摘要
以广东德庆市水稻瘤矮病的典型病株为材料,RT-PCR扩增并克隆了水稻瘤矮病毒(Rice gall dwarf virus,RGDV)的第9组分全长核苷酸序列。生物信息学分析表明,其基因结构特征与已报道的其他分离物基本一致。Pns9蛋白为细胞膜外蛋白,在植物呼肠孤病毒属内没有发现同源蛋白,与霍乱弧菌(Vibrio cholerae)的入口蛋白(portal protein)及姜氏军团菌(Legionella drancourtii LLAP12)的假设蛋白(hypothetical protein LDG_3259)分别有33%和24%的氨基酸序列相似性,功能尚待确定。将S9基因克隆至原核表达载体pET-28b(+)得到高效表达,融合蛋白经亲和层析纯化后,免疫新西兰大白兔制备抗血清。Western blot和ELISA分析表明制备的兔抗为特异抗血清,效价为1∶13 1072。
The full length cDNA of S9 sequence of Rice gall dwarf virus Guangdong isolate was cloned into pMD18-T vector and the complete nucleotide sequence was determined.Its gene organization and features were basically in accordance with other isolates reported before.Bioinformatics analysis of RGDV Pns9 was performed by using biology softwares and analysis tools online.Analysis results showed that Pns9 was an extramembranous protein,and no equivalent protein was found in other members of the genus Phythoreovirus.Pns9 shared 33% and 24% amino acid sequence identities respectively with portal protein of Vibrio cholera and hypothetical protein LDG3259 of Legionella drancourtii LLAP12.The RGDV S9 cDNA was cloned to pET28b（＋）and the protein was highly expressed in Escherichia coli Rosetta（DE3）III,and the expressed recombinant protein was purified and used to immunize the rabbit to prepare antiserum.The antiserum titer above 1∶131 072 measured by ELISA was obtained,and Western blot analysis indicated that the antiserum could serologically react with Pns9.
出处
《热带作物学报》
CSCD
2010年第12期2153-2158,共6页
Chinese Journal of Tropical Crops
基金
国家自然科学基金项目(No.30771407
No.30370929)
广东省自然科学基金项目(No.C036845)资助
关键词
水稻瘤矮病毒
S9
原核表达
抗血清制备
Rice gall dwarf virus
Pns9
Prokaryotic expression
Antiserum preparation
