期刊信息
- 刊名： 河北师范大学学报（自然科学版）Journal of Hebei Normal University (Natural Science)
- 主办： 河北师范大学
- ISSN： 1000-5854
- CN： 13-1061/N
- 中国科技核心期刊
- 中国期刊方阵入选期刊
- 中国高校优秀科技期刊
- 华北优秀期刊
- 河北省优秀科技期刊
猪繁殖与呼吸综合征病毒N蛋白的表达与纯化
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1. 河北师范大学生命科学学院, 河北 石家庄 050024;
2. 石家庄市畜牧水产局, 河北 石家庄 050056 -
DOI：
Expression and Purification of Nucleocapsid Protein of Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)
摘要/Abstract
根据GenBank报道的PRRSV VR2332基因序列设计引物,经RT-PCR扩增,得到大小约为390 bp的阳性产物;利用BamHⅠ,EcoRⅠ位点将N蛋白基因片段克隆到pET-28a载体,构建原核重组表达质粒pET-N,转化BL21(DE3)并进行SDS-PAGE,Western blot分析.结果表明:克隆的N蛋白基因与GenBank报道的VR2332基因同源性为93.83%;重组菌株经IPTG诱导后,N蛋白基因得到了高效表达;经筛选得到最佳诱导条件,即1 mmol/L IPTG诱导6 h,蛋白表达量可达菌体蛋白总量的52.635%,SDS-PAGE后切胶回收可得到纯化的N蛋白,为进一步制备免疫胶体金试纸条或ELISA试剂盒提供基础.
On the basis of the PRRSV VR2332 sequences reported by GenBank,ORF7 gene of porcine reproductive and respiratory syndrome virus(PRRSV) were obtained by RT-PCR after designing one pair of primers and the cDNA fragment was 390 bp approximately.cDNA fragment was cloned into the multiple cloning site(Bam H iv&EcoR iv) of pET28a(+) vectort and then transformed into E.coli BL21(DE3).The results were tested by SDS-PAGE and Western blot.The experimental results demonstrated that the cDNA sequence had 93.83% homology compared to VR2332's.The optimum condition of recombinant strain was induced by 1 mmol/L IPTG in 6 h and accumulation of the N protein was about 52.635% of total cellular protein.The N protein which has been purified after SDS-PAGE will be used for the further preparation of immunocolloidal gold test strip or the provision of basic ELISA Kit.