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猪生殖与呼吸综合征病毒非结构蛋白nsp4基因
的表达及其产物的纯化
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胡涛，才学鹏
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(中国农业科学院 兰州兽医研究所 家畜疫病病原生物学国家重点实验室
甘肃省动物寄生虫病重点实验室，甘肃 兰州730046)
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摘要：
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根据GenBank中已登录的猪生殖与呼吸综合征病毒(PRRSV)全基因组序列,设计合成了1对特异性引物,对PRRSV非结构蛋白nsp4基因进行了RT-PCR扩增，将回收的目的基因片段克隆到大肠埃希氏菌表达载体pET28a中,构建了重组质粒pET-nsp4，测序结果证实了重组质粒pET-nsp4的可靠性。将pET-nsp4转化至大肠埃希氏菌表达菌株BL21(DE3)，用IPTG诱导表达，SDS-PAGE结果表明，重组菌可表达分子质量约23ku的蛋白。经镍离子亲和层析柱(Ni-NTA)纯化，获得了高纯度的重组蛋白，Western-blot分析结果表明，nsp4重组蛋白在大肠埃希氏菌系统中获得了正确表达。 |
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关键词：
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猪生殖与呼吸综合征病毒；nsp4基因；表达；纯化 |
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中图分类号：S 852.659.6：Q 786 文献标识码：
A 文章编号：1673-4696(2006)09-0687-05 |
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Expression and product purification of the nonstructural protein 4 gene
of porcine reproductive and respiratory syndrome virus |
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HU Tao, CAI Xue-peng |
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(Key Laboratory of Animal Parasitology of Gansu Province/State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China)
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Abstract: The nonstructural protein 4 gene of porcine reproductive and respiratory syndrome virus was amplified by RT-PCR with a pair of specific primers, and then the product was cloned into pET28a vector and sequenced. The full length of PRRSV nsp4 gene was inserted into the NcoⅠ-SalⅠ sites of pET28a vector with a correct open reading frame (ORF).The plasmid pET-nsp4 was transformed into Escherichia coli BL21 (DE3) for expression under induction of IPTG. The results of SDS-PAGE analysis revealed that the molecular weight of the expressed product was 23ku. The expressed protein was purified in one step using Ni-NTA affinity chromatography. The results of Western-blot analysis showed that the purified protein could be specifically recognized by anti-His tag antibody. |
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Key words: PRRSV; nsp4 gene; expression; purification |
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