摘要
采用RT-PCR方法自猪繁殖与呼吸综合征病毒基因组分离出核衣壳蛋白基因(orf 7),克隆到pMD18-T载体构建成重组质粒pMD18N并进行测序比较,结果表明,所克隆的核衣壳蛋白基因序列与PRRSV美洲型ATCC VR-2332株的同源性为100%,表明orf 7 是PRRSV基因组内很保守的序列;将orf 7亚克隆到原核表达载体pGEX-KG,构建成重组质粒pGEX-KGN,用pGEX-KGN转化表达菌株BL21,经SDS-PAGE和 Western-blot分析表明:克隆在谷胱苷肽转移酶(Glutathione S-transferase(GST)下游的核衣壳蛋白基因与GST获得了高效融合表达,表达的融合蛋白GST-N分子量约为41kDa,并且有免疫学反应活性;这为猪繁殖与呼吸综合征的血清学诊断方法的建立打下了基础。
The nucleocapsid protein gene (orf 7) of Porcine reproductive and respiratory syndrome virus (PRRSV) was isolated from PRRSV genome by RT-PCR. The gene was cloned into pMD18-T vector, the recombinant plasmid pMD18N containing orf 7 was sequenced and compared with other PRRSV isolates. The result shown that the homologies between the cloned orf 7 and America type PRRSV ATCC VR-2332 reached 100%, the orf 7 was highly conservative sequence in PRRSV genome. The orf 7 was subcloned into prokaryotic expressing vector pGEX-KG, the recombinant plasmid named pGEX-KGN was constructed. The pGEX-KGN was used to transform into E.coli BL21. The results of SDS-PAGE and Western-blot indicated that the nucleocapsid protein gene cloned in downstream of Glutathione S-transferase (GST) was expressed in a high level and the recombinant fusion protein, which was about 41kDa, had immunologically reactive activity. This study lay on foundation for the development of the diagnosis methods in serology for PRRSV.
出处
《中国病毒学》
CSCD
2003年第2期146-151,共6页
Virologica Sinica
基金
湖北省"十五"科技攻关资助项目(2001AA202A04)
关键词
猪
繁殖呼吸综合征病毒
核衣壳蛋白
基因克隆
原核表达
血清学诊断
Porcine reproductive and respiratory syndrome virus (PRRSV)
Nucleocapsid protein gene (orf 7)
Cloning and prokaryotic expression
