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

Abstract

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

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.

References

[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.