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_2 8b ( +)中 ,转化大肠杆菌BL2 1 (DE3 )plyss,于 3 0℃ 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 5 6kDa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 2 3 %。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。
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.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2003年第1期5-8,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国家"863"资助项目( 2 0 0 1AA2 1 3 1 41 )
