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浙江省科技计划项目(011102120)

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番鸭呼肠孤病毒ZJ99株σC基因在甲醇酵母中的表达被引量:3
2006年
将番鸭呼肠孤病毒ZJ99株σC基因插入到酵母表达载体pPIC9K内,转化大肠杆菌TG1,挑取阳性克隆,获得酵母表达重组质粒pPIC9K-σC。用DraⅠ酶切线性化重组表达质粒pPIC9K-σC和对照质粒pPIC9K,电转化至甲醇酵母GS115感受态细胞内,经G418抗性筛选和PCR鉴定,获得了2株pPIC9K-σC酵母阳性整合子和2株pPIC9K空载体对照酵母阳性整合子。分别挑取1株进行甲醇诱导表达,SDS-PAGE检测发现pPIC9K-σC酵母整合子在不同时段的诱导表达上清液中均有可见的目的蛋白条带,分子质量与预计大小相符,而对照pPIC9K酵母整合子的诱导表达上清液中没有相应的蛋白条带。说明番鸭呼肠孤病毒ZJ99株σC基因真核甲醇酵母表达体系构建成功,目的σC蛋白在酵母中能够有效地分泌表达。
余旭平刘慧芳何世成郑新添沈琴芳张秀亮刘江梅
关键词:番鸭呼肠孤病毒甲醇酵母
番鸭呼肠孤病毒ZJ99株σC蛋白基因的克隆与序列分析被引量:2
2005年
参考已发表的番鸭呼肠孤病毒(mDRV)的S4序列设计了1对扩增σC蛋白基因的引物,对mDRVZJ99株的RNA抽提物进行RTPCR扩增,获得了特异性的扩增片段。将PCR产物克隆测序,序列经同源性比较,发现mDRVZJ99株σC蛋白基因序列与福建MW9710株对应基因的同源性为99.5%,与法国89026株对应基因的同源性为94.2%,与法国89330株对应基因的同源性为95.0%;相应的氨基酸序列同源性分别为98.9%、94.1%、93.7%。基于σC基因及其推导氨基酸序列的蛋白质结构及物理化学性质预测结果,与国外学者对mDRVσC蛋白的报道相似。
何世成朱军莉金俊杰郑新添沈琴芳张秀亮余旭平
关键词:番鸭呼肠孤病毒克隆
番鸭“新鸭疫”病原中呼肠孤病毒的初步确证被引量:3
2005年
A so called "new duck disease" with the character of necrosis of spleen and pancreas broke out in flocks of muscovy ducks at the end of 1999 in Zhejiang province. Viruses with different conformation and size, one of which was similar to reovirus were found under electronic microscope in allantoic fluid for virus isolation. Three pairs of primers were designed and synthesized based on the published S1 sequences of muscovy duck reovirus (mDRV). Specific RT-PCR products were obtained from both diseased tissues and allantoic fluid for virus isolation. Cloning and sequencing of the fragment and BLAST analysis result further verified the existence and participation of mDRV in the disease. A 1 260 bp S1 full-length sequence of the virus (ZJ99) was further amplified and sequenced. Phylogenic analysis result based on S1 sequences showed that the ZJ99 was in the same group with mDRVs of the French strain and the Fujian strain, while it had some distance to the group of the chicken reoviruses and was far more distant to that of mammalian. On the other hand, the nucleotide sequence of S1 of the ZJ99 strain had a homology of 98% with that of Fujian strain (300 bp partial sequence), while only had 90% homology to that of French strain. It was speculated that mDRVs isolated from different area of China, which had some differences with French strains, were the same strain but spreading in different area.
余旭平何世成朱军莉金俊杰郑新添沈琴芳张秀亮
关键词:番鸭病原呼肠孤病毒S1基因
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