virus coat protein affect virus pathogenicity and propagationcharacteristics of mediator, the protein encoded by the S1may be Rdrp, function ofother genes is in the research.
This paper reports polyclonal antibody preparation of fragment SRBSDV-S2&SRBSDV-S8&SRBSDV-S10of Fujian Nanping isolates, they can be used in thefield as a rapid method of detection of SRBSDV, but also to provide the material andthe basis for further study of protein function and mechanism of transfering disease.
Bioinformatics software for analysis SRBSDV-S2, S8, S10gene, then we selecta period gene of more antibody antigen regional distribution for preparation.
Sequences were amplified by RT-PCR, then inserted into the Prokaryoticexpression vector pDEST17, resulting pDEST17-SRBSDV-P2&pDEST17-RRSV-P8and pDEST17-RRSV-P10respectively. Later we transferred the Recombinants toRosseta, express the target protein under various IPTG induction conditions.
With Freund's adjuvant to emulsify target protein, multi-point injected themuscles of immunized rabbits, after4to5immunization, Obtained resistantSRBSDV-P2, RRSV-P8and RRSV-P10three antiserum. Elisa and Western blot testresults show that the SRBSDV-P8and SRBSDV-P10have a strong antiserumspecificity, and the optimal dilution factor of the virus detection were1:400and1:100~1:200. Antiserum specificity were detected by Western blot and using Elisa to testtheir potency. Antibody titer and specificity of SRBSDV-P2should be further study.
引文
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2.陈声祥,关惠玲,廖璇刚,吕永平,沈升法,王藕芳,金梅松.2000.水稻黑条矮缩病毒在浙中的回升流行原因分析.浙江农业科学,33(6):287-289
3.陈声祥,张巧艳.2005.我国水稻黑条矮缩病和玉米粗缩病研究进展.植物保护学报,32(1):97-102
4.曹杨,潘峰,周倩,李冠华,刘双清,黄志农,李有志.2011.南方水稻黑条矮缩病毒介体昆虫白背飞虱的传毒特性.应用昆虫学报,48(5):524-530
5.丁新伦.2005.水稻齿叶矮缩病毒基因组S8和S10的原核表达及其抗血清制备硕士学位论文.福建:福建农林大学
6.郭荣,周国辉,张曙光.2010.水稻南方黑条矮缩病发生规律及防控对策初探.中国保护导刊,30(8):17-20
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