高粱花叶病毒外壳蛋白的原核表达及其抗血清制备

生物技术通报 ›› 2013, Vol. 0 ›› Issue (1): 166-171.

高粱花叶病毒外壳蛋白的原核表达及其抗血清制备

王洪星 ,龚殿, 孙玉娟 ,张雨良, 王健华, 刘志昕

中国热带农业科学院热带生物技术研究所农业部热带作物生物学与遗传资源利用重点实验室，海口 571101

收稿日期:2012-08-08 修回日期:2013-01-31 出版日期:2013-01-30 发布日期:2013-01-30
作者简介:王洪星，男，助理工程师，硕士，研究方向：分子植物病理学; E-mail ：hxingw@163.com
基金资助:
中央级公益性科研院所基本科研业务专项（ITBB110303）, 现代农业产业技术体系建设专项资金（nycytx-24）

Prokaryotic Expression of CP Gene of Sorghum mosaic virus and Preparation of Polyclonal Antibody

Wang Hongxing ,Gong Dian, Sun Yujuan ,Zhang Yuliang ,Wang Jianhua, Liu Zhixin

Key Laboratory of Biology and Genetic Resources of Tropical Crops，Ministry of Agriculture，P.R. China，Institute of Tropical Bioscience and Biotechnology，Chinese Academy of Tropical Agricultural Science，Haikou 571101

Received:2012-08-08 Revised:2013-01-31 Published:2013-01-30 Online:2013-01-30

摘要/Abstract

摘要： 高粱花叶病毒（Sorghum mosaic virus，SrMV）是世界上分布最广的侵染甘蔗的病毒之一，引起甘蔗花叶病，对甘蔗产业危害很大。根据SrMV 外壳蛋白（coat protein，CP）基因序列合成一对引物，以海南（SrMV-HN）染病植株总RNA 为材料，采用RT-PCR 方法克隆了长约987 bp 的目的片段。将CP 基因与质粒pET32a（+）连接，构建了含SrMV CP 基因的融合蛋白原核表达载体pET32a-SrMVCP。然后用正确的重组质粒转化大肠杆菌Rosetta（DE3），经IPTG 诱导后，SDS-PAGE 检测出一条约36kD 的特异融合蛋白表达谱带。融合蛋白主要以可溶性蛋白形式稳定表达。通过优化诱导条件，确立了CP 基因表达的最佳条件：IPTG 终浓度为0.1 mmol/L，诱导时间为4 h，诱导温度为30℃。用Ni2+-NTA 琼脂糖亲和层析纯化融合蛋白，免疫家兔制备出抗血清。通过酶联法（ID-ELISA）测定本试验制备的SrMV CP 抗血清工作浓度为1∶1 000，Western blotting 检测结果表明，抗血清与SrMV-HN 诱导表达的CP 蛋白发生特异性反应。对田间25 个甘蔗样品进行检测，结果表明该抗血清的效价高、灵敏度高、特异性强，可以应用于田间样品的检测。

关键词: 高粱花叶病毒 , 外壳蛋白 , 原核表达 , 抗血清

Abstract: Sugarcane mosaic disease caused by Sorghum mosaic virus(SrMV)is now recognized as one of the most widely distributedviral diseases of Saccharum. It is an important limiting factor for sugarcane production. According to the sequence of coat protein(CP)gene ofSrMV, two primers were designed, and a DNA fragment about 987 bp was amplified by RT-PCR. The PCR product, after digested with restrictionenzymes, was inserted into prokaryotic expression vector pET32a and the recombinant plasmid pET32a-SrMVCP was transferred into E.coliRosetta(DE3). Induction with IPTG, recombinant protein was got and SDS-PAGE analysis and western blot showed that CP was expressedsteadily in soluble proteins. With addition of IPTG up to 0.1 mmol/L and culturing for 4 h more in 37℃ is thought to be the best inducingconditions to express CP. The fusion protein was then purified with Ni2+-NTA agarose affinity chromatography, and used to immune rabbits forantiserum preparation. The optimal titer of the antiserum was determined to be 1∶1 000 tested by indirect enzyme-linked immunosorbent assay(ID-ELISA). Western blotting confirmed that the antiserum reacted strongly and specifically to the CP of SrMV-HN field sugarcane sampleswere detected using the antiserum by a methed of ID-ELISA.

Key words: Sorghum mosaic virus , Coat protein , Prokaryotic expression , Antiserum

王洪星, 龚殿, 孙玉娟, 张雨良, 王健华, 刘志昕. 高粱花叶病毒外壳蛋白的原核表达及其抗血清制备[J]. 生物技术通报, 2013, 0(1): 166-171.

Wang Hongxing, Gong Dian, Sun Yujuan, Zhang Yuliang, Wang Jianhua, Liu Zhixin. Prokaryotic Expression of CP Gene of Sorghum mosaic virus and Preparation of Polyclonal Antibody[J]. Biotechnology Bulletin, 2013, 0(1): 166-171.

参考文献

[1] 周国辉, 许东林, 蔡艳清, 等. 我国南方甘蔗病毒种类初步鉴定[J]. 广西农业生物科学, 2006, 25（3）：226-228.
[2] Viswanathan R, Balamuralikrishnan M, Karuppaiah R. Characterizationand genetic diversity of sugarcane streak mosaic virus causingmosaic in sugarcane[J]. Virus Genes, 2008（36）：553-564.
[3] Viswanathan R, Balamuralikrishnan M, Karuppaiah R. Duplexreversetranscription polymerase chain reaction（D-RT-PCR）-a techniquefor the simultaneous detection of viruses causing sugarcane mosaic[J]. Sugar Tech, 2008, 10（1）：81-86.
[4] Grisham MP, Johnson RM, Zimba PV, et a1. Detecting Sugarcaneyellow leaf virus infection in asymptomatic leaves with hyperspectralremote sensing and associated leaf pigment changes[J]. VirolMethods, 2010, 167（2）：140-145.
[5] 王建南, 许莉萍, 陈如凯. 甘蔗褪绿线条病致病机理研究初报[J]. 甘蔗, 1995, 2（4）：10-13.
[6] 王洪星, 张雨良, 刘志昕, 等. 应用多重RT-PCR 检测甘蔗黄叶病毒和高粱花叶病毒[J], 广东农业科学, 2011, 38（10）：128-131.
[7] Shukla DD, Frenkle MJ, Mckern NM. Present status of sugarcanemosaic subgroup ofotyviruses[J]. Archive of Virology, 1992（l5）：363-373.
[8] Shukla DD, Ward CW. Structure of potyvirus coat proteins and itsapplication in the taxonomy of the potyvirus group[J]. Adv VirusRes, 1989, 36 ：273-314.
[9] Van Regenmorte MHV, Fauquet CM, Bishop DHL, et al. Virus Taxonomy[M]. Seventh Report of the International Committee on Taxonomyof Viruses. New York ：Academic Press, 2000 ：691-701.
[10] Chen J, Chcn J, Adams MJ. Characterization of potyviruses fromsugarcane and maize in China[J]. Archives of Virology, 2002,147（6）：1237-12.
[11] Harlow E, LanD. Antibodies[M].1st Edition. New York ：ColdSpring Harbor Laboratory Press, 1998.
[12] 张婷婷, 叶波平. 包涵体蛋白质的复性研究进展[J]. 药物生物技术, 2007, 144 ：306-309.
[13] 王洪星, 张雨良, 刘志昕, 等. 以ScYLV 和SrMV 为材料进行常见PCR 引物优化设计[J]. 中国糖料, 2012（1）：16-20.
[14] 余乃通, 冯团诚, 刘志昕, 等. 香蕉束顶病毒海口分离物核穿梭蛋白（NSP）的纯化及抗血清制备[J]. 基因组学与应用生物学, 2010, 29（2）：375-378.
[15] 余乃通, 冯团诚, 刘志昕, 等. 香蕉束顶病毒海口分离物CP基因的原核表达、纯化及其抗血清制备[J]. 热带作物学报,2010, 31（5）：767-771.
[16] 李向东, 范在丰, 石鹏君. 甘蔗花叶病毒CP 基因的高效表达和抗血清制备[J]. 生物技术通讯, 2003, 14（4）：271-273.