摘要
根据已报道的甘薯病毒G(SweetPotatoVirusG,SPVG)外壳蛋白(CP)基因的核苷酸序列合成引物,利用RT-PCR方法克隆了SPVG河南分离物(SPVG-HN)的CP基因,序列分析表明,SPVG-HNCP基因由1065个核苷酸组成(GenBank登录号为DQ399861),编码355个氨基酸残基。与GenBank中Egypt1(AJ515380)、LSU-1(AY178991)和LSU-3(AY178990)分离物的核苷酸序列相似性为98%左右,与中国广东分离物SPVG-CH(X76944)和SPVG-CH2(Z83314)的相似性分别为98.1%和85.5%。将CP基因克隆到原核表达载体pET-30a(+)上,SDS-PAGE分析表明,经IPTG诱导,CP基因在大肠杆菌BL21(DE3)pLysS中得到了高效表达。以表达的蛋白为抗原,免疫家兔,制备了SPVG外壳蛋白的特异性抗血清。间接ELISA检测结果表明,制备的抗血清可用于田间甘薯样品的检测。
According to published nucleotide sequence, coat protein （CP） gene of sweet potato virus G （SPVG） isolated from Henan province was cloned and sequenced. The results of sequence analysis showed that the CP gene consisted of 1065 nt encoding 355 amino acid residues＇ The CP gene nucleotide sequence were about 98% identical to Egypt1 （AJ515380）, LSU-1 （AY178991）, LSU-3 （AY178990） and SPVG-CH （X76944） isolates, respectively, and were 85.5% identical to SPVG-CH2 （Z83314） isolated from Guangdong province. The CP gene was constructed into expression vector pET30a （＋） for overexpres- sion in prokaryotic cells. The result of SDS-PAGE showed that about 39 kDa specific fusion protein was produced after induction by IPTG. The expressed protein was purified from SDS-PAGE and the antiserum against the protein was raised in rabbit. The antiserum was used for specific detection of SPVG from field samples of sweet potato by ACP-ELISA.
出处
《中国农学通报》
CSCD
2007年第1期38-41,共4页
Chinese Agricultural Science Bulletin
基金
河南省自然科学基金项目"侵染甘薯的Potyvirus病毒种类及其分子变异研究"(0311031500)
河南省省属科研院所科研专项资金项目"植物脱毒及病毒快速检测技术研究与开发"(1050114)。
关键词
甘薯病毒G
外壳蛋白基因
序列分析
原核表达
抗血清
Sweet potato virus G, Coat protein gene, Sequencing, Prokaryotic expression, Antiserum
