| 狂犬病病毒SRV9克隆株核蛋白基因的克隆、表达与特性分析 |
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| 引用本文： | 袁慧君,扈荣良,张守峰,张茂林,涂长春.狂犬病病毒SRV9克隆株核蛋白基因的克隆、表达与特性分析[J].中国预防兽医学报,2003,25(1):5-8. |
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| 作者姓名： | 袁慧君 扈荣良 张守峰 张茂林 涂长春 |
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| 作者单位： | 解放军军需大学军事兽医研究所病毒学研究室,吉林,长春,130062 |
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| 基金项目： | 国家“863”资助项目( 2 0 0 1AA2 1 3 1 41 ) |
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| 摘 要： | 根据已发表的狂犬病病毒核蛋白基因序列，设计并合成了一对引物，从SAD株驯化的SRV9。蚀斑株中提取病毒RNA，通过RT-PCR扩增出核蛋白的全长cDNA序列，测序结果显示，其序列与国外报道的SAD母源株序列一致。将核蛋白的cDNA克隆至原核表达载体pET-28b( )中，转化大肠杆菌BL21（DE3）plyss，于30℃1mmol/LIPTG条件下诱导表达，大肠杆菌菌体裂解产物经SDS-PAGE分析，在分子量约为56kDa处出现一新的蛋白带。和预期的目的蛋白分子量相符，Western-blotting检测表明，表达产物能与狂犬病病毒阳性血清发生特异性反应，出现单一反应带，扫描分析显示，表达产物占菌体总蛋白的23%，包涵体分离，纯化后，纯度达89%，上述结果为核蛋白在狂犬病基因免疫和免疫检测中的进一步应用奠定了基础。
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| 关 键 词： | 特性分析 狂犬病病毒 核蛋白 RT－PCR 基因克隆 基因表达 |
| 文章编号： | 1008-0589(2003)01-0005-04 |
| 修稿时间： | 2002年5月9日 |
Cloning, Sequencing and Expression of N Gene of Rabies Virus Subclone Strain SRV9 |
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| YUAN Hiu-jun,HU Rong-liang,ZHANG Shou-feng,ZHANG Mao-lin,TU Chang-chun.Cloning, Sequencing and Expression of N Gene of Rabies Virus Subclone Strain SRV9[J].Chinese Journal of Preventive Veterinary Medicine,2003,25(1):5-8. |
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| Authors: | YUAN Hiu-jun HU Rong-liang ZHANG Shou-feng ZHANG Mao-lin TU Chang-chun |
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| Abstract: | Genomic RNA was isolated from subcloned rabies virus strain SRV 9 and was used as template for cDNA synthesis of its N gene.The cDNA was amplified by PCR,sequenced and cloned into expression vector pET-28b.The recombinant plasmid was then transoformed into E.coli BL21(DE3).Expression of the protein was induced by IPTG.SDS-PAGE and Western-blotting were performed to analyze the N gene production.Results showed that sequence of the N gene of SRV 9 was completely consistent with its original parental strain SDA.SDS-PAGE results showed that the protein was highly expressed in E.coli .It accounted for 23% of the total proteins.Moleclar weight of the expressed protein was 56 kDa.The expressed protein was specific to antisera against RV by Western-blotting analysis,being presented as a single reaction band.All above demonstrated that the cloned gene was nucleoprotein cDNA of rabies virus and the protein was rabies nucleoprotein specific.It laid a foundation for construction of nucleoprotein genetic vaccine was well for diagnostic reagent development. |
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| Keywords: | RV 9 Nucleoprotein RT-PCR Sequencing expression |
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